CN104471075B

CN104471075B - Nucleic acid amplification

Info

Publication number: CN104471075B
Application number: CN201380031868.1A
Authority: CN
Inventors: C-Y·李; D·拉夫; S-M·陈; J·奥尼尔; R·卡辛斯卡斯; J·罗恩伯格
Original assignee: Life Technologies Inc
Current assignee: Life Technologies Inc
Priority date: 2012-04-19
Filing date: 2013-03-15
Publication date: 2018-06-22
Anticipated expiration: 2033-03-15
Also published as: SG10201802883UA; CN109486902B; CN104471075A; SG11201406717RA; CN116064734A; CN114854832A; CN109486902A; WO2013158313A1

Abstract

In some embodiments, this teachings provides the method for nucleic acid amplification, including forming reaction mixture and reaction mixture experience being made to be suitable for the condition of nucleic acid amplification.In some embodiments, include nucleic acid to be amplified is made to undergo partial denaturation condition for the method for nucleic acid amplification.In some embodiments, it is expanded in the case of being included in the nucleic acid for being not exclusively denaturalized and being amplified for the method for nucleic acid amplification.In some embodiments, the enzyme and polymerase of catalysis homologous recombination are used for the method for nucleic acid amplification.In some embodiments, the method for nucleic acid amplification can be carried out in single reaction vessel.In some embodiments, the method for nucleic acid amplification, the fixation of compartmentation or reactive component without reaction mixture can be carried out in the single Continuous Liquid Phase of reaction mixture.In some embodiments, include optionally depositing in the case of polymers for the method for nucleic acid amplification, expand at least one polynucleotides on the surface under the conditions of isothermal duplication.Polymer may include that sieving agent and/or diffusion reduces agent.

Description

Nucleic acid amplification

Background of invention

Nucleic acid amplification is highly useful in molecular biology and actually in biology, acology, diagnostics, legal medical expert It learns and each aspect of research has wide applicability.Normally, it is generated using one or more primers from starting template Amplicon, wherein amplicon, which are corresponded to or are complementary to from it, generates the template of the amplicon.Multiplex amplification can also make process simple Change and reduce expense.The application is related to method and reagent for nucleic acid amplification and/or analysis.

Summary of the invention

Method, reagent and the product of nucleic acid amplification and/or analysis is provided herein.Amplification can utilize it is fixed and/or The primer of dissolving.Single group primer can be mixed from different templates or can connect single template and a variety of different primers It touches or a variety of different templates can be contacted with a variety of different primers.The amplicon generated from methods provided herein It is the substrate for being suitable for further analyzing such as sequencing.

In some embodiments, this teachings provides the composition for nucleic acid amplification, system, method, apparatus And kit.

Attached drawing is described in detail

Fig. 1 provides the schematic diagram of the embodiment of indicating template walking (template walking).Selectable In embodiment, fixed primer, which includes, to be named as (A)_nThe sequence rich in adenosine, such as (A)₃₀, and needle in template The complementary sequence rich in T, such as (T) are included to the primer binding site of immobilized primer₃₀。

Fig. 2 describe the amplification walked on globule by template and globule planar array on accumulation for The sketch plan of sequencing.

Fig. 3 describes some selectable embodiments of the detection based on semiconductor using synthesis order-checking.Template row It walks and can be used for generating clonal expansion subgroup on globule or in the substrate of reative cell or bottom.In selectable embodiment In, fixed primer, which includes, to be named as (A)_nThe sequence rich in adenosine, such as (A)₃₀, and immobilized primer is directed in template Primer binding site include the complementary sequence rich in T, such as (T)₃₀。

Fig. 4 describe the fixation site in the form of primer lawn (primer lawn) on a planar substrate some are optional The embodiment selected.It can be considered solid that the array in discontinuous fixed site or the single continuous lawn of primer, which can be used, The random array of anchor point.Optionally, position of one or more fixed sites in the continuous lawn of primer can be also not Determining, wherein position is expert at when the attachment of starting template until then and is determined or as the space occupied by the cluster expanded To determine.In selectable embodiment, fixed primer, which includes, to be named as (A)_nThe sequence rich in adenosine, such as (A)₃₀, and the complementary sequence rich in T is included in template for the primer binding site of immobilized primer, such as (T)₃₀。

Fig. 5 shows influence of the temperature to template walking reaction.Template walking is calculated and depicts for reaction temperature to expand The chart of Δ Ct before and after increasing.

Fig. 6 provides the table of the Ct values of 96 dual TaqMan qPCR reactions.

Fig. 7 describes the data of about 100,000 times of amplifications for showing and being carried out on globule by template walking.For Reaction time calculates and depicts the Δ Ct before and after template walking reaction and the expansion before and after template walking reaction Double number.

Fig. 8 provides exemplary chain overturning and the schematic diagram of Running strategy describes.(A) template walk, (B) chain overturning with The chain of overturning is generated, (C) adds new primer binding sequence Pg ' on final overturning chain.

Fig. 9 is described from the Ion of polynucleotide template that the amplified reaction of recombinase-mediated is used to expand Torrent^TMThe exemplary reading length histogram of PGM sequencing operations.

Figure 10 is described from the Ion of polynucleotide template that the amplified reaction of recombinase-mediated is used to expand Torrent^TMThe exemplary reading length histogram of Proton sequencing operations.

Figure 11 is described from the Ion of polynucleotide template that the amplified reaction of recombinase-mediated is used to expand Torrent^TMThe exemplary reading length histogram of Proton sequencing operations.

Figure 12 is described from the Ion of polynucleotide template that the amplified reaction of recombinase-mediated is used to expand Torrent^TMThe exemplary reading length histogram of Proton sequencing operations.

Figure 13 includes the diagram of exemplary measurement system.

Figure 14 includes the diagram of exemplary measurement assembly.

Figure 15 includes the diagram of the array of exemplary measurement assembly.

Figure 16 includes the diagram of exemplary bore structure.

Figure 17 includes the diagram of exemplary bore and sensor structure.

Figure 18, Figure 19, Figure 20 and Figure 21 include the diagram of the work package during being handled by illustrative methods.

Figure 22, Figure 23 and Figure 24 include the diagram of the work package during being handled by illustrative methods.

Figure 25, Figure 26 and Figure 27 include the diagram of the work package during being handled by illustrative methods.

Figure 28 shows the exemplary block of the component of the system for nucleic acid sequencing according to exemplary implementation Figure.

Figure 29 shows the exemplary cross of the part of the IC apparatus and flow cell according to exemplary implementation Figure.

Figure 30 is shown according to the exemplary of the representative chemical sensor of exemplary implementation and corresponding reaction zone Sectional view.

Detailed description of the invention

Nucleic acid-templated conventional amplification generally includes the weight of the template (and/or its filial generation) using appropriate synthesis system It replicates.In such conventional method, each example of duplication usually is waited to be expanded by using extreme Denaturing denaturation The template of increasing starts, so that template is substantially single-stranded.Some for the extreme Denaturing of conventional amplification are logical The thermal denaturation of the temperature far above nucleic acid-templated melting temperature to be amplified often is included the use of with widely used example (for example, Standard PCR is included the use of far above 90 DEG C, the normally about thermal cycle of 94-95 DEG C of denaturation temperature) or template are to strong Exposure of the denaturant of power such as NaOH, guanidine salt reagent.Such method usually requires (such as the thermal cycle of special equipment Instrument), and need during amplification procedure additional operation (such as annealing steps for Standard PCR；For removing chemistry Washing step of denaturant etc.), so as to increase cost relevant with such amplification, workload and time and limitation The yield that can finally be obtained using such method.In addition, such extreme Denaturing is usually so that mould to be amplified Plate is substantially single-stranded, so as to which to be related to multiple clonal expansion, (i.e. multiple different templates are in identical reaction mixture Clonal expansion) extensive application propose challenge.For such multiple application, the uses of these extreme Denaturings can be with It runs counter to desire, because this typically results in a chain of template from the release of its position of associating, so that the chain of release is certainly Migrated in solution by ground and pollute it is other develop in close proximity to amplicon.Such cross contamination typically results in reduction Monoclonal amplification group yield and increase the yield of polyclonal pollutant (be not generally available to many downstream application).It needs It is related to conventional amplification method to eliminate to want improved nucleic acid amplification method (and relevant composition, system and kit) The defects of.

In some embodiments, present disclosure generally relates to the method for nucleic acid amplification and relevant combination Object, system and device, the method includes amplification of nucleic acid templates to generate the expansion for including the substantially polynucleotides group of monoclonal Increase son.It has been generally acknowledged that monoclonicity is desired in nucleic acid determination, because the difference of different polynucleotides is special in polyclonal group Property may be such that determination data explanation complicate.One example is related to nucleic acid sequencing application, wherein the presence of polyclonal group can So that the explanation of sequencing data complicates；It is detected however, many sequencing systems are not enough to sensitive arrive from single polynucleotide template Nucleotide sequence data, therefore need before sequencing the clonal expansion of template.

In some embodiments, amplification method in the present disclosure can be used for optionally mixing using identical reaction Object and expand to clone that two or more are different nucleic acid-templated in identical reaction mixture, to generate at least two The substantially nucleic acid group of monoclonal.Optionally, the expansion that the group of at least one substantially monoclonal passes through single polynucleotide template Increase and formed.

Optionally, two or more different nucleic acid-templated simultaneously and/or are parallelly expanded.

In some embodiments, present disclosure generally relates to (and the relevant combination of nucleic acid synthetic method Object, system and kit), the method includes：At least two double-stranded nucleic acid templates are provided in the reactive mixture；And basis Any method clone ground disclosed herein expands at least two double-stranded nucleic acid template, to form at least two substantially The nucleic acid group of monoclonal.

In some embodiments, the amplification of clone ground optionally includes to form reaction mixture.Reaction mixture may include Continuous liquid phase.In some embodiments, continuous liquid phase includes single continuous aqueous phase.Liquid phase may include two or more A polynucleotide template, the polynucleotide template is optionally with identical nucleotide sequence or can be with different from each other Nucleotide sequence.In some embodiments, at least one of the two or more polynucleotide templates may include At least one at least one other polynucleotide template in reaction mixture is not substantially identical or substantially not complementary Nucleic acid sequence.

In some embodiments, two or more different nucleic acid-templated be confined to before amplification, be placed in or be located at Different sites.

In some embodiments, in the solution, two or more are expanded to clone optionally in single reaction mixture Multiple and different is nucleic acid-templated, and after such clonal expansion, two or more substantially monoclonals of gained Nucleic acid group is then confined to, is placed in or positioned at different sites.

Different sites is optionally the member of site array.Array may include surface (such as flow cell, electronics dress Put, the surface of transistor chip, reative cell, slot etc.) on site two-dimensional array or matrix or other mediums (such as solid, Semisolid, liquid, fluid etc.) in site cubical array.

Optionally, two or more different nucleic acid-templated Continuous Liquid Phases in same reaction mixture, normally connect It is amplified in continuous water phase, so as to generate two or more different polynucleotides groups with substantially monoclonal, wherein each A polynucleotides group is generated by being present in the amplification of single polynucleotide template in reaction mixture.

Optionally, Continuous Liquid Phase is included within single phase or the identical phase of reaction mixture.

In some embodiments, present disclosure generally relates to (and the relevant combination of nucleic acid synthetic method Object, system and kit), the method includes：Double-stranded nucleic acid template is provided；With by expand the double-stranded nucleic acid template come Form the nucleic acid group of substantially monoclonal.Optionally, it is nucleic acid-templated to include clone ground amplifying doulbe-chain for amplification.

Optionally, amplification, which is included under substantially isothermy, carries out at least one amplification bout.

Optionally, amplification, which is included under substantially isothermy, carries out at least two continuous nucleic acid synthesis cycles.

In some embodiments, amplification includes recombinase polymeric enzymatic amplification (RPA).For example, amplification may include carrying out extremely A few RPA bout.

In some embodiments, amplification includes template walking.For example, amplification may include carrying out at least one template row Walk bout.

In some embodiments, amplification, which is optionally included in site or reative cell, carries out two different expand back It closes.For example, amplification may include carrying out at least one RPA bouts in site or reative cell with any sequence of bout or combination And at least one template walking bout is carried out in site or reative cell.In some embodiments, any one or more are expanded At least two continuous circulate under substantially isothermy in bout carry out.In some embodiments, bout is expanded It is at least one to be carried out under substantially isothermy.

Optionally, it is to be amplified nucleic acid-templated to be double-strand or cause the mould using appropriate program before amplification Plate is at least part double-strand.(herein template to be amplified with nucleic acid-templated or polynucleotide template is interchangeable makes With).In some embodiments, template is linear.Alternatively, template can be cricoid or comprising linear and annular section Combination.

Optionally, double-stranded nucleic acid template includes positive chain.Double-stranded nucleic acid template also may include reverse strand.Positive chain is optional Ground includes the first primer binding site.Reverse strand optionally includes the second primer binding site.

In some embodiments, template has included the first and/or second primer binding site.Alternatively, template is optionally Primer binding site is not included initially, primer binding site is connected to or drawn before being optionally included in amplification by disclosed method Enter template.For example, method optionally includes being connected to or be otherwise directed into mould by the connector comprising primer binding site Plate.Can connector be connected to or is otherwise directed into the end or the linear or main body of circular template of linear die.Appoint Selection of land, or can circularized template after introducing connector in connection.In some embodiments, the first connector can be connected to or be drawn Enter the first end of linear die, and can the second connector be connected to or be introduced the second end of template.

In some embodiments, amplification is included by the template of partial denaturation and the first primer, with the second primer or with the One primer and the second primer are contacted with random order or combination.

In some embodiments, the first primer includes the first primer sequence.The first primer is optionally comprising extendible End (such as 3 ' ends containing OH).The first primer is optionally connected to compound (such as " resistance label (drag Tag) ") or it is connected to support (such as globule or site or surface of reative cell).

In some embodiments, the second primer includes the second primer sequence.Second primer is optionally comprising extendible End (such as 3 ' ends containing OH).The second primer is optionally connected to compound (such as " resistance label ") or connection To support (such as globule or site or surface of reative cell).

Optionally, the first primer is bound to the first primer binding site to form the first primer-template double-strand.Second draws Object can be bound to the second primer binding site to form the second primer-template double-strand.

In some embodiments, amplification includes extending the first primer to form the first primer of extension.For example, amplification It may include extending the first primer of the first primer-template double-strand to form the first primer of extension.

Optionally, extended through polymerase.Polymerase can be strand displacement polymerase.

In some embodiments, amplification includes contacting template to be amplified with recombinase.

In some embodiments, amplification includes forming the template of partial denaturation.For example, amplification may include partial denaturation Double-stranded nucleic acid template.

Optionally, partial denaturation includes double-stranded nucleic acid template is made to undergo partial denaturation condition.

In some embodiments, partial denaturation condition includes the temperature less than nucleic acid-templated Tm, including such as less than The temperature of 5 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C or 50 DEG C nucleic acid-templated of Tm.In some embodiments, partial denaturation item Part is included higher than (for example, at least 5 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C or 50 DEG C of highers) the first primer, the second primer or the One and second primer Tm temperature.In some embodiments, partial denaturation condition include higher than (for example, at least 5 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C or 50 DEG C of highers) the first primer binding site, the second primer binding site or the first primer combine The temperature of the Tm of position and the second primer binding site.In some embodiments, it is nucleic acid-templated can one or two end End includes joint sequence, and partial denaturation condition may include the temperature of the Tm higher than joint sequence.In some embodiments In, partial denaturation condition (particularly partial denaturation temperature) for during " template walking " selective amplification it is nucleic acid-templated, As being described further herein.

In other embodiments, partial denaturation condition include with it is one or more can be optionally with sequence-specific Or nucleic acid-templated to be amplified nucleic acid-templated of enzymatic treatment of sequence oriented approach partial denaturation or make its contact.In some realities It applies in scheme, at least one enzyme optionally with sequence-specific fashion catalysis chain intrusion and/or untwists.Optionally, it is a kind of or more Kind enzyme includes one or more enzymes selected from recombinase, topoisomerase and unwindase.In some embodiments, part becomes Property template may include template with recombinase is contacted to and formed the nucleoprotein complex comprising recombinase.Optionally, there are Template is contacted with recombinase in the case of one primer, the second primer or the first and second primers.Partial denaturation may include using Recombination enzymatic chain is exchanged and is hybridized the first primer with the first primer binding site (or by the second primer and the second primer knot Close position hybridization).In some embodiments, partial denaturation include the use of recombinase carry out chain exchange and by the first primer with The first primer binding site and the second primer is hybridized with the second primer binding site.

In some embodiments, the template of partial denaturation includes single stranded portion and double stranded section.In some embodiments In, single stranded portion includes the first primer binding site.In some embodiments, single stranded portion includes the second primer engaging portion Position.In some embodiments, single stranded portion includes the first primer binding site and the second primer binding site.

In some embodiments, partial denaturation template includes contacting template with one or more nucleoprotein complexs. At least one of nucleoprotein complex may include recombinase.At least one of nucleoprotein complex may include primer (for example, The primer of one primer or the second primer or sequence comprising primer binding sequence complementation corresponding with template).In some realities It applies in scheme, partial denaturation template may include contacting template with the nucleoprotein complex comprising primer.Partial denaturation can wrap It includes and hybridizes the primer of nucleoprotein complex primer binding site corresponding with template, so as to form primer-template double-strand.

In some embodiments, partial denaturation template may include template and the first nucleoprotein comprising the first primer Complex contacts.Partial denaturation may include being combined the first primer of the first nucleoprotein complex with the first primer of positive chain Position hybridizes, so as to form the first primer-template double-strand.

In some embodiments, partial denaturation template may include template and the second nucleoprotein comprising the second primer Complex contacts.Partial denaturation may include being combined the second primer of the second nucleoprotein complex with the second primer of reverse strand Position hybridizes, so as to form the second primer-template double-strand.

In some embodiments, disclosed method (and relevant composition, system and kit) may also include one Or multiple primer extension procedures.For example, method may include that being incorporated to nucleotide by using polymerase carrys out extension primer.

In some embodiments, polymerase is strand displacement polymerase.

Optionally, extension primer is included primer and polymerase and the nucleotide of one or more types in nucleotide simultaneously It is contacted under the conditions of entering.In some embodiments, the nucleotide of one or more types does not include exogenous marker, especially Ground optically detectable label, such as fluorescence part or dyestuff.Optionally, reaction mixture includes nucleotide, is natural Existing nucleotide.Optionally, nucleotide does not include group (such as double deoxidation group, the reversible termination for terminating nucleic acid synthesis Son etc.).Normally, extension primer is occurred with template dependent manner.

Optionally, disclosed method (and relevant composition, system and kit) including by using polymerase by one A or multiple nucleotide are incorporated to the first primer of the first primer-template double-strand to extend the first primer, so as to form the of extension One primer.

Optionally, disclosed method (and relevant composition, system and kit) is including passing through any appropriate method Second primer is bound to the second primer binding site of the primer of the first extension by (such as connection or hybridization).

Optionally, disclosed method (and relevant composition, system and kit) including by using polymerase by one A or multiple nucleotide are incorporated to the second primer of the second primer-template double-strand to extend the second primer, so as to form the of extension Two primers.

In some embodiments, extension the first primer leads to the formation of the primer of the first extension.The primer of first extension It may include some or all of the reversed chain-ordering of template.Optionally, the primer of the first extension includes the second primer engaging portion Position.

In some embodiments, the second primer of extension leads to the formation of the primer of the second extension.The primer of second extension It may include some or all of the positive chain-ordering of template.Optionally, the primer of the second extension includes the first primer engaging portion Position.

In some embodiments, in the situation that double-stranded nucleic acid template is not made to undergo extreme Denaturing during amplification Lower carry out method.For example, the nucleic acid-templated experience during amplification can not made more than or equal to the situation of the temperature of the Tm of template Lower carry out method.In some embodiments, can not make template during amplification with chemical denaturant such as NaOH, urea, Method is carried out in the case of the contacts such as guanidine salt.In some embodiments, amplification includes isothermal duplication.

In some embodiments, do not make it is nucleic acid-templated at least two, three, four or continuous more than four Nucleic acid synthesis cycle during undergo extreme Denaturing in the case of carry out method.For example, method may include two, three, Four or the continuous nucleic acid synthesis cycle more than four nucleic acid-templated are contacted without making with chemical denaturant.In some implementations In scheme, method may include that carrying out two, three, four or the continuous nucleic acid synthesis more than four recycles without making nucleic acid Template experience (or the reality of template or template group or is computed flat higher than the reality of template or template group or the Tm that is computed Equal Tm) under 25,20,15,10,5,2 or 1 DEG C of temperature.Two, three, four or the continuous nucleic acid synthesis more than four Cycle may include intervening partial denaturation and/or primer extension procedures therebetween.

In some embodiments, disclosed method (and relevant composition, system and kit) may also include one The primer strand of a or multiple extensions is connected to support.It is completed optionally during amplification or selectively in amplification laggard Row connection.In some embodiments, support includes multiple second primers, and method may include at least one extension The first primer chain and support the second primer hybridization.

In some embodiments, disclosed method (and relevant composition, system and kit) may also include one Second primer strand of a or multiple extensions is connected to support.In some embodiments, support is connected to the first primer. For example, support may include multiple the first primers, and method may include the second primer and support of at least one extension The first primer hybridization, so as to which the second primer extended is connected to support.For example, the first primer can be hybridized to extension The first primer binding site in second primer.

In some embodiments, support is connected to the second primer.For example, support may include multiple second primers, And method may include the second primer hybridization by the first primer of at least one extension and support, the so as to extend One primer is connected to support.For example, the first primer can be hybridized to the second primer binding site in the first primer of extension.

In some embodiments, support includes at least one the first primer and at least one second primer, and public The method (and relevant composition, system and kit) opened is including the first primer extended and the second primer of extension are connected It is connected to support.

Optionally, support is connected to target specific primer.Target specific primer optionally hybridizes (or can hybridize) extremely First subgroup of reaction mixture inner template, but the second subgroup of reaction mixture inner template cannot be bound to.

Optionally, support is connected to universal primer.Universal primer optionally hybridizes (or can hybridize) to reaction and mixes All or substantially all templates in object.

Optionally, reaction mixture include be covalently coupled to the first target specific primer the first support and covalently Ground is connected to the second support of the second target specific primer, and wherein the first and second target specific primers are different from each other.

Optionally, the first target specific primer is essentially complementary the first target nucleic acid sequence and the second target-specific draws Object is essentially complementary the second target nucleic acid sequence, and wherein the first and second target nucleic acid sequences are different.

In some embodiments, disclosed method is included optionally in the identical continuous phase of reaction mixture, the On one support the first amplicon is formed by expanding the first template and on the second support by expanding the second template shape Into the second amplicon.First amplicon optionally connects or is attached to the first support, and the second amplicon optionally connects Or it is attached to the second support.

Disclosed method, which is optionally included, by cloning expands two or more polynucleotide templates to generate two Or more monoclonal or the substantially amplicon of monoclonal.Optionally cloned in the Continuous Liquid Phase of amplification reaction mixture Ground expands two or more polynucleotide templates.The Continuous Liquid Phase of amplification reaction mixture may include continuous aqueous phase.At some In embodiment, amplification includes the polynucleotides group of the substantially amplification of monoclonal of generation at least two, the polynucleotides Each of group is formed by the amplification of single polynucleotide template.Optionally, the amplification of clone ground includes at least one RPA Bout.Optionally, the amplification of clone ground includes at least one template walking bout.

In some embodiments, amplification optionally includes to form the amplification reaction mixture comprising Continuous Liquid Phase.One In a little embodiments, Continuous Liquid Phase is single continuous aqueous phase.Liquid phase may include two or more polynucleotide templates, institute It is optionally different from each other to state polynucleotide template.For example, two or more polynucleotide templates may include it is at least one with At least one other polynucleotide template in amplification reaction mixture is not substantially identical or substantially not complementary core Acid sequence.

In some embodiments, amplification optionally includes to be formed comprising with two or more polynucleotide templates Single continuous aqueous phase amplification reaction mixture.Amplification optionally includes two by cloning expanded in single water phase Or more polynucleotide template form the nucleic acid group of two or more substantially monoclonals.Optionally, clone ground amplification Including at least one RPA bouts.Optionally, the amplification of clone ground includes at least one template walking bout.

In some embodiments, present disclosure generally relates to use the optionally parallel expansion of partial denaturation condition Increase one or more nucleic acid-templated methods (and relatively composition, system and kit).In some embodiments, appoint Selection of land expands two or more templates using such method in the form of an array.Optionally, before in distributing to array Large quantities of amplification templates in the solution.Alternatively, template is distributed to the site into array first, then at the site of array (within or) amplification template in situ.

Optionally, template is single-stranded or double-strand.Template is optionally comprising one or more primer binding sites.

In some embodiments, method may include making including the double-strand core of primer binding site at least one chain Acid template undergoes at least one replication cycle based on template using polymerase.

Optionally, at least one replication cycle based on template includes partial denaturation step, annealing steps and extension step Suddenly.

In some embodiments, method is included by the way that template is made to undergo at least two continuously duplications based on template It is nucleic acid-templated that cycle carrys out amplifying doulbe-chain.

In some embodiments, method includes partial denaturation template.Optionally, method includes being formed comprising single stranded zone Partial denaturation template.Partial denaturation template also may include double stranded region.Single stranded zone may include primer binding site.

Optionally, partial denaturation includes template is made to undergo lower than the Tm of primer binding site at least 20,15,10,5,2 or 1 DEG C temperature.

Optionally, partial denaturation includes making template experience identical with the Tm of primer binding site or higher than the Tm Temperature.

Optionally, partial denaturation includes contacting double-stranded template with recombinase and primer.Recombinase and primer can form core The part of albumen composition, and partial denaturation is included template and complex contacts.

In some embodiments, method is included by the way that primer is made to hybridize to be formed with the primer binding site of single stranded zone Primer-template double-strand.In some embodiments, template pending includes double stranded region.Optionally, double stranded region does not include primer Binding site.

In some embodiments, method includes the primer of extension primer-template double-strand.Optionally, method includes being formed The primer of extension.

In some embodiments, can expand on clone ground on different discontinuous supports (flowing into globule or particle) Increase different templates without being divided before amplification.In other embodiments, by template subregion or distribution before amplification To emulsion.Optionally, template is distributed to droplet, forms the emulsion with discontinuous aqueous favoring and continuous hydrophobic phase The part of aqueous favoring.In some embodiments, the emulsion droplets of aqueous favoring are also required comprising one or more progress PRA institutes Component.For example, emulsion droplets may include recombinase.Optionally, droplet includes strand displacement polymerase.In some embodiments In, droplet includes the fixed primer of support and/or molten phase primers.Optionally, primer can be bound to template or be bound to it Amplified production.

In some embodiments, the part disclosed herein that be included in template for being used for the amplification using emulsion-based Composition, system, method, apparatus and the kit that the nucleic acid amplification of nucleic acid synthesis is carried out after denaturation are provided compared to routine The advantageous aspect of extended method (including the PCR or emPCR of the emulsion-based for involving conventional thermocy).E.g., including based on breast The nucleic acid amplification reaction of the template walking of the RPA (" emRPA ") or emulsion-based of agent can generate the polynucleotides of longer amplification Template has less amplification step, the time for preparing the polynucleotide template expanded of reduction and/or increased sequencing number According to quality.Some appropriate emulsion compositions for being used together with amplification method disclosed herein are found in for example beautiful In state's patent No. 7622280,7601499 and 7323305, above-mentioned patent is incorporated herein by reference in their entirety.

In some embodiments, method includes providing comprising the positive chain containing the first primer binding site, contains the The double-stranded template of the reverse strand of two primer binding sites；Partial denaturation template and formation, which include, contains the first primer binding site Single stranded zone and at least one double stranded region partial denaturation template；By the way that the first primer is made to be combined with the first primer of single stranded zone Position hybridizes to form the first primer-template double-strand；Using the first primer of polymerase extension the first primer-template double-strand with shape Into the first primer of the extension comprising the second primer binding site, wherein the first primer extended at least partly hybridization is in template Positive chain；Partial denaturation carrys out the first chain of the extension of self-template to form the single stranded zone for including the second primer binding site；Make Second primer hybridizes with the second primer binding site of single stranded zone and forms the second primer-template double-strand and extend second and draws Second primer of object-template double-strand, so as to form the second primer of extension.

In some embodiments, present disclosure generally relates to the side of the method from nucleic acid-templated nucleic acid Method (and relevant composition, system and kit), including：The first nucleic acid double chain comprising positive chain and reverse strand is provided, Middle forward direction chain includes forward primer binding site and reverse strand includes reverse primer binding site and wherein the first double-strand With the first melting temperature (" template Tm "), forward primer binding site has the second melting temperature (" forward primer Tm "), Reverse primer binding site has third melting temperature (" reverse primer Tm ")；The first double-strand of partial denaturation, which part denaturation The first double-strand include the single stranded zone containing forward primer binding site and at least one double stranded region；By make forward primer with The forward primer binding site of first double-strand of partial denaturation hybridizes to form the first double-strand pending；By by pending One double-strand contacts to extend forward primer under primer extension conditions with strand displacement polymerase and nucleotide, has so as to be formed Second double-strand of the 4th melting temperature (" the 4th Tm "), second double-strand include one containing forward primer binding site Chain and a chain containing reverse primer binding site；The second double-strand of partial denaturation, the second double-strand of which part denaturation include Single stranded zone containing reverse primer binding site and at least one double stranded region；By make reverse primer and partial denaturation second The reverse primer binding site of double-strand hybridizes to form the second double-strand reversely pending；By by reversely the second double-strand pending It contacts to extend reversely drawing for the second double-strand reversely pending under primer extension conditions with strand displacement polymerase and nucleotide Object.

In some embodiments, method (and relevant composition, system and kit) may also include sequencing amplification Template or the primer (such as the first primer of extension or second primer of extension) of sequencing extension.Sequencing may include in this field Known any appropriate sequencing approach.In some embodiments, sequencing includes the sequencing by synthesizing or is examined by electronics The sequencing (such as nano-pore sequencing) of survey.In some embodiments, sequencing is included through polymerase-mediated nucleotide simultaneously The sequencing primer that the template or extension for entering to extend template or amplification hybridize with the template of template or amplification.In some embodiment party In case, sequencing is included by the way that the primer of template or extension and the nucleotide of sequencing primer, polymerase and at least one type are connect It touches to be sequenced to being connected to the template of support or the template of amplification.In some embodiments, sequencing is included mould The primer of the template or extension of plate or amplification does not include exogenous marker with sequencing primer, polymerase and with only one kind of Or the nucleotide of chain termination group is contacted.

Optionally, template (or product of amplification) can be placed in, be confined to or positioned at site.In some embodiments, it is more The first primer of template/extension of kind template/amplification is placed in or different sites in site array.In some embodiment party It in case, placed, positioned or is localized before template amplification.In some embodiments, it is put after amplification It puts, position or localizes.For example, the template of amplification or the first primer of extension can be placed in, are located at or be confined to array not Same site.

Method disclosed herein leads to the generation of multiple amplicons, and at least some amplicons include the amplification of clone ground Nucleic acid group.The group expanded to the clone generated by method in the present disclosure can be used for a variety of purposes.In some embodiment party In case, the group that disclosed method (and relevant composition, system and kit) expands with optionally further including clone (expands Son) analysis and/or processing.For example, in some embodiments, it is detectable to go out certain expectation spies with optionally quantitative display The quantity of the amplicon of property.

In some embodiments, which discontinuous support (such as globule) is method may include measuring comprising amplification Son.Similarly, method may include which site for measuring array includes amplicon.Optionally use the detection journey based on DNA Sequence such as UV absorption, with DNA specific dyes dye,Measure, qPCR, hybridize with fluorescence probe Etc. the presence for carrying out the amplicon on detection assay support or site.In some embodiments, method may include which is measured A little globule supports (or site of array) have obtained the amplicon of substantially monoclonal.For example, globule support can be analyzed (or array site) can generate detectable and relevant (coherent) with which determining support or site (can analyze ) sequence dependent signal.

In some embodiments, the sequencing of product expanded after amplification.The product for the amplification being sequenced can wrap Include the amplicon for including the substantially nucleic acid group of monoclonal.In some embodiments, disclosed method is included multiple amplifications The single member of son forms or is positioned at different sites.Different sites is optionally formed the part of site array.At some In embodiment, the site in site array is included in the hole (reative cell) on the showing of isFET arrays.

In some embodiments, the method for downstream analysis includes at least some of multiple amplicons are parallelly sequenced.Appoint Selection of land, parallelly sequencing is positioned at the first primer of template/extension of multiple template/amplification of different array sites.

In some embodiments, sequencing may include sequencing primer being bound at least two different amplicons or extremely The nucleic acid of the group of few two different substantially monoclonals.

In some embodiments, sequencing may include nucleotide is incorporated to sequencing primer using polymerase.Optionally, and Enter and be incorporated to by-product including forming at least one nucleotide.

Optionally, nucleic acid to be sequenced is located at site.Site may include reative cell or hole.Site can be it is similar or The part of the array of same loci.Array may include surface (such as flow cell, electronic device, transistor chip, reative cell, The surface of slot etc.) on site two-dimensional array or matrix or other mediums (such as solid, semisolid, liquid, fluid etc.) in Site cubical array.

In some embodiments, site is operationally coupled to sensor.Method may include detecting core using sensor Thuja acid is incorporated to.Optionally, site and sensor are located in the array in site for being coupled to sensor.

In some embodiments, site includes the parent for being conformally placed in and being operationally coupled within the hole of sensor Aqueous polymer matrix.

Optionally, hydrophilic polymer matrix includes aquogel polymer matrix.

Optionally, hydrophilic polymer matrix is in-situ solidifying polymer substrate.

Optionally, hydrophilic polymer matrix includes polyacrylamide, its copolymer, its derivative or combination.

Optionally, polyacrylamide is conjugated in Oligonucleolide primers.

Optionally, hole has 0.1 micron to 2 microns of characteristic diameter.

Optionally, hole has 0.01 micron to 10 microns of depth.

In some embodiments, sensor includes field-effect transistor (FET).FET may include ion-sensitive FET (ISFET)。

In some embodiments, method (and relevant composition, system and kit) may include optionally using FET detects one or more nucleotide and is incorporated to presence of the by-product at array site.

In some embodiments, method may include what is optionally occurred at least one reative cell using FET detections PH changes.

In some embodiments, disclosed method includes nucleotide introducing site；With detection because of nucleotide to sequencing Output signal from sensor caused by being incorporated to of primer.Output signal is optionally based on the threshold voltage of FET.One In a little embodiments, FET includes the floating gate conductor for being coupled to site.

In some embodiments, FET is included comprising electrical coupling each other and by the floating of multiple conductors of dielectric layer separation Moving grid structure, and floating gate conductor is uppermost conductor in multiple conductors.

In some embodiments, floating gate conductor includes the upper surface for the bottom surface for defining site.

In some embodiments, floating gate conductor includes conductive material, and the upper surface of floating gate conductor includes and leads The oxide of electric material.

In some embodiments, floating gate conductor is coupled at least one reative cell by sensing material.

In some embodiments, sensing material includes metal-oxide.

In some embodiments, sensing material is to hydrogen ion sensitivity.

In some embodiments, amplification reaction mixture may include recombinase.Recombinase may include it is any it is appropriate can Promote the reagent of the recombination between polynucleotide molecule.Recombinase can be the enzyme for being catalyzed homologous recombination.For example, amplified reaction Mixture may include recombinase, and the recombinase includes or the weight derived from bacterium, eucaryote or virus (such as bacteriophage) Group enzyme.

In some embodiments, amplification reaction mixture, which includes, can combine primer and polynucleotide template to be formed again It closes object or the chain intrusion of polynucleotide template can be catalyzed to form the enzyme of D- ring structures.In some embodiments, amplified reaction Mixture includes one or more albumen for being selected from UvsX, RecA and Rad51.

In some embodiments, amplification reaction mixture may include recombinase auxilin such as UvsY.

In some embodiments, amplification reaction mixture may include single strand binding protein (SSBP).

In some embodiments, amplification reaction mixture may include polymerase.Polymerase optionally has or lacks outer Cut nuclease.In some embodiments, polymerase has 5 ' to 3 ' exonuclease activities, 3 ' to 5 ' exonucleases Enzymatic activity is active with above two.Optionally, it polymerize any or more of exonuclease activity as azymia Kind.

In some embodiments, polymerase has strand-displacement activity.

In some embodiments, amplification reaction mixture may include one or more solid or semisolid supports.Branch Holding at least one of object may include one or more the first primers for including the first primer sequence.In some embodiments, instead At least one of mixture polynucleotides is answered to include the first primer binding sequence.The first primer binding sequence can draw with first Object sequence is substantially the same or is substantially complementary.In some embodiments, support it is at least one, some or All supports include multiple the first primers substantially identical to one another.In some embodiments, all on support draw Object is that substantially the same or all primers include substantially the same the first primer sequence each other.

In some embodiments, at least one support includes two or more different primers for being connected to it. For example, at least one support may include at least one the first primer and at least one second primer.

In some embodiments, the water phase of reaction mixture includes multiple supports, and plurality of support is at least Two supports are connected to the primer for including the first primer sequence.In some embodiments, reaction mixture include two or More different polynucleotide templates with the first primer binding sequence.

In some embodiments, amplification reaction mixture may include diffusion limitation agent.Diffusion limitation agent can be effective Ground prevents or slows down the one or more of polynucleotide template and/or the one or more of amplified reaction product anti-by expanding Any reagent that mixture is answered to spread.

In some embodiments, amplification reaction mixture may include sieving agent.Screening agent can be that effectively screening is deposited The one or more polynucleotides (such as amplified reaction product and/or polynucleotide template) being in amplification reaction mixture Any reagent.In some embodiments, screening agent limits or slows down polynucleotide amplification product is moved by reaction mixture It moves.

In some embodiments, amplification reaction mixture may include crowding agent.

In some embodiments, amplification reaction mixture includes crowding agent and screening agent.

In some embodiments, two or more are more including clone ground amplification by the following method for disclosed method At least two of oligonucleotide template：(a) amplification reaction mixture for including single Continuous Liquid Phase is formed, the liquid phase includes two A or more polynucleotide template, one or more surfaces or support and amplification component；(b) it is propped up in one or more It holds and expands at least two polynucleotide templates to clone on object.Optionally, the amplification of clone ground includes forming at least two The amplicon of different substantially monoclonals.In some embodiments, the amplification of clone ground includes passing through amplification reaction mixture Go through amplification condition.In some embodiments, two or more described amplicons are each attached to surface or support.Example Such as, amplification reaction mixture may include single support or surface, so as to each polynucleotide template be connected to support or The given area on surface.

In some embodiments, the method for nucleic acid amplification can be carried out in single reaction vessel.

In some embodiments, the method for nucleic acid amplification, the single company can be carried out in single Continuous Liquid Phase Continuous liquid phase does not provide the division of multiple nucleic acid amplification reactions occurred in single reaction vessel.In some embodiments, may be used The method for nucleic acid amplification is carried out in the water-in-oil emulsion (micro- reaction vessel) divided is provided.

In some embodiments, it can carry out for the method for nucleic acid amplification so that multiple polynucleotides are connected to support Object or surface.For example, method may include forming the reaction mixture comprising at least one surface and undergo reaction mixture Amplification condition.In some embodiments, the plane surface or inner wall of surface of the surface including globule, slot or pipe.

In some embodiments, include for the method for nucleic acid amplification：(a) expansion for including single Continuous Liquid Phase is formed Increase reaction mixture, the liquid phase includes multiple globules, multiple and different polynucleotides and recombinase；(b) make amplified reaction Mixture undergoes isothermal duplication condition, so as to generate the small of multiple nucleic acid groups for being connected to the substantially monoclonal for being attached to it Pearl.

In some embodiments, present disclosure generally relates to carry out nucleic acid mould directly on the surface of array The amplification based on array of plate includes amplicon (the amplification production comprising substantially monoclonal so as to cause its any personal feature Object group) array formation method (and relevant composition, system and kit).These embodiments with retouching herein The other embodiments stated form comparison, nucleic acid-templated optionally in the solution in not in other embodiments It is amplified on continuous support (such as globule), is then assigned into array.

In some embodiments, method (and relevant composition, the system for the amplification based on array are provided And kit).In some embodiments, different polynucleotide templates is distributed into site array and then carried out former Position amplification.Then using amplification subarray caused by appropriate downstream program analysis.

In some embodiments, include for the method for nucleic acid amplification：A) by the way that single polynucleotides are introduced each other At least two sites being in fluid communication distribute at least two different polynucleotides to site；(b) by described in amplification Polynucleotides at least two sites form the nucleic acid group of at least two substantially monoclonals.Site optionally includes Surface, hole, ditch, flow cell, reative cell or slot.In some embodiments, in the case where site is not made to close each other into Row amplification.For example, at least two sites can each other keep being in fluid communication during amplification.

In some embodiments, include for the method for nucleic acid amplification：A) by the way that single polynucleotides are introduced at least Two sites distribute at least two different polynucleotides to site；(b) by expanding at least two site Polynucleotides form the nucleic acid group of at least two substantially monoclonals.Site optionally includes surface, hole, ditch, flowing Pond, reative cell or slot.In some embodiments, it is expanded in the case where site is not made to close each other.For example, at least Two sites can each other keep being in fluid communication during amplification.

In some embodiments, site includes reative cell, and includes for the method for nucleic acid amplification：A) pass through by At least two reative cells that single polynucleotides introducing is in fluid communication with each other distribute at least two polynucleotide templates to anti- Answer room；(b) at least two substantially Dan Ke are formed by expanding the indoor polynucleotide template of at least two reaction Grand nucleic acid group.In some embodiments, it is expanded in the case where reative cell is not made to close each other.For example, at least Two reative cells can each other keep being in fluid communication during amplification.

In some embodiments, site includes reative cell, and includes for the method for nucleic acid amplification：A) pass through by At least two reative cells that the introducing of single polynucleotides is in fluid communication with each other by least two different polynucleotides distribute to Reative cell；(b) at least two substantially monoclonals are formed by expanding at least two indoor polynucleotides of reaction Nucleic acid group.In some embodiments, it is expanded in the case where reative cell is not made to close each other.For example, at least two A reative cell can each other keep being in fluid communication during amplification.

In some embodiments, the in the present disclosure arbitrary and methodical amplification step of institute can be during amplification not It is carried out in the case of complete denatured polynucleotide.For example, disclosed method may include expanding at least two by isothermal duplication Different polynucleotides.Amplification may include expanding at least two different polynucleotides under conditions of substantially isothermal.Optionally Ground expands in the case that during amplification polynucleotides are not contacted with chemical denaturant.

In some embodiments, amplification, which is optionally included in site or reative cell, carries out two different amplifications time It closes.For example, amplification may include carrying out at least one RPA bouts in site or reative cell with any sequence of bout or combination And at least one template walking bout is carried out in site or reative cell.In some embodiments, any one or more are expanded At least two continuous circulate under substantially isothermy in bout carry out.In some embodiments, bout is expanded It is at least one to be carried out under substantially isothermy.

In some embodiments, amplification includes contacting polynucleotides to be amplified with reaction mixture.Contact can Optionally carry out before a distribution or later；It will be understood that present disclosure is included polynucleotides and reaction mixture wherein The embodiment and wherein mix reaction that each component (or combination of component) is continuously contacted in different times Any one or some the component polynucleotides different at least two for closing object contact before dispensing and by reaction mixture The embodiment that remaining component polynucleotides different at least two contact after dispensing.

At least two assigned polynucleotides are used as optionally in its respective reative cell for nucleic acid synthesis Template.In some embodiments, at least two polynucleotides include different sequences.In some embodiments, multinuclear glycosides Acid is double-strand before a distribution or is used as single-stranded.In some embodiments, polynucleotides are linear, cricoid or two The combination of person.In a typical implementation, polynucleotides be at least part double-strand (or with single stranded form distribute and it is subsequent Make it in site or reative cell after the distribution at least part double-strand).It is double-strand that can make polynucleotides before amplification (in the embodiment for particularly including RPA or template walking in wherein amplification).

In some embodiments, at least two different polynucleotide templates to be amplified respectively contain primer knot Position is closed, and expands and includes primer being bound to primer binding site to form primer-template double-strand.

Optionally, amplification includes the primer of extension primer-template double-strand.Extension be optionally happened at array site or At reative cell or within.Optionally, extension primer is included primer and polymerase and the nucleotide of one or more types in core Thuja acid is contacted under the conditions of being incorporated to.In some embodiments, the nucleotide of one or more types does not include external source mark Note, particularly optically detectable label, such as fluorescence part or dyestuff.Optionally, reaction mixture includes nucleotide, It is naturally occurring nucleotide.Optionally, nucleotide do not include terminate nucleic acid synthesis group (such as double deoxidation group, can Inverse terminator etc.).Normally, extension primer is occurred with template dependent manner.

Optionally, at least two different polynucleotides (template i.e. to be amplified) respectively contain and the first primer At least certain First ray (being referred to as " the first primer binding site ") that is a part of substantially the same or being substantially complementary.

In some embodiments, reaction mixture includes the first primer for including the first primer sequence.The first primer is appointed Selection of land includes extendible end (such as 3 ' ends containing OH).Optionally by the first primer be connected to compound (such as " resistance label ") or it is connected to support (such as globule or site or surface of reative cell).

In some embodiments, at least two different polynucleotides include at least certain part with the second primer The second sequence (being referred to as " the second primer binding site ") that is substantially the same or being substantially complementary.

In some embodiments, method is drawn including one or more nucleotide are incorporated to second by using polymerase Second primer of object-template double-strand extends the second primer, so as to forming the second primer of extension.

In some embodiments, do not make it is nucleic acid-templated at least two, three, four or continuous more than four Nucleic acid synthesis cycle during undergo extreme Denaturing in the case of carry out method.For example, method may include two, three, Four or the continuous nucleic acid synthesis step more than four nucleic acid-templated are contacted without making with chemical denaturant.In some implementations In scheme, method may include that carrying out two, three, four or the continuous nucleic acid synthesis more than four recycles without making nucleic acid Template experience (or the reality of template or template group or is computed flat higher than the reality of template or template group or the Tm that is computed Equal Tm) under 25,20,15,10,5,2 or 1 DEG C of temperature.Two, three, four or the continuous nucleic acid synthesis more than four Cycle may include intervening partial denaturation and/or primer extension procedures therebetween.

In some embodiments, disclosed method (and relevant composition, system and kit) may also include one Second primer strand of a or multiple extensions is connected to support.In some embodiments, support is connected to the first primer. For example, support may include multiple the first primers, and method may include the second primer and support of at least one extension The first primer hybridization, so as to which the second primer extended is connected to support.For example, the first primer can be hybridized to extension The first primer binding site in second primer.Support may include the surface of for example any array.

In some embodiments, disclosed method include optionally in the identical continuous phase of reaction mixture and At the different loci on surface (such as in array), on the first support by expand the first template formed the first amplicon and On the second support the second amplicon is formed by expanding the second template.First amplicon optionally connects or is attached to One support, and the second amplicon optionally connects or is attached to the second support.

Two or more different sites that disclosed method is optionally included in site array by cloning expand Two or more polynucleotide templates generate two or more monoclonals or the substantially amplicon of monoclonal, so as to At least two sites are formed as respectively containing to the nucleic acid group of substantially monoclonal.Two or more polynucleotide templates are appointed Selection of land be placed in or positioned at different sites and then with the Continuous Liquid Phase of the amplification reaction mixture of array contact in be cloned Ground expands.The Continuous Liquid Phase of amplification reaction mixture may include continuous aqueous phase.

In some embodiments, amplification includes the polynucleotides group of the substantially amplification of monoclonal of generation at least two, Each of the polynucleotides group is formed by the amplification of single polynucleotide template.

Optionally, the amplification of clone ground includes at least one RPA bouts.

Optionally, the amplification of clone ground includes at least one template walking bout.

In some embodiments, multiple and different polynucleotide templates expand it is preposition in or positioned at different site. For example, the template of amplification or the first primer of extension can be placed in, are located at or be confined to the different loci of array.

In some embodiments, amplification causes to form at least two bases in at least two different sites on surface The nucleic acid group (such as amplicon) of monoclonal, may then use that nucleic acid group described in appropriate program in-situ study in sheet.

In some embodiments, present disclosure generally relates to prepare method (and the relevant combination on surface Object, system and kit).Optionally, surface includes multiple sites comprising the first site and the second site.

In some embodiments, method includes forming nucleic acid array on the surface, wherein described formed is included first Nucleic acid is connected to the first site and the second nucleic acid is connected to the second site.The connection is optionally by using herein Disclosed any method, including being for example covalently attached to the primer on surface by the way that nucleic acid is connected to and carrying out.

In some embodiments, method is included at least the first and second nucleic acid with including the examination synthesized for nucleic acid The single reaction mixture contact of agent.Reaction mixture is optionally including any one or more components described herein. In some embodiments, reaction mixture includes all components carried out needed for RPA.In some embodiments, reaction mixing Object includes all components for carrying out template walking.

In some embodiments, method includes answering by using the reagent for nucleic acid synthesis in reaction mixture First or second nucleic acid processed to form the first amplicon in the first site and forms the second amplicon in second point.Duplication can Including primer extend.Duplication may include one or more RPA cycles.Duplication may include that one or more template walkings follow Ring.

In some embodiments, it replicates and includes at least one RPA cycles.

In some embodiments, it replicates and includes at least one template walking cycle.

In some embodiments, it replicates and includes at least one RPA cycles and at least one template walking cycle.

In some embodiments, it replicates and includes at least one RPA bouts.

In some embodiments, it replicates and includes at least one template walking bout.

In some embodiments, it replicates and includes at least one RPA bouts and at least one template walking bout.

In some embodiments, present disclosure generally relates to prepare method (and the relevant combination on surface Object, system and kit), the method includes：(a) it provides with multiple sites comprising the first site and the second site Surface；(b) nucleic acid array is formed on the surface, wherein described formed includes the first nucleic acid being connected to the first site and by the Two nucleic acid are connected to the second site；(c)；(d) the is replicated by using the reagent for nucleic acid synthesis in reaction mixture One or second nucleic acid come the first site formed the first amplicon and second point formed the second amplicon.

In some embodiments, present disclosure generally relates to the method for preparing surface, the method includes： Surface with multiple sites comprising the first site and the second site is provided；Nucleic acid array is formed on the surface, wherein described It is formed and includes the first nucleic acid is connected to the first site and the second nucleic acid is connected to the second site；To at least first and second Nucleic acid is contacted with the single reaction mixture comprising the reagent synthesized for nucleic acid；With by using the use in reaction mixture In nucleic acid synthesis reagent amplification first or second nucleic acid come the first site formed first substantially the amplicon of monoclonal and In the amplicon of the substantially monoclonal of second point formation second.Optionally, the first and second sites keep stream during amplification Body connects.Optionally, it is expanded in the case of incomplete denatured polynucleotide during amplification.For example, disclosed method can Including expanding at least two different polynucleotides by isothermal duplication.Amplification may include under conditions of substantially isothermal Expand at least two different polynucleotides.Optionally, the feelings not contacted polynucleotides with chemical denaturant during amplification It is expanded under condition.

In some embodiments, at least one site in multiple sites includes reacting hole, trough or room.

In some embodiments, at least one site in multiple sites is connected to sensor.

In some embodiments, sensor can detect the nucleotide occurred at or near at least one site simultaneously Enter.

In some embodiments, sensor includes field-effect transistor (FET).

In some embodiments, at least the first site or the second site or the first and second sites, which include, is connected to table The primer in face.

In some embodiments, at least one site in multiple sites includes conformally to be placed in and operationally be coupled to Hydrophilic polymer matrix within the hole of sensor.

Optionally, hydrophilic polymer matrix includes aquogel polymer matrix.

Optionally, polyacrylamide is conjugated in Oligonucleolide primers.

Optionally, hole has 0.1 micron to 2 microns of characteristic diameter.

Optionally, hole has 0.01 micron to 10 microns of depth.

In some embodiments, sensor includes field-effect transistor (FET).FET may include ion-sensitive FET (ISFET), chemFET, bioFET etc..

In some embodiments, FET can detect nucleotide and be incorporated to presence of the by-product at least one site.

In some embodiments, FET can detect the chemical part selected from hydrogen ion, pyrophosphate, hydroxidion etc..

In some embodiments, method may include optionally detecting at site or at least one reaction using FET The pH variations of indoor generation.

In some embodiments, disclosed method includes introducing nucleotide at least one site in multiple sites；With Detect the output signal from sensor because of caused by being incorporated to of nucleotide to sequencing primer.Output signal is optionally based on The threshold voltage of FET.In some embodiments, FET includes the floating gate conductor for being coupled to site.

In some embodiments, sensing material includes metal-oxide.

In some embodiments, sensing material is to hydrogen ion sensitivity.

In some embodiments, reaction mixture includes all components carried out needed for RPA.

In some embodiments, reaction mixture includes all components carried out needed for template walking.

In some embodiments, reaction mixture may include one or more solid or semisolid supports.Support At least one may include one or more the first primers for including the first primer sequence.In some embodiments, support It is at least one include two or more different primers for being connected to it.For example, at least one support may include at least One the first primer and at least one second primer.

Alternatively, in some embodiments, reaction mixture does not include any support.In some embodiments, directly It is connected at least two different polynucleotide templates of amplification on the site of array or the surface of reative cell.

In some embodiments, reaction mixture may include recombinase.Recombinase may include any appropriate promote The reagent of recombination between polynucleotide molecule.Recombinase can be the enzyme for being catalyzed homologous recombination.For example, reaction mixture can Comprising recombinase, the recombinase includes or the recombinase derived from bacterium, eucaryote or virus (such as bacteriophage).

Optionally, reaction mixture includes the nucleotide for not carrying out exogenous marker.For example, nucleotide can naturally be deposited Nucleotide or optically detectable label not comprising fluorescence part, dyestuff or other external sources synthetic analogues. Optionally, reaction mixture includes nucleotide, is naturally occurring nucleotide.Optionally, nucleotide, which does not include, terminates nucleic acid The group (such as double deoxidation group, reversible terminator etc.) of synthesis.

Optionally, reaction mixture includes nucleotide, is naturally occurring nucleotide.Optionally, nucleotide does not include Terminate the group (such as double deoxidation group, reversible terminator etc.) of nucleic acid synthesis.

In some embodiments, reaction mixture, which includes, can combine primer and polynucleotide template to form compound Or the chain intrusion of polynucleotide template can be catalyzed to form the enzyme of D- ring structures.In some embodiments, reaction mixture packet Containing one or more albumen for being selected from UvsX, RecA and Rad51.

In some embodiments, present disclosure generally relates to prepare method (and the relevant combination on surface Object, system and kit), the method includes：(a) surface with multiple sites is provided, wherein each site is connected to core Sour primer；(c) surface with multiple polynucleotide templates is contacted and at least one template is connected to surface；On the surface At least one template is expanded, so as to form the target of the amplification of the substantially monoclonal at least one site positioned on surface The group of polynucleotide sequence.

In some embodiments, the method that present disclosure generally relates to nucleic acid amplification, including：(1) tool is provided There is the surface in the first site and the second site, the first site is operationally coupled to first sensor and comprising the first template；The Two sites are operationally coupled to second sensor and comprising the second templates；(2) reaction mixture is distributed to first and second Site；(3) the first amplicon is formed by expanding the first template in the first site；With by the second site expand second Template forms the second amplicon.

In some embodiments, amplification includes at least one RPA cycles.

In some embodiments, amplification includes at least one template walking cycle.

In some embodiments, amplification includes at least one RPA cycles and at least one template walking cycle.

In some embodiments, amplification includes at least one RPA bouts.

In some embodiments, amplification includes at least one template walking bout.

In some embodiments, amplification includes at least one RPA bouts and at least one template walking bout.

In some embodiments, disclosed herein some or all of methods can lead to the generation of multiple amplicons, At least some nucleic acid groups for including the amplification of clone ground of the amplicon.The clone generated by method in the present disclosure The group of amplification can be used for a variety of purposes.In some embodiments, disclosed method (and relevant composition, system and reagent Box) optionally further include the analysis and/or processing of group's (amplicon) expanded with cloning.

In some embodiments, can make to undergo downstream analysis method for example according to the amplicon that present disclosure generates Sequencing.

In some embodiments, the nucleic acid of (such as sequencing) amplification can further be analyzed at the site of distribution and nothing The product of amplification need to be recycled and move to different sites or surface for analysis (such as sequencing).

In some embodiments, the method for downstream analysis includes at least part that multiple amplicons are parallelly sequenced. Optionally, the first primer of template/extension of multiple template/amplification positioned at different array sites is parallelly sequenced.

For example, in some embodiments, it is in situ after amplification that the product expanded is sequenced.The product for the amplification being sequenced It may include including the amplicon of the substantially nucleic acid group of monoclonal.Optionally, the different loci positioned at array is parallelly sequenced The nucleic acid group (amplicon) of monoclonal.

Optionally, hydrophilic polymer matrix includes aquogel polymer matrix.

Optionally, polyacrylamide is conjugated in Oligonucleolide primers.

Optionally, hole has 0.1 micron to 2 microns of characteristic diameter.

Optionally, hole has 0.01 micron to 10 microns of depth.

In some embodiments, sensing material includes metal-oxide.

In some embodiments, sensing material is to hydrogen ion sensitivity.

In some embodiments, reaction mixture may include recombinase auxilin such as UvsY.

In some embodiments, reaction mixture may include single strand binding protein (SSBP).

In some embodiments, reaction mixture may include polymerase.Polymerase optionally has or lacks circumscribed core Phytase activity.In some embodiments, polymerase has 5 ' to 3 ' exonuclease activities, 3 ' to 5 ' exonuclease enzyme activity Property or with above two activity.Optionally, it polymerize any one or more of exonuclease activity as azymia.

In some embodiments, polymerase has strand-displacement activity.

In some embodiments, reaction mixture may include diffusion limitation agent.Diffusion limitation agent can be effective ground resistance Only or slow down the one or more of polynucleotide template and/or the one or more of amplified reaction product passes through reaction mixture Any reagent of diffusion.

In some embodiments, reaction mixture may include sieving agent.Screening agent can be that effectively screening is present in Any examination of one or more polynucleotides (such as amplified reaction product and/or polynucleotide template) in reaction mixture Agent.In some embodiments, screening agent limits or slows down polynucleotide amplification product is migrated by reaction mixture.

In some embodiments, reaction mixture may include crowding agent.

In some embodiments, reaction mixture includes crowding agent and screening agent.

In some embodiments, disclosed method is included by each and recombinase of at least two polynucleotides, even Being connected to the supports of multiple first Oligonucleolide primers, (the first Oligonucleolide primers are at least partially complementary to polynucleotides extremely Certain few part), polymerase and multiple nucleotide contacted in any order and with any combinations.

In some embodiments, at least two different polynucleotides are included comprising the first primer binding site just To chain, and the amplification of (or at least two reative cells) is optionally included in site or reaction at least two sites Interior is combined the first primer with the first primer binding site to form the first primer-template double-strand.Optionally, the first primer The combination of the polynucleotide template different at least two is by recombinase-mediated.For example, amplification may include being formed comprising recombination The nucleoprotein complex of enzyme and the first primer.Optionally, the first primer is connected to site or the surface of reative cell.In some realities It applies in scheme, includes in site or the indoor amplification of reaction：Form the first nucleoprotein complex (or " first nucleoprotein filament "). Amplification optionally further include by least one and the first nucleoprotein filament of the polynucleotides in site or reative cell, polymerase and Multiple nucleotide are in any order or combination is contacted.

Optionally, the support (such as globule) that the dispersion of multiple the first primers will be respectively contained before amplification is respective In distribution to reative cell or site, and expand and be included at least two difference of amplification on site or the indoor support of reaction One of polynucleotides.In some embodiments, can before amplification any one of duplicate allocation and/or contact procedure, Optionally the site of monoclonal product or the quantity and/or yield of reative cell are generated to increase.

Optionally, amplification includes extending the first primer of the first primer-template double-strand using polymerase in reative cell, So as to form the first primer of extension.Optionally, extension the first primer replaces reverse strand from positive chain.The first primer of extension Optionally include the second primer binding site.

Optionally, amplification includes inverse composition step, and the step is drawn including the second primer is bound to the first of extension Second primer binding site of object, and extend the second primer to form the second primer-template double-strand.Optionally, the second primer is extremely The combination of polynucleotide template is by recombinase-mediated.For example, amplification may include being formed the core egg comprising recombinase and the second primer White compound.Optionally, the second primer is connected to site or the surface of reative cell.In some embodiments, in site or Indoor amplification is reacted to include：Form the second nucleoprotein complex (or " second nucleoprotein filament ").Amplification optionally further include by At least one and the second nucleoprotein of at least one or extension the first primer of polynucleotide template in site or reative cell Silk, polymerase and multiple nucleotide are in any order or combination is contacted.

Optionally, amplification includes the use of polymerase extension the first primer-template double-strand, the second primer-template double-strand or prolongs It stretches both above-mentioned.Polymerase can have strand-displacement activity.

In some embodiments, method (and relevant composition, system and kit) may include at least one base The group of monoclonal is placed in, is positioned at or is confined to site in sheet.Site can form the part of site array.

Optionally, site is at least one including reative cell, support, particle, particle, sphere, globule, filter, flowing Pond, hole, ditch, tank therefor (channel reservoir), gel or pipe inner wall.

In some embodiments, at least one site includes the hole for being conformally placed in and being operationally coupled to sensor Within hydrophilic polymer matrix.

Optionally, hydrophilic polymer matrix includes aquogel polymer matrix.

Optionally, polyacrylamide is conjugated in Oligonucleolide primers.

Optionally, hole has 0.1 micron to 2 microns of characteristic diameter.

Optionally, hole has 0.01 micron to 10 microns of depth.

In some embodiments, sensing material includes metal-oxide.

In some embodiments, sensing material is to hydrogen ion sensitivity.

Optionally, the multiple and different polynucleotide template polynucleotides of amplification (or) comprising at least one containing optional The nucleic acid of the part of selecting property cutting.

Optionally, the part of alternative cutting includes uracil.

Optionally, it is further included for the method for nucleic acid amplification and cleavable part is cut with cutting agent.

Optionally, cutting can for example carry out before amplification before reaction mixture is formed.

Optionally, cutting can be carried out for example after nucleic acid-templated be amplified after amplification.

Optionally, reaction mixture includes at least one primer containing cleavable part.

Optionally, multiple and different polynucleotides include multiple amplicons.

Optionally, multiple and different polynucleotides include multiple and different amplicons.

In some embodiments, the U.S. Provisional Patent Application No. 61/ that on March 15th, 2013 submits can be used Any method, composition, system and kit disclosed in 792247 (being incorporated herein by reference in their entirety) are expanded.

In some embodiments, can make to undergo downstream analysis method for example according to the amplicon that present disclosure generates It is quantitative.For example, in some embodiments, the number of the detectable amplicon for going out certain desired characteristics with optionally quantitative display Amount.

In some embodiments, the nucleic acid of amplification is optionally made to undergo other downstream point in the disclosed methods Analyse step.

It is included in the discontinuous embodiment with different polynucleotide templates is expanded on support that is detaching at some In, which discontinuous support (such as globule) is method may include measuring comprising amplicon.Similarly, template exists wherein It is assigned before amplification into the embodiment of array, method may include which site for measuring array includes amplicon, and can Optionally further include the quantity for counting the site comprising amplicon.Optionally use the detection program based on DNA for example purple It is outer absorb, with DNA specific dyes dye,Measure, qPCR, hybridize etc. to detect with fluorescence probe Measure the presence of support or the amplicon on site.In some embodiments, method may include measuring which globule is supported Object (or site of array) has obtained the amplicon of substantially monoclonal.For example, globule support (or array position can be analyzed Point) to determine which support or site can generate detectable and relevant (i.e. analyzable) sequence dependent signal.

In some embodiments, disclosed method includes other downstream analysis step, and the downstream analysis step carries For previously passed routine techniques such as digital pcr or number RPA (as example in Shen 2011Analytical Chemistry 83:3533-3540；(all documents pass through reference to U.S. Patent Application Publication US2012/0264132 and 2012/0329038 It is integrally incorporated herein) described in) information of the same type of acquisition.Digital pcr (dPCR) is Conventional polymerase chain formula React (PCR) improvement, can be used for direct quantitative and clone ground amplification of nucleic acid (including DNA, cDNA, methylate DNA or RNA).Difference lies in the methods for measuring nucleic acid amount by one between dPCR and normal PCR.The every single samples of PCR and dPCR are all A reaction is carried out, dPCR carries out single reaction also in sample, however sample is separated into a large amount of subregion and every It is respectively reacted in a subregion.The sensitive measurement for separately allowing nucleic acid amount.DPCR is proved to can be used for studying gene Variation in sequence, such as copy number variation or point mutation.

Compared with this method, dPCR usually requires subregion of the sample before amplification；On the contrary, several realities disclosed herein The scheme of applying provides parallel amplification of the different templates in the single continuous phase of reaction mixture without subregion.In dPCR In, the positioning of individual nucleic acid molecules and be confined in the region of many separation that sample is usually partitioned in sample.Pass through letter Single dilution method divides the DNA profiling or less that sample includes about 1 copy so as to each part.By detaching individual DNA profiling, this method are effectively enriched in original sample with DNA molecular existing for extremely low level.The subregion of sample has Conducive to using the statistical numerator counts of Poisson.As a result, each subregion will respectively include " 0 " or " 1 " molecule, Or negative or positive reaction.Although the starting copies quantity of molecule is proportional to the quantity of amplification cycles in Standard PCR, It is that dPCR is generally independent of the quantity of amplification cycles to determine the sample size of starting.

The conventional method of dPCR analyses is usually explored using fluorescence and identifies the production of amplification based on the detection method of light Object.Such method needs the abundant amplification of target molecule to generate enough signals being detected, but can lead to additional mistake Mistake or deviation.

It is in the present disclosure those include it is nucleic acid-templated in the hole of isFET arrays distribution and subsequent template exist It, can be quantitative to include expansion after amplification into the downstream analysis step of line option in the embodiment of amplification in the hole of array Increase production the site of object or the quantity in hole.In some embodiments, the product of nucleic acid amplification reaction is can detect to count to include to expand The site of the template of increasing or the quantity in hole.

For example, in some embodiments, present disclosure is usually directed to nucleic acid synthetic method, including：Offer includes The sample of the polynucleotides of first quantity；It is distributed with by the single polynucleotides of sample into the different loci of site array.

Optionally, method may also include is formed substantially by expanding single polynucleotides in their own site The nucleic acid group of monoclonal.

Optionally, site keeps being in fluid communication during amplification.

Optionally, amplification includes partial denaturation template.

Optionally, amplification includes template is made to undergo partial denaturation temperature.In some embodiments, template include comprising The eutectic point sequence of primer binding site, when template experience partial denaturation temperature, the sequence is endowed single-stranded.

Optionally, amplification includes partial denaturation template.

Optionally, amplification is included at least two different templates at two different array sites with being used for core The single reaction mixture contact of acid amplification.

Optionally, reaction mixture includes recombinase.

Optionally, reaction mixture includes at least one primer for including " resistance label ".

Optionally, amplification includes carrying out at least one amplification cycles, and the amplification cycles include：Partial denaturation template, makes Primer hybridizes with template and with template dependent manner extension primer.Optionally, amplification includes isothermal duplication.In some implementations In scheme, expanded under conditions of substantially isothermal.

In some embodiments, the percentage in the site containing one or more template molecules is greater than 50% and small In 100%.

In some embodiments, disclosed method may also include at least one site of detection because of at least one amplification The variation of ion concentration caused by cycle.

In some embodiments, disclosed method may also include the initial amount of quantitative target nucleic acid.

It is found in such as 2012 4 using some examples of the digital pcr based on array of the sensing technology based on ion In the U.S. Provisional Application No. 61/635584 (being incorporated herein by reference in their entirety) that the moon is submitted on the 19th.

In some embodiments, the method that present disclosure generally relates to detection target nucleic acid, including：By sample It is divided in multiple sample sizes, wherein the part more than 50% includes the target nucleic acid molecule that every sample size is not more than 1； Make condition of multiple sample size experience for amplification, wherein the condition includes partial denaturation condition；Wherein there are targets for detection The variation of ion concentration in the sample size of nucleic acid；Count the quantity of the part of the target nucleic acid with amplification；With determining sample The amount of target nucleic acid in this.The variation of ion concentration can be the raising of ion concentration or can be ion concentration reduction.One In a little embodiments, method may also include sample and bead set.In some embodiments, method may include sample It is loaded onto on substrate, wherein the substrate includes at least one hole.

In some embodiments, target nucleic acid experience partial denaturation condition is made to include target nucleic acid molecule in each Sample size in contacted under the conditions of RPA with recombinase and polymerase.

In some embodiments, target nucleic acid experience partial denaturation condition is made to include becoming target nucleic acid molecule experience part Warm-natured degree.

In some embodiments, present disclosure generally relates to the method for carrying out the absolute quantitation of nucleic acid, packet It includes：Nucleic acid-templated sample of the dilution comprising initial amount and by multiple sites of the nucleic acid-templated distribution of sample to array, In the percentage comprising one or more nucleic acid-templated sites be more than 50% and less than 100%；Make multiple site experience at least One amplification cycles, wherein carrying out the amplification cycles according to any amplification method disclosed herein；Detect multiple samples The variation of at least one middle ion concentration because of caused by least one amplification cycles of this capacity；With quantitative nucleic acid template Starting quantity.The variation of ion concentration can be the raising of ion concentration, the reduction of ion concentration, the variation of pH, can relate to sun The detection of property ion such as hydrogen ion, anion such as pyrophosphate molecule or cation and anion.

In some embodiments, present disclosure also generally relates to exchange by using the chain of recombinase-mediated The different supports that the individual member of nucleic acid-templated group is connected in multiple supports or the difference being connected in multiple sites The method (and relevant composition, system and kit) in site.These methods, composition, system and kit can need Individually obtain or distinguish the fixed amplification subgroup for generation suitable for operation in the application of different amplicons.One In a little embodiments, multiple sites in multiple discontinuous supports or array respectively contain capture primer.Each template is extremely The fixation of each support (or each site to array) can be by (" merging and drawing template in primer with support or site Object ") in the presence of contacted to realize.In some embodiments, fusion primer includes the part for being complementary to template Target specific moiety and be complementary to support or site capture primer at least certain part universal primer binding site. Optionally, it is contacted in the presence of RPA components.RPA components may include recombinase.RPA components may include strand displacement Polymerase.In some embodiments, it is exchanged by the chain of recombinase-mediated and recombinates fusion primer into template, so as to shape Into the template for including universal primer binding site:Primer adduct.It in some embodiments, then will capture primer recombination Into universal primer binding site, the fixed template for being connected to support or site is formed.

In some embodiments of the amplification based on globule, different target specific moiety and common will be respectively contained The fusion primed libraries of universal primer binding site gather with multiple template and multiple supports comprising polymerase and strand displacement It is contacted in the reaction mixture of synthase.Then by the way that mixture is made to undergo RPA conditions by template library connection to multiple Object is held, so as to generate the multiple supports for being respectively connected with different templates.

In some embodiments of the amplification based on array, different target specific moiety and common will be respectively contained Fusion primed libraries and the multiple template of universal primer binding site and the surface comprising multiple sites comprising polymerase and It is contacted in the reaction mixture of strand displacement polymerase.At least some of multiple sites include general capture primer.It is then logical Crossing makes mixture experience RPA conditions that multiple sites of template library connection to surface are respectively connected with difference so as to generate Template multiple supports.

In some embodiments, present disclosure, which is usually directed to, includes parallelly to expand using partial denaturation condition Increase the composition (and relevant be used to prepare and the method using the composition) of one or more nucleic acid-templated reagents.

In some embodiments, composition may include described herein for carrying out any group of component of RPA Point.

In some embodiments, composition may include appointing for the component described herein for being used to carry out template walking What component.

In some embodiments, present disclosure generally relates to the composition and system of nucleic acid amplification, including： Surface comprising the first site and the second site；And nucleic acid amplification reaction mixture, wherein the mixture and first and second Site contacts.

In some embodiments, reaction mixture includes recombinase.

In some embodiments, the first site is operationally coupled to first sensor, and the second site is operable Ground is connected to second sensor.

In some embodiments, the first and second sites are operably coupled to identical sensor.

Optionally, the first site includes the nucleic acid group of the first substantially monoclonal.Second site optionally includes the second base The nucleic acid group of monoclonal in sheet.

In some embodiments, disclosed composition includes：Surface comprising the first site and the second site, wherein First site includes the first nucleic acid that substantially the nucleic acid group of monoclonal and the second site include the second substantially monoclonal Group；And nucleic acid amplification reaction mixture, wherein the mixture is contacted with the first and second sites.

In some embodiments, composition includes the array in site, and the array in the site is included comprising (such as connecting Be connected to) first capture primer the first site and include (such as being connected to) second capture primer the second site.

In some embodiments, sensor includes field-effect transistor (FET).

In some embodiments, at least the first site or the second site or the first and second sites, which include, is connected to table The capture primer in face.

Optionally, hydrophilic polymer matrix includes aquogel polymer matrix.

Optionally, polyacrylamide is conjugated in Oligonucleolide primers.

Optionally, hole has 0.1 micron to 2 microns of characteristic diameter.

Optionally, hole has 0.01 micron to 10 microns of depth.

In some embodiments, sensing material includes metal-oxide.

In some embodiments, sensing material is to hydrogen ion sensitivity.

In some embodiments, it is public to may include carrying out the United States Patent (USP) as disclosed in 21 days June in 2012 for the method for amplification " template walking " described in the number of opening 2012/0156728 (it is incorporated herein by reference in their entirety).For example, in some implementations In scheme, present disclosure expands one or more nucleic acid-templated to expand with forming clone with generally relating to clone Method, composition, system, device and the kit of nucleic acid-templated group.Any amplification method described herein optionally wraps Include the nucleic acid amplification cycle of repetition.Amplification cycles optionally include：(a) primer to template strand hybridization, (b) primer extend with Form the chain of the first extension, the chain of (c) extension is from the partially or incompletely denaturation of template strand.Be hybridized to template strand primer (for For the sake of convenient, it is named as " forward direction " primer) optionally fix on the support or be fixed to support.Support is, for example, solid Body or semisolid.Optionally, the denaturation portion of the template strand from step (c) in next amplification cycles can freely with Different forward primer hybridization.In embodiments, in subsequent amplification cycles primer extend include first extension chain from The displacement of template strand.It may include such as second " reversed " primer, be hybridized to 3 ' ends of the chain of the first extension.Reverse primer Optionally it is revocable.

In embodiments, using being fixed on one or more solid or semisolid supports or be fixed to drawing for its Object expands template.Optionally support includes the primer of the fixed first part for being complementary to template strand.Optionally, support The primer of fixed the second non-overlapping homeologous with identical template strand is not included significantly.If two parts are not wrapped Containing any subdivision hybridized each other or be complementary to object hybridization, then they are non-overlapping.In another example, it supports Object does not include the fixed primer that can hybridize with the complement of template strand significantly optionally.

Optionally, in single Continuous Liquid Phase in the presence of one or more supports, wherein each is supported Object includes one or more fixed sites, simultaneously expands multiple nucleic acid-templated.In embodiments, each template is expanded To generate the amplification subgroup of clone, wherein each clone group is fixed on the support or fixed bit different from the group of other amplifications Point within or on.Optionally, the group of amplification keeps substantially monoclonal after amplification.

Template be for example amplified with generate clone group, it includes the homologous chain of template (referred to herein as " template strand " or " reverse strand ") and/or template complementary strand (referred to herein as " primer strand " or " positive chain ").In embodiments, in institute Keep Clonal by maintaining the association between template strand and its primer strand in the nucleic acid group of the amplification of generation, so as to effectively Ground relevant clone generation joint or " constraint " together and are reduced into the different possibilities for cloning cross contamination between group. Optionally, the nucleic acid of one or more of clone group amplification is connected to support.The clone group of substantially the same nucleic acid can Optionally there are macro manifestations spatially limit to or discontinuous.In embodiments, clone group can be similar to the point of separation Or colony (for example, when being assigned into support, optionally on the outer surface of support).

In some embodiments, present disclosure is usually directed to the new of the clone group for the limitation for generating clonal expansion Type method, the clone group are optionally fixed to one or more supports or are fixed in which or on which.Support can example Solid or semisolid (such as gel or hydrogel) in this way.The clone group of amplification be optionally connected to support outer surface or It can also be in the inner surface of support (such as when support has porous or matrix structure).

In some embodiments, prolonged by the primer along purpose template strand (also referred to as " reversed " chain) of multiple cycles It stretches to realize amplification.For convenience, the primer for being hybridized to purpose template strand is referred to as " forward direction " primer, and optionally with mould Plate dependence mode extends it, to form " forward direction " chain for being complementary to purpose template strand.In certain methods, positive chain is in itself With the second primer hybridization referred to as " reversed " primer, it is (also referred to as anti-to form new template strand to extend second primer To chain).Optionally, at least part of new template chain and original purpose template (" reversed ") chain are homologous.

As mentioned, one or more primers can be fixed to one or more supports or be fixed in which or on which. Optionally, a primer is fixed by being connected to support.Second primer can be it is existing and be optionally not fixed or It is connected to support.It can simultaneously be expanded not on different supports or fixed site for example in single Continuous Liquid Phase With template to form the nucleic acid group of monoclonal.If any part of liquid phase and any other part of liquid are that fluid connects It touches or connects, then it is believed that liquid phase is continuous.In another example, if not part and liquid rest part It is fully segmented or is divided or in other ways by fully physically separate, then it is believed that liquid phase is continuous.Optionally, Liquid phase is flowable.Optionally, Continuous Liquid Phase is not in gel or Medium Culture.In other embodiments, Continuous Liquid Phase is solidifying Glue or Medium Culture.Such as Continuous Liquid Phase occupies the hole, gap or other gaps of solid or semisolid support.

When liquid phase is in gel or Medium Culture, one or more primers are optionally fixed on the support.Optionally support Object be gel or matrix in itself.Alternatively, support be not gel or matrix in itself.In instances, a primer, which is fixed on, includes On solid support in gel and it is unsecured to gel molecular.Support be, for example, plane surface form or one Or multiple particles.Optionally plane surface or multiple particles include the forward primer with substantially the same sequence.In reality It applies in scheme, support does not include the second different primer of significant quantity.Optionally, the second on-fixed primer is in gel In solution.Second on-fixed primer is for example bound to template strand (i.e. reverse strand), and fixed primer is bound to positive chain.

The embodiment of template walking includes the method for primer extend, including：(a) primer-hybridization step, (b) extension step Suddenly and (c) walking step.Optionally, primer-hybridization step includes hybridizing the first primer molecule (" the first forward primer ") Complementary forward primer binding sequence (" positive PBS ") on to nucleic acid chains (" reverse strand ").Optionally, extension step includes The first positive chain of extension is generated, which is the overall length complement of reverse strand and is hybridized to it.Such as by using reverse strand The first forward primer molecule is extended to generate the first of extension the positive chain with template dependent manner as template.Optionally, row It walks step to include the second primer (" the second forward primer ") being hybridized to positive PBS, wherein reverse strand is also hybridized to the first forward direction Chain.For example, walking step includes at least part (" free fraction ") from positive chain denaturation forward direction PBS, and reverse strand is another A part is still hybridized to positive chain.

In embodiments, primer extension method is to include the amplification method that template is walked, wherein primer-hybridization, extension And/or any one or more steps of walking are repeated at least once.For example, method may include expanding by one or more Cyclic amplification forward direction chain.Amplification cycles optionally include extension and walking.Exemplary amplification cycle is included or substantially by extending It is formed with subsequent walking.Optionally, the second forward primer of the first amplification cycles is used as the first of subsequent amplification cycles Forward primer.For example, the extension that the second forward primer of step of walking in the first amplification cycles is used as subsequent amplification cycles walks The first rapid forward primer.

Optionally, primer extend or the method for amplification further include extends or expands reverse strand as follows：(a) will First reverse primer molecule hybridizes with the complementary reverse primer binding sequence (" reversed PBS ") on the positive chain extended；(b) First reverse primer molecule is extended to generate the first of extension instead with template dependent manner as template by using positive chain To chain, which is the overall length complement of positive chain and is hybridized to it；(c) the second primer (" the second reverse primer ") is hybridized To reversed PBS, wherein positive chain is also hybridized to the first reverse strand.Optionally carry out the one or many heavy of step (b)-(c) Multiple, the second reverse primer of wherein step (c) is first reverse primer of (b) the step of repetition；Wherein described primary or The major part of institute's having time forward direction chain during or between multiplicating is hybridized to reverse strand.In embodiments, it is most of to appoint Selection of land is at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%.

Optionally, reverse strand and/or positive chain are not exposed to complete Denaturing, the complete denaturation during amplification Condition can cause multiple chains of signal portion (such as more than 10%, 20%, 30%, 40% or 50%) with its extend and/or The complement of overall length is kept completely separate.

In embodiments, in (such as the amplification of 1,5,10,20 or all of progress of one or more amplification cycles Cycle) during or between institute having time is positive and/or the major part of reverse strand is optionally hybridized to extension and/or overall length Complement.In embodiments, the major part of chain be optionally chain at least 60%, 65%, 70%, 75%, 80%, 85%th, 90%, 95% or 99%.In embodiments, this passes through the primer that is maintained above not extending by amplified reaction T_mAnd less than the T of primer-complementary strand_mTemperature realize.For example, amplification condition is maintained in such temperature, it is higher than The T for the forward primer not extended_mAnd less than the T of extension or overall length reverse strand_m.Similarly, for example, amplification condition is kept In such temperature, higher than the T of reverse primer not extended_mAnd less than the T of extension or overall length positive chain_m。

Optionally, one or more forward primers and/or one or more reverse primers are respirable (breathable), such as with low T_m.In instances, can breathe the nucleotide base of primer at least 60%, 65%, 70%th, 75%, 80%, 85%, 90%, 95% or 99% be adenine, thymidine or uracil or be complementary to adenine, Thymidine or uracil.

In some embodiments, present disclosure generally relates in amplified reaction solution on the support gram Method, composition, system, device and the kit of grand ground amplification of nucleic acid template.Optionally, it is nucleic acid-templated to be wrapped with support It is contacted in solution containing Continuous Liquid Phase.Support may include including the primer group of at least the first primer and the second primer.It can example Such as primer group is fixed on the support by the covalent linkage to support.In some embodiments, nucleic acid-templated packet Containing the primer binding sequence close to target sequence.Primer binding sequence can be complementary to the sequence of the first primer and optionally second and draw The sequence of object.Target sequence can not be complementary to the primer in primer group.In some embodiments, nucleic acid-templated primer knot It closes sequence and is hybridized to the first primer.Polymerase switching templates extension the first primer can be used, so as to form the first primer of extension. At least part of the primer binding sequence of template can detach (such as denaturation or unwinding) with the first primer of extension.Optionally into Row separation maintains hybridizing between the part of template and the first primer of extension simultaneously.The part of the primer binding sequence of separation The second primer then can be hybridized to.Optionally, the other parts and the first of extension that such hybridization maintains template simultaneously are carried out Hybridization between primer.Can be used polymerase switching templates extend the second primer, so as to formed include extension the first primer and The support of second primer of extension.The part of the extension of the first primer of extension and/or the second primer of extension may include mutually Mend the sequence in target sequence.

In some embodiments, present disclosure generally relates in amplified reaction solution on the support gram The method of grand ground amplification of nucleic acid template, including：It is contacted nucleic acid-templated in liquid solution with support, wherein support packet The fixed primer group for including at least the first primer and the second primer is included, and wherein nucleic acid-templated comprising close to target sequence Primer binding sequence, wherein primer binding sequence are complementary to the sequence of the first primer and the sequence of the second primer, and target sequence Row are not complementary to the primer in primer group；Nucleic acid-templated primer binding sequence is hybridized to the first primer；Use polymerase edge Template extends the first primer, so as to form the first primer of extension；By at least part of the primer binding sequence of template and extension The first primer denaturation, while maintain hybridizing between another part of template and the first primer of extension；By the primer of denaturation The partial hybridization of binding sequence remains miscellaneous between the other parts of template and the first primer of extension to the second primer It hands over；With use polymerase switching templates extend the second primer, so as to formed include extension the first primer and extension the second primer Support, wherein the extension of the second primer of the first primer extended and extension respectively contains the sequence for being complementary to target sequence. Primer group may include substantially the same primer, and the substantially the same primer has not more than 1,2,3,4 or 5 in sequence The difference of a nucleotide.In some embodiments, primer group includes different primers, and at least some of the primer include It is complementary to the sequence of the primer binding sequence of template.In some embodiments, the primer of primer group is not complementary to the 5 ' of template Hold half edge sequence.In some embodiments, the primer of primer group is not complementary to 3 ' ends of the primer of any extension of support Half edge sequence.In some embodiments, the primer of primer group is not complementary to any in addition to primer binding sequence of template Sequence.

In some embodiments, present disclosure is generally related in amplified reaction solution on support group The method of clone ground amplification of nucleic acid template group, including：According to any method disclosed herein on the first support first Nucleic acid-templated upper clone ground expands the first template and expands the second core with cloning on the second support according to identical method Acid template, wherein all supports are included in during amplification in single Continuous Liquid Phase.

Among other things, provide the clone group's of the limitation for fixed clonal expansion for generating single-stranded template sequence Method, including：(a) single-stranded template sequence (" template 1 ") is connected to fixed site (" IS1 "), wherein IS1 includes multiple copy The fixed primer (" IS1 primers ") that can be substantially hybridized to template 1 of shellfish, and template 1 by with IS1 primer hybridizations and It is bound to IS1 and (b) and expands template 1 in the solution using IS1 primers and revocable primer (" SP1 primers "), wherein mutually It mends chain in the amplification of single-stranded template 1 and cannot substantially hybridize with the primer on IS1 when being single-stranded, wherein expanding in template 1 The clone group of the limitation of fixed clonal expansion is generated around to the initial cross point of IS1.

Additionally provide the separation and fixation for generating the first template sequence (" template 1 ") and the second template sequence (" template 2 ") Clone group method, the method includes amplification the first and second template sequences to be substantially connected to the first fixation to generate Site (" IS1 ") rather than second fix site (" IS2 ") template 1 clonal expansion subgroup or be substantially connected to IS2 and The clonal expansion subgroup of the template 2 of non-IS1, wherein：(a) two templates and all amplicons are all contained in identical continuous liquid In phase, wherein Continuous Liquid Phase and first and second fix site (respectively, " IS1 " and " IS2 ") contact, and wherein IS1 with IS2 is spatially separated, and (b) template 1 includes the first subsequence (" T1-FOR ") simultaneously when for single stranded form an end The second subsequence (" T1-REV ") is included in its opposing end portions, and (c) template 2 includes the when for single stranded form an end One subsequence (" T2-FOR ") simultaneously includes the second subsequence (" T2-REV ") in its opposing end portions, and (d) IS1 includes multiple copies Fixed nucleic acid primer (" IS1 primers "), the primer T1 and T2 for it is single-stranded when can substantially be hybridized to T1-FOR and T2-FOR, (e) IS2 include the fixed primer (" IS2 primers ") of multiple copies, and the primer can when T1 and T2 is single-stranded Substantially it is hybridized to T1-FOR and T2-FOR, the reverse complemental object of (f) T1-REV cannot be substantially hybridized to when being single-stranded Primer on IS1, but can substantially be hybridized to revocable primer (" SP1 ") in Continuous Liquid Phase；(g) T2-REV's is reversed Complement cannot substantially be hybridized to the primer on IS2 when being single-stranded, but can substantially be hybridized to non-solid in Continuous Liquid Phase Fixed primer (" SP2 ")

Optionally, in any method being described herein, any nucleic acid dissociated from a fixed site being capable of base The nucleic acid that described two fixed sites are hybridized in sheet and are dissociated described in Continuous Liquid Phase is appointed to another fixation site What mobile (such as passing through diffusion, the movement of convection current) is substantially unobstructed.

Optionally, in any method being described herein, Continuous Liquid Phase contacts simultaneously with IS1 and IS2.

Optionally, in any method being described herein, the first part of the template that fixed primer combines not with The second part overlapping of template, the complement of the second part are combined by revocable primer.

Optionally, in any method being described herein, at least one template to be amplified is being incited somebody to action from input nucleic acid It is generated after nucleic acid and at least one fixed bit point contact.

Optionally, any method described herein includes the following steps：(a) support that fixed primer will be included It is contacted with single stranded nucleic acid template, wherein：By the primer binding sequence (PBS) on the first fixed primer hybridization to template；(b) The first primer of hybridization is extended to form the chain of extension with Template Dependent extension, the chain of the extension is complementary to template simultaneously And at least partly hybridization is in template；(c) it is at least partially single-stranded so as to PBS from the complementary strand partial denaturation template of extension Form (" free fraction ")；(d) free fraction is hybridized to second primer do not extend, fixed；(e) with Template Dependent Extension extends the second primer to form the chain for the extension for being complementary to template；(f) optionally, by the fixed of the extension of annealing Nucleic acid chains are separated from each other.

Optionally, in any method being described herein, (a) forms nucleic acid double chain thus comprising rising during amplification Beginning template and/or the chain of amplification；The double-strand does not suffer from the complete denaturation for the double-strand that can lead to substantial amounts during amplification Condition.

Optionally, in any method being described herein, by obtaining multiple input double-strands to be amplified or single-stranded Nucleic acid sequence (sequence can be known or unknown) and addition or generation on the end of at least one input nucleic acid First universal linker sequence and the second universal linker sequence generate single-stranded template；Wherein described first universal linker sequence is miscellaneous Hand over to IS1 primers and/or IS2 primers, and the reverse complemental object of second universal linker sequence be hybridized to it is at least one Revocable primer.Connector can be double-strand or single-stranded.

Optionally, in any method being described herein, in the first and second ends of the single-stranded template sequence First and second nucleic acid linker sequences are provided.

Optionally, in any method being described herein, label is also added to one or more nucleic acid sequence (examples Such as template or primer or amplicon), the label makes it possible to the nucleic acid that identification includes label.

Optionally, in any method being described herein, all at least one fixed site or support draw Object has identical sequence.Optionally, fixed site or support include, and there are at least two the multiple of different sequences to draw Object.In some embodiments, fixed site or support include at least one target specific primer.

Optionally, in any method being described herein, continuous media is flowable.Optionally, revocable core Acid molecule is blended in Continuous Liquid Phase during at least part of amplification procedure, such as is retouched herein at any one or more It is substantially uncrossed during the step of stating or cycle.

Optionally, in any method being described herein, in the period of during amplification in mixing be not to be obstructed substantially Hinder.For example, mixing is substantially uncrossed during the entire duration of amplification.

In embodiments, (see, for example, WO2003072805, passed through using RPA, that is, recombinase-polymeric enzymatic amplification It is incorporated herein by reference) it is expanded to realize.Optionally carried out in the case of no temperature or the substantial variation of reagent conditions RPA.In embodiments herein, recombinase and/or single strand binding protein can be used to realize partial denaturation and/or amplification, Including any one or more steps described herein or method.Optionally combined with single strand binding protein (SSB), appropriate Recombinase includes RecA and its protokaryon or eukaryon analog or its functional fragment or variant.In embodiments, recombinase agent Single stranded DNA (ssDNA) such as amplimer is optionally coated with to form nucleoprotein filament chain, the nucleoprotein filament chain invades template The upper double stranded region with homology.This optionally generates short hybrid and alternative chain bubble (displaced strand bubble) (being referred to as D- rings).In embodiments, free 3 '-end of silk chain extends to synthesize through archaeal dna polymerase in D- rings New complementary strand.Complementary strand replaces the marriage chain of the original pair of template in extension.In embodiments, it is one or more Amplimer with before the template contacts of optionally double-strand with one or more recombinase agent to contacting.

In any method being described herein, the amplification of template (target sequence) include by recombinase agent with it is at least one One or more contacts of amplimer pair, so as to form one or more " forward direction " and/or " reversed " RPA primers.Optionally Remove any recombinase agent do not associated with one or more primers.Optionally, then by one or more forward direction RPA primers It is contacted with template strand, the template strand optionally has the region at least one RPA Primers complementaries.It can be by template strand To RPA primers and the contact position of complementary template, the contact optionally leads to hybridizing between the primer and template.Optionally Ground, using one or more polymerases (such as in the presence of dNTP) along 3 ' ends of template extension primer to generate Double-strandednucleic acid and displacement template strand.Amplified reaction may include such contact and the repetitive cycling extended until that can it is expected Amplification degree.Optionally, the displacement chain of amplification of nucleic acid is reacted by parallel RPA.Optionally, the displacement chain of nucleic acid passes through It is contacted to expand with one or more complementary primers successively；(b) it is complementary by any strategy extension described herein Primer.

In embodiments, one or more primers include " forward direction " primer and " reversed " primer.By two kinds of primers and mould Plate contact optionally leads to the first duplex structure in the first part of first chain and is led in the second part of second chain Cause duplex structure.Optionally, extend positive and/or reverse primer 3 ' ends with one or more polymerases to generate first With the second double-strandednucleic acid and the first and second displacement chains of nucleic acid.Optionally, the second displacement chain is at least partly complementary to each other And it can hybridize to form sub- double-strandednucleic acid, double stranded template nucleic acid can be used as in subsequent amplification cycles.

Optionally described first and described second, which replaces chain, is at least partially complementary to the described first or described second primer simultaneously And the described first or described second primer can be hybridized to.

In any method or step or composition or the alternative embodiment of array that are described herein, support is appointed Selection of land includes the fixed primer with more than one sequence.After template nucleic acid chain is hybridized to the first complementary immobilized primer, It then may extend away the first primer and template and primer can partially or even wholly be detached to each other.It then can drawing extension Object is annealed to the second immobilized primer with the sequence different from the first primer, and extensible second primer.It is then separable The primer of (such as being completely or partially denaturalized each other) two kinds of extensions simultaneously by it and then can be used as extending other fixation The template of primer.Repeatable process is to provide amplification, fixed nucleic acid molecules.In embodiments, which causes to have The fixed primer extension product of two different sequences complimentary to one another, wherein all primer extension products are solid in 5 ' ends Determine to support.

In some embodiments, disclosed method includes amplification, wherein amplification includes chain overturning.It is described below In " overturning " embodiment, extend two or more primers to form the chain of two or more corresponding extensions.Optionally Ground, two or more extended primers include substantially the same sequence or consisting essentially of, and corresponding prolong The part of the extension for the chain stretched is at least partly different and/or complimentary to one another.

One exemplary implementation of overturning is as described below.Such as walked by template and expand starting template, to generate The chain (for convenience of rising, seeing that it will be referred to as " forward direction " chain) of multiple primer-extensions.Optionally, positive chain is complementary to starting mould Plate.Optionally, positive chain is fixed on the support.Optionally, positive chain includes substantially the same sequence, such as positive chain It is substantially identical.In embodiments, by extending one or more fixed primers on the support, (" forward direction " draws Object) form positive chain.Forward primer and/or positive chain are optionally connected in its 5 ' end or nearby support.Optionally Ground, the one or more of the positive chain of primer-extension include 3 ' sequences being referred to as from hybridization sequences, and the 3 ' sequence is not deposited Be in the primer not extended and 5 ' sequences can be hybridized under selected conditions (this process will be referred to as " from hybridizing "). 5 ' sequences are optionally the parts for the forward primer not extended.In instances, positive extension products are formed after such hybridization " stem-loop " structure.Optionally, the forward primer not extended includes susceptible " cleavable to cutting in its 3 ' end or nearby " nucleotide.In embodiments, cleavable nucleotide is connected at least one by being connected between " scissile " nucleosides A other nucleotide are connected and can be cut under conditions of it will not substantially cut phosphodiester bond between the nucleosides.

After extension, optionally allow forward direction-primer extension product (i.e. positive chain) from hybridization.In other embodiments, Allowing from after hybridizing, (such as the nucleosides of scissile connection is being formed with adjacent nucleotide in cleavable nucleotide Acid) the positive chain of scissile junction cutting.Cutting leads to two pieces of primer-extension products (the positive chain extended) Section.In embodiments, the first segment includes at least part of the original forward primer not extended.Optionally, first Section does not include the sequence of any extension.Optionally, the first segment is fixed (for example, because of the forward primer not extended It is fixed).In embodiments, the second segment includes the sequence of extension.Optionally, the second segment is drawn comprising what is do not extended Any 3 ' part of the object except cleavable nucleotide or any part not comprising the primer not extended.Optionally, Two segments are hybridized to first part by it from hybridization sequences.

In instances, cleavable nucleotide is the nucleotide removed by one or more enzymes.Enzyme may, for example, be glycosyl Change enzyme.According to N- glycosylases activity, cleavable nucleotide is optionally discharged from double-stranded DNA for glycosylase.Optionally, The removal of cleavable nucleotide is generated without base, without purine or without pyrimidine site.Optionally for example pass through another enzyme activity Property further modifies abasic site.Optionally, abasic site is modified by lyase to generate base notch.Split conjunction Enzyme for example cuts the 3 ' and/or 5 ' of abasic site.5 ' and 3 ' ends are optionally happened at by the cutting of lyase, are caused It removes abasic site and leaves base notch.Exemplary cleavable nucleotide such as 5- hydroxyls-uracil, 7,8- dihydros- 8- hydroxyls guanine (8- hydroxyls guanine), 8- hydroxyls adenine, fapy- guanines, methyl-fapy- guanines, fapy- adenines, Aflatoxin B1-fapy- guanines, 5- hydroxy-cytosines can be identified and be gone divided by formed without fast by a variety of glycosylases Purine site.A kind of suitable enzyme is formamido group pyrimidine [fapy]-DNA glycosylases, also referred to as 8- hydroxyls guanine DNA glycosyls Change enzyme or FPG.FPG can be used as N- glycosylases and AP- lyases.N- glycosylases activity is optionally discharged from double-stranded DNA Impaired purine, so as to generate no purine (AP site), wherein phosphodiester backbone is optionally complete.AP- lyases 3 ' and the 5 ' of activity cutting AP site are so as to remove AP site and leave single base notch.In instances, cleavable nucleotide It is 8- hydroxyl adenines, single base notch is converted to by the FPG with glycosylase and lyase activity.

In another embodiment, cleavable nucleotide is uridine.Optionally, uridine is cut by " USER " reagent, The reagent includes uracil dna glycosylase (UDG) and DNA glycosylase-lyases endonuclease VIII, wherein UDG The excision of uracil base is catalyzed, no base (no pyrimidine) site is formed and keeps the complete of phosphodiester backbone simultaneously, and The lyase activity for wherein cutting nuclease VIII destroys phosphodiester backbone in the 3 ' of abasic site and 5 ' sides to discharge nothing The deoxyribose of base is subsequently optionally converted the phosphate group on 3 ' ends of cleaved products to-OH bases using kinases.

Segment optionally by least one cutting is contacted with polymerase.Optionally first fixed segment can be by polymerizeing Enzyme extends.If it is expected in this way, the segment of the second hybridization can be used as the template of the extension of the first segment.In embodiments, Form " overturning " double-strand extension products.The product of the overturning optionally by it is described herein it is any in a manner of undergo template Walking.When overturning and unturned experience template walking, two different extension products groups are formed, two of which is prolonged Stretching product, there is identical part (corresponding to the primer not extended) and part complimentary to one another (to correspond to prolonging for extension products The part stretched).

In embodiments, optionally by the way that the positive chain extended and single-stranded " montage " joint sequence are tried in extension It is contacted in the presence of agent (such as polymerase and dNTP) by aim sequence for example from hybridization sequences or new primer Binding site is added in 3 ' ends of the positive chain of extension.The montage sequence optionally includes and is being essentially complementary extension just Partly and 5 ' parts of aim sequence to be added are essentially complementary to the 3 ' of 3 ' end sections of chain.It is connect by montage After the 3 ' ends of positive chain that head is hybridized to extension, positive chain experience Template Dependent is made using montage connector as template Polymerase extends.Such extension leads to the addition of 3 ' ends of the aim sequence to the positive chain extended.

Therefore, primer extend described herein and/or any method of amplification may include following steps any one or It is multiple：(a) fixed forward primer is extended by template walking to generate the positive chain of multiple extensions, the forward direction chain is optional Ground is identical；(b) optionally using montage connector be hybridized to extension positive chain 3 ' ends and use montage connector as mould Plate makes positive chain experience template dependent polymerase extension, so as to which other 3 ' sequences are added to the positive chain further extended, The part of 3 ' sequences wherein added is to be complementary to the part of the forward primer that does not extend and be hybridized to it to form stem ring Structure；(c) cleavable at or near tie point of the forward primer sequence not extended with the positive chain-ordering extended Nucleotide the positive chain of easy cut-out junction cutting；Cleavable nucleotide is optionally removed, so as to generate two cuttings Segment, wherein the first segment includes the forward primer not extended of 3 ' primers-complementary series that is hybridized in the second segment Part；(d) the experience polymerase extension of the first segment is optionally made to generate the forward direction of overturning using the second segment as template Chain；(e) the second montage connector is optionally hybridized to 3 ' ends of the positive chain of overturning, and uses montage connector as template Make positive chain experience Template Dependent extension, so as to which other 3 ' sequences to be added to the positive chain of overturning, wherein add 3 ' The part of sequence is the new primer binding sequence being not present in the chain of overturning；(f) it is connect optionally through with new primer It touches and (such as described in this article) extends or expand to extend or expand comprising new primer combination sequence in any method The chain of the overturning of row.New primer will not be incorporated into unturned chain or the overturning not being extended still further in step (e) Chain.

Fig. 8 shows that the schematic diagram of exemplary chain overturning and Running strategy describes.(A) template walk, (B) chain overturning with The chain of overturning is generated, (C) adds new primer binding sequence Pg ' on final overturning chain.

Optionally, by any amplification method of single support in this article, can hybridize wherein single support has In multiple primers of template.In such embodiments, the adjusting template before template gleanings and solid support into contact The concentration of gleanings is so that the individual template molecule in gleanings is at least 10²、10³、10⁴、10⁵、4x 10⁵、5x 10⁵、6x 10⁵、8x 10⁵、10⁶、5x 10⁶Or 10⁷A molecule/mm²Density be connected or associate and (such as be fixed on by being hybridized to Primer on body support).

Optionally, the individual template molecule of amplification in situ, generation are spatially confined to the miscellaneous of starting template on the support Clone group around intersection point.Optionally, it expands from the template of single amplification and generates no more than about 10²、10³、10⁴、10⁵、10⁶、 10⁷、10⁸、10⁹,10¹⁰、10¹¹、 10¹²、10¹⁵Or 10²⁰Amplicon.Optionally, the colony of clonal expansion is at least 10²、 10³、10⁴、10⁵、4x 10⁵、5x 10⁵、6x 10⁵、8x 10⁵、10⁶、5x 10⁶Or 10⁷A molecule/mm²Density be located at solid branch It holds on object.

It in some embodiments, can multiple nucleic acid be combined wherein with one or more supports by nucleic acid gleanings It is contacted under conditions of to identical support.Such contact can be particularly used for being related to nucleic acid in identical support The method of parallel clonal expansion in different zones.The ratio that adjustable nucleic acid quantity accumulates support surface, with for example, by Ensure to be properly spaced nucleic acid in support to promote the monoclonal group of the nucleic acid of amplification substantially in different clones It is formed that monoclonal is promoted to be formed in the case of no cross contamination between group.Such as when using single support, it will wait to be expanded The gleanings of the nucleic acid of increasing are adjusted to such dilution so that the clone of the amplification of the gained group generated from individual nucleic acid is universal It is discontinuous or separation, it is such as non-overlapping.For example, the clone group of 50%, 70%, 80% or 90% or more amplification Do not have to spread the nucleic acid being not substantially identical in interior individual nucleic acid.Optionally, different amplification groups does not expand with others The detection method of group's contact or completely overlapped or usable selection is distinguished from each other.

In some embodiments, nucleic acid is connected to the surface of support.In some embodiments, nucleic acid can be attached It in support.For example, for the support comprising hydrogel or other porous matrixes, nucleic acid can be attached to support Whole volume include surface on and support in.

In some embodiments, support (or at least one of support group support) can be connected at least One primer, is optionally connected to primer group.For example, support (or at least one support) may include primer group.Primer group Primer be substantially identical or may include substantially the same sequence.One of primer, some or all can Include the sequence for being complementary to one or more nucleic acid-templated interior sequences.In some embodiments, primer group may include to The primer of few two incomplementarities.

Primer can be connected to support by its 5 ' end, and with 3 ' free ends.Support can be glass slide Surface or globule surface.Primer has low melting temperature, such as few (dT)₂₀, and the low of gleanings connector can be hybridized to T_mRegion.Distance between primer needs shorter than joint length template to be allowed to walk or selectively, and there is 5 ' ends length to connect The chance that the long primer of head will increase walking.

In some embodiments, by support and primer and template (or reverse strand), primer and template be each other wherein Hybridization is attached and/or contacts under conditions of nucleic acid double chain to be formed.Double-strand may include the complementary sequence comprising template and primer At least one nucleotide residue of the double stranded section of row, wherein complementary series is base pairing each other.In some embodiments In, double-strand also may include single stranded portion.Double-strand also may include single stranded portion.Single stranded portion may include in template (or primer) The sequence of any any other sequence not being complementary in primer (or template).

The non-restrictive illustrative method for the nucleic acid amplification cloned on the support is as described below.It clones on the support Ground amplification of nucleic acid (for convenience, will be referred to as reverse strand), and the complementation that multiple copies are connected on the support is positive Primer.Exemplary nucleic acid is one of multiple DNA gleanings molecules, such as multiple nucleic acid members are in its 5 ' and/or 3 ' end tool There is the sequence of one or more common (" connectors ") and there is variable sequence, such as gDNA or cDNA therebetween.Implementing In scheme, 3 ' common grounds such as connector has respirable (such as low T_m) region, and 5 ' consensus (such as connect Head) optionally there is less respirable (such as higher T_m) region, or vice versa.In another embodiment, 5 ' All it is respirable with 3 ' consensus.Respirable (such as low T_m) the region e.g. region rich in A, T and/or U, example Such as it is rich in the sequence of AT (or U), such as polyT, polyA, polyU and A, T and U bases or the alkali for being complementary to such base Any combinations of base.Illustrative methods describe in this article.

One non-restrictive illustrative method of the nucleic acid amplification of the clone carried out on the support by " template walking " It is shown in Fig. 1.The non restrictive description of the illustrative methods of template walking is as described below.

Double-stranded DNA library molecule is denaturalized and supports single stranded DNA to be connected to by the primer being hybridized on support Object.DNA molecular quantity is provided to the ratio of support area or globule quantity monoclonal is promoted to be formed.

Primer 5 ' is connected on primer and with free 3 ' by it.Support can be the surface or small of glass slide The surface of pearl.Primer has low melting temperature, such as few (dT)₂₀It is or few (dA)₃₀, and the low T of library connector can be hybridized to_m Region.Distance between primer can be shorter than joint length template to be allowed to walk or selectively, has 5 ' end lengthening joints Long primer will increase walking chance.

Ground amplification of nucleic acid is cloned on the support, and the primer of multiple copies is connected on the support.Exemplary nucleic acid It is one of multiple DNA library molecules, the multiple DNA library molecule for example has a kind of or more in its 5 ' and/or 3 ' end The sequence of kind common (such as " connector ") simultaneously has variable sequence, such as gDNA or cDNA therebetween.In embodiments, 3 ' connectors have low T_mRegion, and 5 ' connectors optionally have higher T_mRegion, or vice versa.Low T_mRegion is for example It is the region rich in pyrimidine, such as the sequence rich in AT (or U), such as polyT, polyA, polyU and A, T and U bases or mutually It mends in any combinations of the base of such base.Illustrative methods describe in this article.

Using DNA polymerizations or extension mixture, any one or more reagents such as enzyme, dNTP are optionally included And buffer solution, one or more primers are incubated, no matter it is dissolved form or is connected to support.Extension primer (such as Forward primer).Optionally, extension is that primer extends along the Template Dependent of template, continuous in template including being respectively complementary to The continuous of the nucleotide of nucleotide is incorporated to, so that forward primer that is extension or not extending is complementary to reverse strand (also referred to as instead To parallel or complementary).Optionally, prolonged by the enzyme such as polymerase with polymerase activity or other extension activity to realize It stretches.Optionally there are enzyme other activity to include 3 ' -5 ' exonuclease activities (proofreading activity) and/or 5 ' -3 ' exonucleases Enzymatic activity.Alternatively, in some embodiments, enzyme can lack the one or more of these activity.In embodiments, it polymerize Enzyme has strand-displacement activity.The example of useful strand displacement polymerase includes bacteriophage Ф 29DNA polymerases and Bst Archaeal dna polymerase.Optionally, enzyme is in raised temperature, for example, at 45 DEG C or on, at 50 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C It is or active on 85 DEG C.

Exemplary polymerases are Bst archaeal dna polymerases (exonuclease are negative), are 67kDa stearothermophilus gemma bars Bacterium (Bacillus stearothermophilus) archaeal dna polymerase albumen (large fragment) (being illustrated in accession number 2BDP_A), Lack 3 ' -5 ' exonuclease activities with 5 ' -3 ' polymerase activities and strand-displacement activity list.Other polymerases include coming Taq DNA polymerase is (being illustrated in accession number 1TAQ) from thermus aquaticus (Thermus aquaticus), from large intestine The Eco DNA polymerase is (accession number P00582) of bacillus (Escherichia coli), from hyperthermophile (Aquifex Aeolicus Aea DNA polymerase is (accession number 067779) or its functional fragment or variant), such as in nucleosides sour water Flat upper functional fragment or variant at least 80%, 85%, 90%, 95% or 99% sequence identity.

Normally, extension step generates nucleic acid, and it includes the double-stranded duplex portions that two of which complementary strand hybridizes each other Point.In one embodiment, walking includes nucleic acid is made to undergo partial denaturation condition, the partial denaturation condition denaturing nucleic acid The part of chain but it is not enough to fully denaturing nucleic acid over the whole length.In embodiments, be expert at walking program part or Nucleic acid is not made to undergo complete Denaturing during the entire duration.

In embodiments, design in this way the sequence of minus strand and/or normal chain so as to primer binding sequence or part thereof be can Breathing, i.e., it is susceptible to being denaturalized under the condition of selection (such as amplification condition).Respirable part is optionally than most of phase The nucleic acid with random sequence like length it is more susceptible or than comprising can at least another part of chain of respiration sequence be more easy to Sense.Optionally, can respiration sequence shown under the amplification condition of selection significant quantity denaturation (for example, at least 10%, 20%, 30%th, 50%, 70%, 80%, 90% or 95% molecule can be completely denaturalized in respiration sequence).Such as it can breathe Sequence design be under the condition (such as amplification condition) of selection 30,35,40,42,45,50,55,60,65 or 70 DEG C It is denaturalized completely in 50% chain molecule.

When by heating or raised temperature come when realizing partial denaturation, can illustratively breathe PBS can be rich in it is phonetic (such as A and/or T and/or U with high-content) of pyridine.PBS includes such as poly-A, poly-T or poly-U sequence or more Poly- pyrimidine track.Optionally one or more amplification or other primers (such as fixed primer) are designed as accordingly being complementary to These primer binding sequences.The exemplary PBS of nucleic acid chains include poly-T sequences, for example, at least 10,15,20,25 or 30 The section of thymidine nucleotide, and corresponding primer has the complementary series of PBS, for example, at least 10,15,20,25 or 30 adenosines The section of nucleotide.Exemplary low melting point primer optionally have it is such at high proportion (for example, at least 50%, 60%, 65%, 70%th, 75%, 80%, 85%, 90%, 95% or 100%) nucleobase：Its usual (example when primer hybridizes with complementary template Such as under the amplification condition of selection) and complementary base formation not more than two hydrogen bonds.The example of such nucleobase includes A (glands Purine), T (thymidine) and U (uracil).Exemplary low melting point primer optionally has a high proportion of A (adenine), T (thymidine) and/or U's (uracil) is any one or more or derivatives thereof.In embodiments, derivative includes complementation In the nucleobase of A (adenine), T (thymidine) and/or U (uracil).The part for being hybridized to the primer of PBS optionally has There are A (adenine), the T (thymus gland of at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% Pyrimidine) or U (uracil) nucleotide or its arbitrary combine.In another example, the part for being hybridized to the primer of PBS includes PolyA sequences (for example, at least 5,10,15,20,25 or 30 nucleotide are long).Other Exemplary primers include (NA_x)_nWeight Complex sequences.Optionally, n (lowercase) is 2-30, such as 3-10, such as 4-8." N " (uppercase) is arbitrary nucleosides Sour-and optionally, N is C or G." A " is the abbreviated notation of adenine, and " x " represents the adenine residue in repetitive sequence Quantity, such as 2,3,4,5,6,10 or more.Exemplary primers include (CAA)_n、CA)_n、(CAAA)_nOr even (GAA)_n Multiple repetitive sequences.

Optionally only a chain (such as chain forward or backwards) has respirable PBS.In another embodiment, Forward and reverse chain all has respirable PBS.Respirable PBS, which is optionally complementary to, is fixed to support or revocable (such as dissolved form) primer.Optionally, the chain comprising respirable PBS be fixed to support or it is revocable (such as Dissolved form).Optionally two kinds of primers are fixed or two chains are fixed.Optionally primer is not solid Fixed or chain is not fixed.

Amplification cycles optionally include breathing (breathing), annealing and extension.Optionally make nucleic acid to be amplified Undergo for these at least one steps be suitable or optimal conditions.In embodiments, it is suitable for nucleic acid experience The condition of these more than one steps (for example, annealing and extension or breathing and extension).In some instances, all three this A little steps can occur simultaneously at identical conditions.

In illustrative methods, it can allow nucleic acid experience or promote the condition of breathing.In embodiments, work as double-strand Two chains of double helix is substantially hybridize but are when being denaturalized in purpose Part portions each other, then it is assumed that " breathing " hair It is raw.The respirable sequence (such as positive and/or reversed PBS with low Tm parts) of one or more of nucleic acid hybridizes from it The first complementary strand (such as chain forward or backwards) partial denaturization (" breathing ") extremely, so as to be used to be hybridized to another second Chain.Exemplary first chain is the primer extension product from the first primer.Exemplary second chain be, for example, second do not extend draw Object (for example, including the PBS complementary oligonucleotides of such as dT or dA sequences).Optionally, the first and second chains are fixed on support On, and can be (for example, sufficiently close adjacent to allow to walk) closely placed.Condition for breathing is optional Ground partial denaturation condition, PBS is substantially denaturalized under the described conditions but another part of nucleic acid keeps hybridization or double-strand shape State.Optionally, DNA helicase may include in the reactive mixture promoting partial denaturation.

Optionally, nucleic acid experience is then made to promote the condition of annealing, such as reduces temperature so that respirable PBS with It can hybridize between second chain.In embodiments, using identical condition to promote to breathe and extend.In another implementation In scheme, annealing conditions are different from breathing condition-for example, annealing conditions are non-Denaturings or promote to be less than breathing condition The condition of denaturation.In instances, annealing conditions include the temperature (such as 37 DEG C) lower than breathing condition, in the breathing item Higher temperature (such as 60-65 DEG C) has been used in part.Optionally, in (such as the most of amplification of one or more amplification cycles Cycle or essentially all of amplification cycles) during avoid complete Denaturing.

Optionally, one or more PBS- breathings and primer extension procedures are repeated as many times to expand initial nucleic acid.When one When kind or multiple nucleic acids reagent (such as primer) are fixed to support, primer-extension products before amplification for example due to not prolonging The primer for the extension stretched is to the connection of support or by hybridizing to be kept substantially to the company of support with such primer It connects.

Optionally, the sample of one or more nucleic acid groups to be amplified is prepared.Nucleic acid group can be single-stranded or double-stranded shape Formula；Optionally one or more nucleic acid respectively contain the nucleic acid chains with known 3 ' end sequence and known 5 ' end sequence, institute It is substantially the same or complementary that end sequence, which is stated, with one or more primers for amplification.Partly may be used the 3 ' of nucleic acid chains Such as with fixed Primers complementary, and 5 ' partly can be identical with the primer of dissolving.Between intragroup individual nucleic acid 5 ' and/ Or 3 ' can be partly common (" general ") or constant.Optionally, intragroup nucleic acid is respective between common ground Comprising different (such as unknown) sequences, for example, it is genomic DNA, cDNA, mRNA, pairing (mate-pair) segment, outer aobvious Subgroup etc..Gleanings for example can ensure corresponding heredity of the covering higher than 50%, 70% or 90% with enough members Source.

In some embodiments, present disclosure generally relates to including for nucleic acid amplification and is used for nucleic acid amplification The composition of reaction mixture and relevant system, device, kit and method.

In some embodiments, present disclosure generally relates to the group for including reaction mixture of nucleic acid amplification Object and relevant system, device, kit and method are closed, the reaction mixture includes Continuous Liquid Phase, the Continuous Liquid Phase Comprising (i) polymerase and (ii) multiple supports, at least one nucleic acid group for being connected to substantially monoclonal of support.

In some embodiments, present disclosure generally relates to the group for including reaction mixture of nucleic acid amplification Object and relevant system, device, kit and method are closed, the reaction mixture includes Continuous Liquid Phase, the Continuous Liquid Phase Include (i) polymerase and the multiple supports of (ii) including the first support and the second support.

In some embodiments, present disclosure generally relate to nucleic acid amplification composition (and relevant system, Device, kit and method), it includes：Reaction mixture comprising Continuous Liquid Phase, the Continuous Liquid Phase include (i) and include the Multiple supports of one support and the second support, (ii) comprising the first polynucleotides and the second polynucleotides it is multiple not With polynucleotides and (iii) for isothermal nucleic acid amplification reagent.In some embodiments, for the examination of nucleic acid amplification Agent includes polymerase and the nucleotide (such as multiple nucleotide) of one or more types.Optionally, for isothermal nucleic acid amplification Reagent include recombinase.

Optionally, the first and second polynucleotides have different sequences.

Optionally, at least one at least one end of multiple and different polynucleotides is connected at least one few core Thuja acid connector.

Optionally, at least some of at least one end of multiple and different polynucleotides includes common sequence.

Optionally, at least two of polynucleotides different in reaction mixture include common sequence.

Optionally, the first and second polynucleotides are different.

In some embodiments, liquid phase includes one or more of multiple supports comprising primer.

Optionally, reaction mixture includes Continuous Liquid Phase.

Optionally, can reaction mixture be subjected to isothermal or thermal cycle nucleic acid amplification.

Optionally, Continuous Liquid Phase includes any of (i) one or more polymerases and/or (ii) at least one support A or any combinations.

Optionally, Continuous Liquid Phase includes multiple supports.

Optionally, Continuous Liquid Phase includes the first support.

Optionally, Continuous Liquid Phase includes the second support.

Optionally, at least one of multiple supports can be connected to the nucleic acid group of substantially monoclonal.

Optionally, the first support can be connected to the nucleic acid group of the first substantially monoclonal.

Optionally, the second support can be connected to the nucleic acid group of the second substantially monoclonal.

Optionally, the nucleic acid group of the first and second substantially monoclonals includes different sequences or substantially the same sequence Row.

Optionally, first and second substantially monoclonals nucleic acid group under stringent hybridization conditions each other hybridization or it is not miscellaneous It hands over.

Optionally, the nucleic acid group of the first and second substantially monoclonals is different.

Optionally, the nucleic acid group of the first and second substantially monoclonals is non-complementary.

Optionally, reaction mixture includes the nucleotide for not carrying out exogenous marker.For example, nucleotide can naturally be deposited Nucleotide or optically detectable label not comprising fluorescence part, dyestuff or other external sources synthetic analogues.

Optionally, reaction mixture is included in single reaction vessel.

Optionally, reaction mixture includes isothermal or thermal cycle reaction mixture.

Optionally, multiple supports include the inner wall of globule, particle, particle, sphere, gel, filter or pipe.

Optionally, at least one of multiple supports can be connected to multiple nucleic acid.

Optionally, at least one of multiple supports can be connected to one or more primers.Primer can be identical It is (or including common sequence) or different.

Optionally, at least one support can be connected to multiple the first primers.

Optionally, at least one support can be connected to multiple the first primers and multiple second primers.

Optionally, multiple the first primers include substantially the same sequence.

Optionally, multiple the first primers include at least one include and the polynucleotides of multiple and different polynucleotides The first primer of at least partly identical or complementary sequence.

Optionally, multiple second primers include at least one include and the polynucleotides of multiple and different polynucleotides Second primer of at least partly identical or complementary sequence.

In some embodiments, at least one polynucleotides of multiple and different polynucleotides include and the first primer Interior sequence is substantially the same or the First ray that is substantially complementary.In some embodiments, at least one polynucleotides are also Include the second sequence that is substantially the same with the sequence in the second primer or being substantially complementary.In some embodiments, it is multiple Essentially all polynucleotides in different polynucleotides include First ray and the second sequence.

Optionally, at least one of multiple supports are connected to 2-10 different multiple primers.

Optionally, 2-10 different multiple primers include different sequences.

Optionally, multiple primers different 2-10 include at least one at least part from different polynucleotides into The sequence of row hybridization.

Optionally, 2-10 different multiple primers include at least one consensus from different polynucleotides The sequence that is hybridized of at least part.

Optionally, at least one of support is connected at least one unique identification sequence of barcodes.

Optionally, the nucleic acid group of the first and second substantially monoclonals has substantially the same or different sequence.

Optionally, reaction mixture includes at least one recombinase.

Optionally, recombinase can be catalyzed homologous recombination, chain intrusion and/or D- rings and be formed.

Optionally, recombinase is the part of nucleoprotein filament, and the nucleoprotein filament, which includes to be connected in reaction mixture, to be adhered to In the recombinase of the primer of support.By the primer of recombinase connection connectable to support or in the solution.

Optionally, reaction mixture includes nucleoprotein complex or multiple nucleoprotein complexs.

Optionally, reaction mixture includes the first nucleoprotein complex.

Optionally, reaction mixture includes the second nucleoprotein complex.

Optionally, at least one of multiple nucleoprotein complexs include at least one recombinase for being connected to primer.

Optionally, reaction mixture includes the first nucleoprotein containing at least one recombinase for being connected to the first primer Compound.

Optionally, reaction mixture includes the second nucleoprotein containing at least one recombinase for being connected to the second primer Compound.

Optionally, recombinase is included from T4, T2, T6, Rb69, Aeh1, KVP40, acinetobacter (Acinetobacter) bacteriophage 133, Aeromonas (Aeromonas) bacteriophage 65, cyanophage P-SSM2, indigo plant Green alga bacteriophage PSSM4, cyanophage S-PM2, Rb14, Rb32, Aeromonas bacteriophage 25, vibrio (Vibrio) bacteriophage nt-1, phi-1, Rb16, Rb43, bacteriophage 31, bacteriophage 44RR2.8t, Rb49, bacteriophage Rb3 or The bacteriophage recombinase of bacteriophage LZ2.

Optionally, recombinase includes the uvsX recombinases from T4 bacteriophages or the recA weights from Escherichia coli Group enzyme.

Optionally, reaction mixture also includes polymerase.

Optionally, it polymerize 5 ' to 3 ' exonuclease activity of azymia.

Optionally, polymerase includes strand-displacement activity.

Optionally, polymerase includes thermal stability or heat sensitivity polymerase.

Optionally, polymerase includes archaeal dna polymerase or RNA polymerase.

Optionally, reaction mixture also includes the nucleotide of at least one type.

Optionally, at least one of multiple supports include at least one primer.

Optionally, the first support is connected to the first substantially nucleic acid group of monoclonal and the second support is connected to The nucleic acid group of second substantially monoclonal.

Optionally, the nucleic acid group of the first and second substantially monoclonals has different nucleic acid sequences.

Optionally, the nucleic acid group of the first and second substantially monoclonals does not hybridize each other under stringent hybridization conditions.

Optionally, the nucleic acid group of the first and second nucleic acid substantially monoclonal differs and incomplementarity.

Optionally, reaction mixture includes the polynucleotide templates to be amplified of (i) at least two and/or (ii) at least One nucleoprotein filament compound.

Optionally, reaction mixture include at least one polynucleotides for being connected to resistance compound, primer, template or Amplified production.As used herein, term " resistance compound " and its variant description as any Chemical composition that：It is described Composition is connectable to nucleic acid and hinders its diffusion by reaction mixture, but still allows to use in nucleic acid synthetic reaction Such polynucleotides, primer, template or amplified production carry out nucleic acid synthesis.Such resistance compound in synthetic reaction To nucleic acid connection usually reduce as nucleic acid mobility in the reactive mixture and available for prevent use it is identical The cross contamination of amplified production or template between the different synthetic reactions that reaction mixture occurs.In some embodiments, The connection of resistance component to one or more nucleic acid compositions can increase the quantity or ratio of monoclonal product.

In some embodiments, this introduction provides the method for nucleic acid amplification, and the nucleic acid amplification includes at least one It is a to flow the nucleic acid (such as primer) sexually revised.In some embodiments, the nucleic acid sexually revised is flowed to show increased or subtract The small mobility by aqueous medium.In some embodiments, the nucleic acid of modification is included in along any position of length nucleic acid On be connected to one or more change nucleic acid nucleic acid of the compound (such as resistance compound) of mobility for passing through aqueous medium (such as primer).In some embodiments, the resistance compound for the mobility for changing nucleic acid can be connected to nucleic acid amplification anti- Any primer in answering, including first, second, third, fourth or any other primer.For example, can 5 ' ends, 3 ' ends and/ Or one or more resistance compounds are connected to nucleic acid in any one or any combinations of interior location.In some embodiment party In case, the nucleic acid of modification can be covalently or non-covalently connected to and change the resistance of mobility that nucleic acid passes through aqueous medium Close object.For example, resistance compound when being connected to nucleic acid, can pass through chemical combination of the nucleic acid that change is modified compared to shortage connection Appearance and size, length, radius, shape or the charge of the nucleic acid of object provides aqueous fluid dynamic drag.In some embodiments In, be connected to nucleic acid resistance compound can be changed between nucleic acid and aqueous medium interaction (compared to aqueous medium with lack Interaction between the nucleic acid of the compound of weary connection).In some embodiments, resistance compound can be synthesis, It is recombination or naturally occurring.In some embodiments, resistance compound can be with point, it is uncharged, polarity or It is hydrophobic.In some embodiments, resistance compound can be it is linear, branch or with dendroid paradigmatic structure (dendrimeric structure).In some embodiments, resistance compound may include nucleosides, sugar, fat or amino acid Single part or polymer.

Optionally, resistance compound includes saccharide part, polysaccharide, albumen, glycoprotein or polypeptide.Optionally, resistance compound Including BSA, lysozyme, beta-actin, myosin, lactalbumin, ovalbumin, beta galactosidase, lactic dehydrogenase Or immunoglobulin (such as IgG).

Optionally, change the resistance compound of mobility that nucleic acid passes through aqueous medium and include one or more polyoxyethylenes Alkene (PEO) or polypropylene oxide (PPO) part, the polymer including polyethylene glycol oxide (PEO) or polypropylene oxide (PPO).This The non-limiting examples of the polymer of sample include triblock copolymer (such as PEO-PPO-PEO), Pluronics^TMType polymerize Object and hydrophobically modified PEO polymer.Optionally resistance compound includes one or more amino acid moieties, polypeptide and gathers Class peptide.Optionally, it is fine to include saccharide part, polysaccharide, hydrophobically modified polysaccharide, cellulose derivative, carboxymethyl for resistance compound The plain sodium of dimension, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose or hypromellose.Optionally, resistance It closes object and includes hydrophobically modified alkali solvable united (HASE) polymer, hydrophobically modified polyacrylamide, thermo-responsive polymerization Object or n-isopropyl acrylamide (NTPAAm), optionally, resistance compound include poly(ethylene glycol) methyl ether acrylate (PEGMEA), tetraethylene glycol diacrylate (TEGDA), poly(ethylene glycol) dimethacrylate (EGDMA) or N, N '-methylene Base-two-acrylamide (NMBA).

Optionally, resistance compound include albumen or polypeptide, including BSA, lysozyme, beta-actin, myosin, Lactalbumin, ovalbumin, beta galactosidase or lactic dehydrogenase.In some embodiments, amido or sulfydryl can be passed through Resistance compound is connected to nucleic acid by connection.

In some embodiments, flow the nucleic acid that sexually revises include being connected to binding partners (such as with receptor portion The affinity part of split-phase interaction) nucleic acid.In some embodiments, acceptor portion can be used as resistance compound.One In a little embodiments, affinity can be partially attached to nucleic acid, and affinity part (it is used as resistance compound) and receptor Part interacts.For example, nucleic acid can be connected to the biotin moiety that can combine avidin sample part.Antibiont Fibroin sample part can be used as resistance compound.Avidin sample part includes avidin and antibiotin Any derivative that can be combined with biotin moiety, analog and the other unnatural forms of albumen.Other realities of binding partners Example includes epitope (such as albumin A) and its respective antibody (such as anti-FLAG antibody) and fluorescein and anti-fluorescein resists Body.Those skilled in the art will readily recognize to be combined for resistance compound to be connected to other binding partners of nucleic acid.

Optionally, can by resistance compound and primer are connected to binding partners pair two members each come Resistance compound is connected to primer.

Optionally, at least one of reaction mixture primer includes biotin.

Optionally, resistance compound includes avidin or streptavidin.

Optionally, resistance compound includes saccharide part, polysaccharide, albumen, glycoprotein or polypeptide.

Optionally, resistance compound includes BSA, lysozyme, beta-actin, myosin, lactalbumin, egg white egg In vain, beta galactosidase, lactic dehydrogenase or immunoglobulin (such as IgG).

Optionally, reaction mixture also includes auxilin.

Optionally, auxilin includes unwindase, single strand binding protein or recombinase load factor.

Optionally, unwindase includes the uvsW from T4 bacteriophages.

Optionally, single strand binding protein includes the Sso for carrying out bin cure ore deposit sulfolobus solfataricus (Sulfolobus solfataricus) SSB, the MjA SSB from Methanococcus jannaschii (Methanococcus jannaschii) or Escherichia coli SSB albumen.

Optionally, single strand binding protein includes gp32 albumen from T4 bacteriophages or from T4 bacteriophages through modification Gp32 albumen.

Optionally, recombinase loading albumen includes the uvsY from T4 bacteriophages.

Optionally, reaction mixture also includes ATP.

Optionally, reaction mixture also includes ATP regenerating system.

Optionally, ATP regenerating system includes phosphocreatine.

Optionally, ATP regenerating system includes creatine kinase.

Optionally, reaction mixture also includes to increase the efficiency of nucleic acid amplification reaction or the adduct of yield.

Optionally, adduct includes glycine betaine, DMSO, proline, trehalose, MMNO (4- methylmorpholine N-oxides) Or PEG sample compounds.

Optionally, at least one of reaction mixture polynucleotide template includes at least a certain portion with the first primer Point complementary or identical First ray and at least a certain partial complementarity of the second primer or the second identical sequence.Optionally Ground, reaction mixture include multiple double-stranded polynucleotides, and the polynucleotides are included at least certain part of the first primer mutually Mend or identical First ray and at least a certain partial complementarity of the second primer or the second identical sequence.Optionally, One sequence be located at it is multiple at least one double-stranded polynucleotide end at or near, and the second sequence be located at it is multiple in extremely At or near another end of a few double-stranded polynucleotide.

Optionally, reaction mixture is also comprising diffusion limitation agent.

Optionally, diffusion limitation agent reduces the diffusion rate that polynucleotides leave support.

Optionally, diffusion limitation agent reduction is connected to the level of the polyclonal nucleic acid group of support.

Optionally, diffusion limitation agent includes polymer compound.

Optionally, diffusion limitation agent includes glycopolymers.

Optionally, diffusion limitation agent includes the compound based on cellulose.

Optionally, diffusion limitation agent includes glucose or galactose polymer.

Optionally, glycopolymers include cellulose, glucan, starch, glycogen, agar or agarose.

Optionally, diffusion limitation agent includes block copolymer compound.

Optionally, diffusion limitation agent includes poly- (propyIoxirane) by two poly- (ethylene oxide) hydrophilic chain side joints Center chain.

Optionally, diffusion limits dosage form into micella.

Optionally, diffusion limits dosage form into micella liquid crystal.

Optionally, diffusion limitation agent includes Pluronics^TMCompound.

Optionally, reaction mixture further includes a concentration of about 0.025-0.8%w/v or about 0.05-0.7%w/v or about The diffusion of 0.075-0.6%w/v or about 0.1-0.5%w/v or about 0.2-0.4% w/v reduce agent.

Optionally, packet is further included for the composition of nucleic acid amplification and relevant system, device, kit and method Surface, matrix or medium containing multiple sites, wherein at least one site is operationally coupled to one or more sensors.

Optionally, multiple sites include reative cell, support, particle, particle, sphere, globule, filter, flow cell, hole, Ditch, tank therefor, gel or pipe inner wall.

Optionally, multiple sites can be arranged in random array or organized array.

Optionally, multiple sites can be in fluid communication with each other.

Optionally, multiple sites is at least one including three dimensional chemical matrix.

Optionally, at least one of multiple sites is covalently coupled to three dimensional chemical matrix.

Optionally, multiple sites is at least one including acrilamide layer.Optionally, at least one packet in multiple sites Include the nucleic acid for being covalently coupled to acrilamide layer.

Optionally, hydrophilic polymer matrix includes aquogel polymer matrix.

Optionally, polyacrylamide is conjugated in Oligonucleolide primers.

Optionally, hole has 0.1 micron to 2 microns of characteristic diameter.

Optionally, hole has 0.01 micron to 10 microns of depth.

In some embodiments, sensor includes field-effect transistor (FET).FET may include ion-sensitive FET (ISFET), chemosensitivity field-effect transistor (chemFET) or bioactivity field-effect transistor (bioFET).

Optionally, one or more sensors are configured to detect the by-product that nucleotide is incorporated to.

Optionally, configurable one or more sensors detect chemical part in one or more the multiple sites Presence.

Optionally, one or more sensors include field-effect transistor (FET), ion-sensitive field-effect transistor (ISFET), chemosensitivity field-effect transistor (chemFET) or bioactivity field-effect transistor (bioFET).

In some embodiments, sensing material includes metal-oxide.

In some embodiments, sensing material is to hydrogen ion sensitivity.

Optionally, the by-product for reaction being incorporated to from nucleotide includes pyrophosphate, hydrogen ion or proton.

It additionally provides comprising any one following, any subgroup or all compositions：At least one reversed nucleic acid chains, It is fixed on multiple forward primers at least one support, multiple reverse primers and polymerase in solution.It is positive and/or Reverse primer is optionally low melting point or is rich in adenine, thymidine or uracil as described herein.Exemplary group It closes object to include comprising multiple solid supports of clone group being spatially separated, each comfortable 3 ' end of the clone group includes low Fusing point primer binding sequence simultaneously includes low melting point primer sequence in 5 ' ends.Optionally, composition also includes recombinase.Alternatively, Composition does not include the enzyme that another kind is not polymerase, such as recombinase or reverse transcriptase or unwindase or nickase optionally. Another exemplary composition includes any one or more of following component：(1) reversed nucleic acid chains, (2) are fixed on the support Multiple low melting point forward primers, multiple low melting point reverse primers and (4) polymerase in (3) solution.Optionally, it is positive 3 ' the parts or end of primer and reverse strand are interfertile (such as complementary).Optionally, 5 ' portions of reverse primer and reverse strand Divide or end is substantially the same.Composition may include any one or more reagents described herein and/or can undergo Any one or more programs or condition described herein.

In some embodiments, present disclosure is usually directed to comprising by any method generation in the present disclosure The composition of the nucleic acid of amplification.In some embodiments, clonal expansion is formed around discontinuous site on the support Limitation clone group.Exemplary discontinuous site is tie point of the initial nucleic acid chain to support, and is utilized from the site Initial nucleic acid or its copy directly or indirectly generate other nucleic acid in clone group as template by primer extend.

Optionally, composition includes the gleanings of nucleic acid that can be generated by any one or more methods described herein. For example, gleanings may include occupying the fixed nucleic acid in the region of one or more separation on the surface.In some embodiments In, each region includes multiple identical nucleic acid chains and optionally multiple identical complementary strands for being hybridized to it, wherein complementary strand Do not adhere to solid support or connect or associate, in addition to because caused by hybridizing with fixed nucleic acid.Optionally, so Individual nucleic acid chains as positioning in region so as to another nucleic acid chains on the surface positioned at the distance of the length of the chain it It is interior.Every mm on the surface of nucleic acid is optionally secured on it²On there are at least one separation region.For example, the area of separation Quantity/the mm in domain²The surface for securing nucleic acid on it is more than 10², more than 10³, more than 10⁴, more than 10⁵, more than 10⁶, it is big In 10⁷Or more than 10⁸。

The gleanings of the clone group of amplification can form array, can be that one-dimensional (such as a universal monoclonal of row is micro- Pearl) or two dimension (such as the clone group of amplification is located on plane support) or three-dimensional.Optionally but not necessarily positioning or The individual of arranged array clones group so that it is addressed or addressable.Optionally, different clones group is with appropriately distance It is spaced, the distance is typically enough to that different clone groups is allowed to be distinguished from each other.In embodiments, with orderly or nothing (such as random) mode of sequence spreads the clone group of limitation on a planar substrate.

Exemplary array is characterized in the differentiable nucleic acid clone group of individual, wherein optionally being supported in one or more This feature is distributed on object.In exemplary microballon embodiment, array includes multiple microballons, wherein individual microballon generally comprises The monoclonal group of nucleic acid, different microballons generally comprise different clone groups (for example, it is different in sequence).Optionally, will Microballon is distributed or fills in individual layer on a planar substrate.In other embodiments, array includes single (such as plane) Support, single support include multiple spatially discontinuous nucleic acid clone groups, wherein different clone groups is optionally in sequence It is different on row.

Optionally, one or more nucleic acid in individual clone group can be attached directly to planar substrate.In another reality In example, the nucleic acid of individual clone group is connected to microballon, such as discussed in this article.Optionally on a planar substrate with Machine or orderly mode are tightly packed within microballon is cloned together.Optionally, more than 20%, 30%, 50%, 70%, 80%th, 90%, 95% or 99% microballon with it is at least one, two, the other microballons of four or six contact.Optionally, Microballon less than 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95% or 99% and one, two, four or six A others microballon contact.

In some embodiments, present disclosure generally relates to the method for nucleic acid amplification and relevant combination Object, system, kit and device include the use of any amplification method disclosed herein, composition or system and carry out multi-kernel Acid amplification.

In some embodiments, method includes the use of polymerase to carry out multiplex amplification (such as polymerase-mediated multiple Nucleic acid amplification reaction).

In some embodiments, method may also include to be expanded again using nucleic acid amplification reaction and be expanded from multiple nucleic acid Amplicon.

Optionally, multiple nucleic acid amplification can carry out in single reaction mixture.

Optionally, multiple nucleic acid amplification can be carried out to the sample comprising multiple and different nucleic acid target sequences.

Optionally, multiple and different nucleic acid target sequences can be expanded in single reaction mixture.

Optionally, can be expanded in single reaction mixture it is a small number of beat it is at least hundreds of or at least it is thousands of (or more) Nucleic acid target sequence.

Optionally, at least 50 or at least 100 nucleic acid target sequences can be expanded in single reaction mixture.

Optionally, multiplex amplification may include at least part sample and recombinase, polymerase and/or at least one primer It is any or arbitrary combination contacted.

Optionally, multiplex amplification can be carried out under isothermal or thermal cycle conditions.

In some embodiments, present disclosure generally relates to the method for nucleic acid amplification and relevant combination Object, system, kit and device are expanded including multiple nucleic acid, and the multiple nucleic acid amplification is included in single reaction mixture From the sample comprising multiple and different nucleic acid target sequences expand at least 50 different nucleic acid target sequences (or more), it is described Amplification includes with recombinase and multiple primers contacting at least part sample under the conditions of isothermal duplication.

In some embodiments, present disclosure generally relates to the method for nucleic acid amplification and relevant combination Object, system, kit and device are expanded including multiple nucleic acid, and the multiple nucleic acid amplification is included in single reaction mixture Different nucleic acid target sequences is expanded from the sample comprising multiple and different nucleic acid target sequences, the amplification will at least including passing through Part sample contacted under the conditions of isothermal duplication with recombinase and multiple primers generate at least 50 it is multiple not With amplification target sequence (or more).

In some embodiments, present disclosure generally relates to the method for nucleic acid amplification and relevant combination Object, system, kit and device, including being expanded again from multiple nucleic acid by using nucleic acid amplification reaction (such as recombinase) The amplicon of amplification generates the nucleic acid group of substantially monoclonal.

Optionally, the method for multiple nucleic acid amplification may also include the nucleic acid amplification method of recombinase-mediated, including Expand at least some of the target sequence of at least 50 different amplifications again as follows：(a) formation includes single continuous The reaction mixture of liquid phase, it includes (i) multiple supports, the target sequence of (ii) 50 different amplifications it is at least one (iii) recombinase；(b) reaction mixture is made to undergo amplification condition, so as to generate multiple supports, the multiple support Object is connected to the nucleic acid group for being connected to it of substantially monoclonal.

Optionally, in the method expanded for multiple nucleic acid, can substantially it is non-exhaust under conditions of amplification from sample This different nucleic acid target sequences.

Optionally, in the method expanded for multiple nucleic acid, it can be expanded under conditions of substantially used up and come from sample Different nucleic acid target sequences.

Optionally, in the method expanded for multiple nucleic acid, single reaction mixture includes isothermal or thermal cycle reaction Mixture.

In some embodiments, two or more moulds can be expanded in the room of the separation of array, hole, chamber or site Plate or target, room, hole, chamber or the site of the separation of the array are in fluid communication with each other or are amplified the identical of reaction mixture Single Continuous Liquid Phase occupies.Such embodiment includes the embodiment for the nucleic acid amplification based on array.

For example, in some embodiments, present disclosure is related to the method for nucleic acid amplification, including：By target multinuclear Thuja acid is distributed to the reative cell in reative cell or the array in site or site and that single target is expanded in reative cell or site is more Nucleotide.Optionally, two or more target polynucleotides are distributed to two or more reative cells of array or site In, and two or more the allocated target polynucleotides are parallelly amplified in its respective reative cell or site. Optionally, at least two of reative cell or site respectively receive during distribution single target polynucleotide (reative cell or site It is one or more optionally to receive zero or more than one target polynucleotide during distribution).At least two target polynucleotides It can be expanded with being cloned in its respective reative cell.At least one reative cell comprising target polynucleotide can with it is more comprising target At least one other reative cell of nucleotide is in fluid communication during amplification.

In some embodiments, present disclosure generally relates to method (and the relevant combination of nucleic acid amplification Object, system, device and kit), including：(a) it is anti-by the way that single polynucleotides introducing at least two is in fluid communication with each other Room is answered to distribute at least two different polynucleotides into the array of reative cell；(b) by anti-described at least two Answer the nucleic acid group of indoor amplifying polynucleotides formation at least two substantially monoclonal.Normally, at least two reative cells exist It keeps being in fluid communication each other during amplification.

In some embodiments, present disclosure generally relates to method (and the relevant combination of nucleic acid amplification Object, system, device and kit), including：(a) in the array of the reative cell including the first and second reative cells, by first Template polynucleotide is distributed into the first reative cell and is distributed the second template polynucleotide into the second reative cell and (b) is logical It crosses and expands the first and second template polynucleotides to clone in its respective reative cell to form at least two substantially singly The nucleic acid group of clone, wherein distributing single polynucleotides from the sample of nucleic acid with multiple and different polynucleotides.Optionally, First and second reative cells include the different piece of single Continuous Liquid Phase during amplification.For example, the first and second of array are anti- Answer room that can be in fluid communication during amplification.

In some embodiments, the method that present disclosure generally relates to nucleic acid amplification, will be different including (a) Single polynucleotides distribute into each of multiple reative cells and (b) in multiple reative cells by expanding different lists A polynucleotides to form monoclonal nucleic acid group in each of reative cell, wherein from multiple and different polynucleotides Sample of nucleic acid distribute single different polynucleotides.

In some embodiments, the method that present disclosure generally relates to nucleic acid amplification, including (a) pass through by Single polynucleotides introduce at least two reative cells being in fluid communication with each other by least two different polynucleotides distribute to In the array of reative cell；(b) by least two reative cell amplifying polynucleotides formed at least two substantially The nucleic acid group of monoclonal.

In some embodiments, method, which may also include, draws one or more supports (such as globule or particle etc.) Enter at least one reative cell or the site of array.It can be before, during or after polynucleotides be distributed into array by one Or multiple supports introduce at least one reative cell or site.In some embodiments, at least one reative cell of array or Site receives single support.In some embodiments, most of reative cell or site receive single support.At some In embodiment, support with polynucleotides can be mixed before a distribution and distribute it to array together with polynucleotides In.At least one support is optionally connected to nucleic acid molecules, and the nucleic acid molecules are included with being present in during amplification instead The part of the polynucleotides in room or site is answered to be substantially complementary or substantially the same primer sequence.In some embodiments In, at least one support include nucleic acid molecules, the nucleic acid molecules include with the target polynucleotide in reative cell or hole or The part of template is substantially complementary or substantially the same primer sequence.In some embodiments, at least one support packet Containing nucleic acid molecules, the nucleic acid molecules are included with another primer that is present in during amplification in reative cell or site extremely Small part is substantially complementary or substantially the same primer sequence.

In some embodiments, amplification may include introducing reaction mixture at least one reative cell or the position of array Point in.It optionally, will reaction before, during or after the introducing of the distribution or support of polynucleotides to array to array Mixture is introduced into reative cell or site.It can in any order or any combinations are by reaction mixture, support and multinuclear glycosides Acid is introduced into or distributes into array.In some embodiments, at least one reative cell of array or site receive single branch Hold object, single polynucleotides and enough supporting the reaction mixture of amplification of the polynucleotides in reative cell or site.

In some embodiments, method may include by support is contacted with polynucleotides under hybridization conditions come At least part of polynucleotides is hybridized to support.Hybridization can support and/or polynucleotides to array reative cell or Occur before, during or after the introducing in site.In some embodiments, at least the one of the first primer sequence will be connected to A support introduces at least one reative cell or the site of array, polynucleotides then is introduced reative cell or site, anti- Polynucleotides in room or site is answered to be hybridized to support.Alternatively, support can be hybridized to through the multinuclear in reative cell or site The amplified production that the amplification of thuja acid generates.

Reaction mixture may include any reaction mixture described herein and component.In some embodiments, instead Mixture is answered to may include any one or more of following component：Isothermal duplication reagent is (for example, one or more recombinases, untwist Enzyme, that is, relevant confactor, polymerase etc.), screening agent, nucleotide etc..

In some embodiments, present disclosure generally relates to method (and the relevant combination of nucleic acid amplification Object, kit, system and device), including：(a) in any order or combination is by the first polynucleotide template and the first support The first reative cell or the site of reative cell or the array in site are introduced, and the second polynucleotide template and the second support are drawn Enter the second reative cell or the site of array；(b) it is expanded to clone on the first support in the first reative cell or site First polynucleotide template, and in the second reative cell or site clone the second polynucleotides are expanded on the second support Template, while the first reative cell or site are in fluid communication with the second reative cell or site during amplification.The amplification of clone ground can wrap It includes and generates the first support for being connected to the first amplicon from the first polynucleotide template and be connected to from more than second Second support of the second amplicon of oligonucleotide template.Optionally, the first and second sites (or reative cell) is in the amplification phase Between include the identical Continuous Liquid Phase of same reaction mixture.Such as reaction mixture may include comprising the first and second multinuclear glycosides The single Continuous Liquid Phase of acid template and the first and second supports.Can polynucleotide template and/or support introducing it Before, during or after by reaction mixture introduce array reative cell or site.In some embodiments, disclosed method It is additionally included in introduce after the first and second polynucleotide templates and the first and second supports and reaction mixture is introduced the One and second reative cell or site.In some embodiments, reaction mixture include recombinase or unwindase or recombinase and Unwindase.Recombinase can derive from myovirus (such as uvsX), bacterium, yeast or the recombinase of people or it comes from other objects The analog of kind.In some embodiments, reaction mixture includes polymerase.In some embodiments, reaction mixing Object includes screening agent, such as polyacrylamide, agarose or cellulosic polymer (for example, HEC, CMC or MC or its derivative Object).In some embodiments, reaction mixture includes diffusion limitation agent.

In some embodiments, amplification includes the first primer binding sequence of polynucleotide template being hybridized to support The polynucleotide template is connected to the comprising the first primer sequence by the first primer sequence of the first primer of object The support of one primer or surface (such as particle or globule).

In the embodiment wherein expanded in the reative cell of array or site and wherein in single reaction vessel In the embodiment inside expanded, surface or support optionally include at least the first primer, and it includes the first primer sequences Row.In some embodiments, one or more polynucleotide templates include the first primer binding sequence.The first primer combines Sequence can be identical or substantially the same with the first primer sequence.Alternatively, the first primer binding sequence can draw with first Object sequence is complementary or is substantially complementary.In some embodiments, the first primer sequence and the first primer binding sequence Do not show significant homogeneity or complementarity, but with nucleotide sequence another present in reaction mixture substantially phase Together or it is substantially complementary.In such embodiments, amplification may include the formation of amplified reaction intermediate product, the centre Product includes the nucleosides for having significant homogeneity or complementarity with the first primer sequence, the first primer binding sequence or both Acid sequence.

In some embodiments, in the reactive mixture there are at least two different polynucleotide templates, and expand Increasing leads to the formation of at least two different substantially monoclonal groups, and the monoclonal group respectively derives from the single multinuclear The amplification of thuja acid template.In some embodiments, two or more of at least two substantially monoclonal groups are connected to phase Same support or surface.Two or more substantially monoclonal group each may be connected to identical support or table Different unique locations on face.Alternatively, each of two or more substantially monoclonal groups can be each attached to difference Support or surface.The separation on different monoclonal groups to different supports or surface is needing to detach before analysis It can be advantageous in the application of group.In some embodiments, support or surface are the parts of globule or particle, Can be spherical or sphere in shape.In some embodiments, support or surface form two dimension or cubical array Part.

In some embodiments, it is carried out when agent being reduced comprising screening agent or diffusion in the reactive mixture public The method for nucleic acid amplification opened.These reagents can increase the total quantity and/or ratio of the monoclonal group formed during amplification Example (such as percentage).In some embodiments, method is included the use of provides increased list relative to popular response mixture Clone or the substantially reaction mixture of the yield of the amplicon of monoclonal.

In some embodiments, method includes being expanded by partial denaturation template.For example, amplification includes template Walking.For example, template to be amplified may include including the joint sequence of primer binding site, integrally have compared to template There is relatively low Tm.In some embodiments, as being more fully described herein, it is being substantially higher than joint sequence Tm and expanded at a temperature of the substantially less than Tm of template.In some embodiments, less than nucleic acid-templated Tm It is expanded at a temperature of at least 5 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C or 50 DEG C.In some embodiments, higher than (example Such as at least 5 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C or 50 DEG C of highers) the first primer, the second primer or the first and second primers It is expanded at a temperature of Tm.

In some embodiments, reaction mixture is optionally any one or more including following component：(a) appoint Selection of land includes one or more supports of at least the first primer sequence；(2) recombinase；(3) polymerase；(4) diffusion limitation Agent；(5) agent is sieved；(6) crowding agent；(7) ATP regenerative systems；(8) single strand binding protein (SSBP)；(8) recombinase is auxiliary The factor, such as recombinase is helped to be loaded into albumen.In some embodiments, by existing in diffusion limitation agent and/or screening agent In the case of on the surface amplification (for example, by the way that an amplimer is connected on surface) polynucleotide template increase The yield of the group of monoclonal or substantially monoclonal.Diffusion limitation agent sieves what agent can be expanded by reduction during amplification Product polynucleotides leave diffusion or the migration on surface to provide the yield of increased monoclonal group.

In some embodiments, reaction mixture includes one or more isothermal duplication reagents.Such reagent can wrap Include such as recombinase or unwindase.

In some embodiments, using the enzyme of catalysis homologous recombination, such as can be compound to be formed with reference to the first primer Object can be catalyzed chain intrusion or can form the enzyme of D- ring structures, to carry out the method for nucleic acid amplification.In some embodiments In, the enzyme for being catalyzed homologous recombination includes recombinase.

In some embodiments, amplification condition includes isothermy or thermal cycle conditions.

In some embodiments, include for the method for nucleic acid amplification：(a) it is formed and includes the anti-of single Continuous Liquid Phase Answer mixture, the single Continuous Liquid Phase includes the enzyme of (i) catalysis homologous recombination, (ii) one or more surface and (iii) Multiple and different polynucleotides；(b) reaction mixture experience is made to be suitable for the condition of nucleic acid amplification.

In some embodiments, include for the method for nucleic acid amplification：(a) it is formed and includes the anti-of single Continuous Liquid Phase Mixture is answered, the single Continuous Liquid Phase includes the enzyme of (i) catalysis homologous recombination, and (ii) one or more is each attached to more The globule of a the first primer and (iii) multiple and different polynucleotides；(b) by the way that reaction mixture is made to undergo amplification condition shape Into the nucleic acid group of two or more substantially amplifications of monoclonal.Amplification condition may include isothermal or thermal cycle conditions.One In a little embodiments, the first primer can be hybridized at least part of polynucleotides.

In some embodiments, the method that present disclosure generally relates to nucleic acid amplification, including:(a) packet is formed Include the reaction mixture of single Continuous Liquid Phase, the single Continuous Liquid Phase includes one or more supports (or surface), multiple Polynucleotides and recombinase；(b) by the way that reaction mixture is made to undergo amplification condition at least one support (or surface) on gram At least two of the plurality of different polynucleotides are expanded grandly.In some embodiments, amplification condition may include Temperature or thermal cycling amplification condition.Reaction mixture optionally includes recombinase.In some embodiments, reaction mixture Including polymerase.In some embodiments, reaction mixture includes primer, and the primer can be in the solution.Optionally, Support or at least one of surface may include primer.

In some embodiments, the method that present disclosure generally relates to nucleic acid amplification, including:(a) packet is formed The reaction mixture of single Continuous Liquid Phase is included, the single Continuous Liquid Phase includes (i) recombinase, and (ii) is connected to one or more The multiple and different polynucleotide template of the multiple globules and (iii) of a the first primer comprising the first primer sequence；(b) by institute It states at least one of the first primer and is hybridized at least one of multiple and different polynucleotide templates；(c) pass through reaction mixture It goes through nucleic acid amplification condition and is generated at least by expanding at least one polynucleotide template with forming at least first amplification group The polynucleotides group of one substantially monoclonal.In some embodiments, in the group of at least one substantially monoclonal extremely Few 30%, 90% polynucleotides and original at least one of reaction mixture polynucleotide template that is present in are substantially Identical (or being substantially complementary).In some embodiments, at least part of the first amplification group is connected to multiple globules One globule.

In some embodiments, the formation reaction mixture in step (a) includes：By by recombinase with being connected to At least one of multiple the first primers of multiple globules is contacted to form nucleoprotein complex.

In some embodiments, reaction mixture experience nucleic acid amplification condition is made to include carrying out nucleosides in step (b) Sour polymerisation.For example, nucleotide polymerization reaction may include optionally when the first primer sequence is hybridized in reaction mixture Nucleotide is incorporated to the first primer sequence during one polynucleotide template.

In some embodiments, reaction mixture experience nucleic acid amplification condition is made to include the first primer and multinuclear glycosides Acid template, recombinase, polymerase and nucleotide are contacted in any order or with any combinations.

In some embodiments, nucleic acid amplification condition includes repeating such recycle：It is formed comprising recombinase, first At least part of nucleoprotein complex of at least part of primer and the first polynucleotide template and by nucleoprotein complex with The polymerase being incorporated to for being catalyzed one or more nucleotide to the first primer is contacted.

In some embodiments, the nucleic acid amplification reaction that can be recycled respectively is connected to substantially Dan Ke to generate Multiple globules of grand polynucleotides group.

In some embodiments, the method for nucleic acid amplification can be carried out under isothermy or thermal cycle conditions.

In some embodiments, multiple and different polynucleotides can be single-stranded or double-stranded polynucleotides.In some realities It applies in scheme, the thermal denaturation or chemical modification of double-stranded polynucleotide are non-required, because recombinase can be invaded by being catalyzed chain Generate the chain denaturation of part.

In some embodiments, the method for nucleic acid amplification can be carried out in single reaction vessel.In some implementations In scheme, nucleic acid amplification reaction can be carried out in the single reaction vessel comprising single Continuous Liquid Phase.For example, single continuous liquid Mutually may include amplification mixture, the amplification mixture include multiple globules for being respectively connected to multiple the first primers, it is multiple not Same polynucleotides and multiple recombinases.In some embodiments, amplification mixture may also include polymerase and multiple cores Thuja acid.In some embodiments, amplification mixture may also include ATP, nucleotide and confactor.Single reaction vessel Non-limiting examples include pipe, hole or similar structure.

In some embodiments, combination continuously or substantially simultaneously or both can be included in any order Polynucleotides and reagent are placed into reaction vessel.In some embodiments, reagent includes being connected to multiple the first primers Globule, recombinase, polymerase, nucleotide, ATP, bivalent cation and confactor.

In some embodiments, the method for nucleic acid amplification can be carried out in single Continuous Liquid Phase.Single continuous liquid It mutually may include any given part of wherein single Continuous Liquid Phase or any other portion of region and identical single Continuous Liquid Phase Point or regional fluid connection any liquid phase.Normally, the component being dissolved or suspended in single Continuous Liquid Phase can freely expand It dissipates or migrates any other point into liquid phase.In some embodiments, however, single Continuous Liquid Phase may include diffusion limit Preparation slows down the diffusion rate in single Continuous Liquid Phase.One exemplary implementation of single Continuous Liquid Phase is Water-In-Oil Single aqueous droplet in type emulsion；In such emulsion, each droplet will form the phase detached；Two droplets can close And form single phase.

In some embodiments, single Continuous Liquid Phase is substantially made of single water phase.In some embodiments, Single Continuous Liquid Phase lacks nonaqueous phase；For example, Continuous Liquid Phase does not include oil or organic solvent.In some embodiments, it is multiple Nucleic acid amplification reaction occurs in the water phase of single reaction vessel.In some embodiments, single Continuous Liquid Phase is not divided in Multiple nucleic acid amplification reactions occur in single reaction vessel.

In some embodiments, the method for nucleic acid amplification can be carried out in the water-in-oil emulsion divided is provided.

When multiple polynucleotide templates is used to carry out nucleic acid amplification, the clonal expansion using conventional amplification method is usual Reaction mixture is for example divided to the part of the separation of nonfluid connection or component each other dependent on technology, to keep cloning Property and prevent the cross contamination of different amplification groups and keep the yield of enough monoclonal amplified productions.Using such Conventional amplification method, in identical reaction mixture clone ground amplifying polynucleotides template and without reaction mixture extremely The division or distribution of the compartment or container of separation are typically infeasible, because in such amplification present invention mixture Any polynucleotides (including template and/or amplified production) will tend to pass through mixture because diffusion and/or when Brownian movement Randomly migrate.Such diffusion or migration usually increase the generation of polyclonal amplification, so as to will generate seldom (if there is If) monoclonal group.

In conventional amplification method physical barriers are used to reduce by the generation of polyclonal group appropriate technology Individual amplified reaction is detached into discontinuous compartment.For example, emulsion PCR uses the micro- reaction vessel of water-in-oil type, wherein oil Mutually include the i.e. discontinuous aqueous reaction compartment of many separation.Each compartment is used as independent amplified reaction container, so as to Entire emulsion can be supported in separation (discontinuous) liquid phase in single reaction vessel (such as Eppendorf pipes or hole) The amplified reaction of many separation.Similarly, amplification " Master Mix " can be prepared and distributed to the reative cell (example of separation Such as the array in hole) in, one group of discontinuous and separation phase is generated, each of which limits the amplified reaction of separation.It can expand It is preceding mutually further to close such separation each other.Such closing can be used for preventing between parallel and separation reaction Cross contamination.The exemplary form of closing may include using lid or phase barrier (such as the mineral oil on aqueous reaction Layer) with the be divided to individual and discontinuous compartment by PCR reactions, between the compartment transfer of reactive component will not send out It is raw.

Depend on the one or more during amplification anti-to prevent cross contamination and reduce other technologies of polyclonal property The cross contamination for being fixed against amplified reaction product of component (such as one or more templates and/or primer) is answered to be led with it The reduction of the monoclonicity of cause.One such example includes bridge-type PCR, wherein expanding required all primers (such as just To and reverse primer) be connected to the surface of matrix support.In addition to such fixation, may include in the reactive mixture another Outer fixation component.For example, polynucleotide template and/or amplimer can be suspended in gel or other bases during amplification In matter with prevent amplified reaction product from synthesis site migrate.Such gel and matrix usually require to be removed later, this It needs using appropriate " unwinding " or other recycling steps, so as to lose yield.

In some embodiments, present disclosure is provided for parallel in the single Continuous Liquid Phase of reaction mixture Ground carries out the amplification of the substantially monoclonal of multiple polynucleotide templates, and without multiple reactive components, (such as two kinds drawn Object) division during amplification or fixed method.It alternatively, can be directly by the mixing of the polynucleotide template in solution The appropriate surface or support of object and amplified reaction component and the first primer attached thereto are contacted, can be in phase With Continuous Liquid Phase in provide amplification needed for other components, including polymerase, one or more types nucleotide and optionally The second primer of ground.In some embodiments, reaction mixture further includes recombinase.Optionally, reaction mixture further include to A kind of few reagent selected from diffusion limitation agent, screening agent and crowding agent.It is suitable for reality included in single Continuous Liquid Phase The example of the amplification mixture of the monoclonal amplification of existing template is further described through in this article.It optionally, can be in single table Different templates is expanded on different positions on face or support or can be in the different tables in identical reaction mixture Different templates is expanded on face or different supports.

In some embodiments, reaction mixture may include one or more screening agent.Screening agent optionally includes can Any compound of physical barriers is provided the migration of polynucleotide template or its corresponding amplified production.(migration may include mould Any movement of plate or amplified production in reaction mixture；Diffusion includes involving the migration form moved along concentration gradient). In some embodiments, screening agent includes that any compound of the matrix with multiple apertures can be provided, the multiple small Hole is sufficiently small to reduce the shifting of any one or more specific components of nucleic acid synthesis reaction mixture or nucleic acid reaction mixture It is dynamic.

In some embodiments, screening agent provides molecular sieve.For example, screening agent can reduce polynucleotides (or and surface Or the polynucleotides of globule association) pass through the movement of the reaction mixture comprising screening agent.Agent is sieved optionally with small Hole.

When in the single Continuous Liquid Phase in reaction mixture clone expand two or more template polynucleotides when It waits, sieving including for agent can be advantageous.For example, screening agent can prevent or slow down template or by at least a certain of template Diffusion of the polynucleotides of amplification that partial duplication generates in reaction mixture, so as to prevent the shape of polyclonal pollutant Into without dividing reaction mixture by physical method or packaging method (such as emulsion) during amplification.It is such in list In the single Continuous Liquid Phase of a reaction mixture clone ground amplification template without the method that is divided considerably reduce with Generate the relevant cost in library, time and the workload for being suitable for high throughput method such as digital pcr, next-generation sequencing.

In some embodiments, the average pore size of agent is sieved as so that target component (such as polynucleotides) is mixed in reaction The movement closed in object is hindered or is prevented by selectivity.In an example, screening agent includes that the base with multiple apertures can be provided Any compound of matter, the multiple aperture it is sufficiently small with slow down or hinder polynucleotides by comprising screening agent reaction mix Close the movement of object.Therefore, screening agent can reduce the Brownian movement of polynucleotides.

In some embodiments, screening agent selectively acting is higher than being averaged for certain threshold value or range to hinder to have The migration of molecular size or the molecule of weight, without hinder it is other have less than threshold value or range mean molecule sizes or The migration of the molecule of weight.

In some embodiments, screening agent selectively acting is less than being averaged for certain threshold value or range to hinder to have The migration of molecular size or the molecule of weight, without hinder it is other have higher than threshold value or range mean molecule sizes or The migration of the molecule of weight.

In some embodiments, screening agent may be selected selectively hindering, slowing down, reducing or polynucleotides is prevented to lead to Cross the movement of reaction mixture, but it is sufficiently large with allow smaller component (for example, cation, nucleotide, ATP and auxiliary because Son) pass through the movement of reaction mixture.In some embodiments, screening agent, which has, to sieve agent by increaseing or decreasing Concentration is come the average pore size or average pore size scope that adjust.For example, the molecule of screening agent (or combination of screening agent) may be selected Amount, intrinsic viscosity and concentration prepare nucleic acid reaction mixture to generate such matrix in specific solvent (such as water)：Its With it is desired prevent the target polynucleotide with particular size or length migration ability or desired average pore size or Viscosity.In some embodiments, screening agent can reduce bulk flow by increasing the viscosity of nucleic acid reaction mixture. In some embodiments, screening agent can be water-soluble.It in some embodiments, can be by the way that agent and solvent (example will be sieved Such as aqueous solvent, such as water) it mixes to prepare the matrix with multiple apertures.In some embodiments, screening agent is not interfered The formation of recombinase nucleoprotein complex or nucleotide polymerization.

In some embodiments, present disclosure generally relates to the method for carrying out nucleic acid amplification reaction, including logical Cross there are one or more screening agent, optionally there are recombinase, polymerase or any other it is appropriate can be catalyzed or Promote to expand target polynucleotide on surface or support in the case of the reagent of nucleic acid amplification to generate two or more bases The group of monoclonal in sheet.

In some embodiments, polynucleotides can be reduced including screening agent in the reactive mixture and leaves given support The movement (such as reduce come off) on object or surface simultaneously can increase polynucleotides and will be hybridized to support or surface and provide nucleotide The possibility at the starting position of polymerization, so as to increase the ratio of the amplicon of substantially monoclonal generated during amplified reaction Example.

In some embodiments, in multiple and different globule supports in the case that amplification is included in the presence of screening agent The globule support of the multiple and different polynucleotide template of upper amplification and the substantially monoclonal for recycling certain percentage, it is each The globule that the globule support of a such substantially monoclonal includes being connected to the polynucleotides group of substantially monoclonal is supported Object.In some embodiments, the percentage of the globule support of the substantially monoclonal of recycling is substantially higher than from reaction The globule support (i.e. including polyclonal or monoclonal group total globule support) of total amplification of mixture recycling 5%th, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 89%, 90% or 95%.In some embodiments, the percentage of the globule support of the substantially monoclonal of recycling is substantially high In there is no screening agent in the case of but amplification condition essentially similar or identical in other aspects under expanded after The percentage of the globule support of the substantially monoclonal of recycling.

In some embodiments, screening agent includes polymer compound.In some embodiments, screening agent includes Crosslinked or noncrosslinking polymer compound.By way of non-limiting example, screening agent may include polysaccharide, polypeptide, organic Polymer etc..

In some embodiments, screening agent includes linear or branch polymer.In some embodiments, it sieves Agent includes with point or neutral polymer.

In some embodiments, screening agent may include one or more respectively poly- with average molecular weight and viscosity Close the mixture of object.

In some embodiments, screening agent include with about 10,000-2,000,000 or about 12,000-95,000 or The polymer of the average molecular weight of about 13,000-95,000.

In some embodiments, screening agent can be displayed in when being dissolved in the water with 2 weight percent at about 25 DEG C About 5 centipoises of measurement are to about 15,000 centipoises or with about 10 centipoises that 2% aqueous solution measures at about 25 DEG C to about 10,000 Centipoise or with about 15 centipoises that 2% aqueous solution measures at about 25 DEG C to the average viscosity range of about 5,000 centipoises.

In some embodiments, screening agent includes about 25 to about 1,5000kM_vOr about 75-1,000kM_vOr about 85- 800kM_vViscosity-average molecular weight (M_v).In some embodiments, reaction mixture include about 0.1 to about 20% weight/ The screening agent of volume or about 1-10%w/v or about 2-5%w/v.

In some embodiments, screening agent includes acrylamide polymer such as polyacrylamide.

In some embodiments, screening agent includes the polymer of one or more amino acid.For example, screening agent can wrap Include polylysine, polyglutamic acid, actin, myosin, keratin, tropomyosin (tropmyosin) etc..At some In embodiment, screening agent may include the derivative of any polypeptide of these polypeptides.

In some embodiments, screening agent includes polysaccharide polymer.In some embodiments, screening agent includes Portugal The polymer of grape sugar or galactolipin.In some embodiments, screening agent include it is one or more selected from cellulose, glucan, Starch, glycogen, agar, chitin, pectin or agarose polymer.In some embodiments, screening agent includes pyrans Portugal Grape glycopolymers.

In some embodiments, it is polarity or band under amplification reaction condition that screening agent, which includes having one or more, The polymer of the group of electricity.For example, polymer may include one or more cation groups, one or more anionic groups Or both.In some embodiments, screening agent is to include the polysaccharide of one or more charged groups.In some embodiments In, screening agent is to include the polysaccharide of one or more carboxyls, and the carboxyl is or is intended to band under amplification reaction condition Negative electricity.Such as screening agent may include carboxymethyl cellulose (CMC) polymer.In some embodiments, screening agent may include Spermine and/or spermidine.In some embodiments, screening agent includes polylysine and/or poly arginine.For example, screening agent can Including poly-L-Lysine, poly- D-Lys, poly- D-Glu etc..In some embodiments, screening agent include one or Multiple histones or histone-nucleic acid complexes or derivatives thereof.Histone is high alka albumen, can combine nucleic acid And including albumen H1, H2A, H2B, H3 and H4.In some embodiments, such as by methylating, it is acetylation, phosphorylation, general Histone is modified in elementization, SUMOization, citrullinated, ribosylation (including ADP- ribosylation) etc..

In some embodiments, screening agent includes polymer, the polymer including chemical substitute.Polymer may include Reactive group (for example, reactive group can react to generate substituted polymer with appropriate substituent).In some embodiment party In case, polymer include fluorescence-, carboxyl-, the polymer of amino-or alkoxy-substituted.In some embodiments, pass through It methylates, acetylation, phosphorylation, ubiquitination, carboxylated etc. carry out modified polymer.Substituent may include charged group, such as Anion or cation group, the substitution in the group to polymer chain can lead to the generation of electropolymer.Substituted degree Can about 0.2 between about 1.0 derivatives/monomeric unit, usually between about 0.4 to about 1.0, more generally about 0.6 to Change between about 0.95.

In some embodiments, screening agent includes cellulose derivative, such as sodium carboxymethylcellulose, carboxymethyl 2- Sodium hydroxyethyl cellulose, methylcellulose, hydroxyethyl cellulose, 2- hydroxypropyl celluloses, carboxymethyl cellulose, hydroxypropyl are fine Tie up element, hydroxyethylmethylcellulose, hydroxy butyl methyl cellulose, (hydroxypropyl) methylcellulose or hydroxyethyl ethylcellulose Or any one or more mixtures comprising such polymer.

In some embodiments, nucleic acid reaction mixture includes the mixture of different screening agent, such as different fibres The mixture of the plain derivative of dimension, starch, polyacrylamide etc..

In some embodiments, screening agent includes one or more complexing polymers, the complexing polymer packet Subdivision containing the different polymer of any two (including any polymer described herein).For example, complexing polymer It may include being connected to the polysaccharide polymer of polynucleotides polymer, such as be connected to the polysaccharide of DNA or RNA.In some embodiment party In case, screening agent may include the polymer comprising cellulosic sections and nucleic acid moiety, such as DNA- celluloses.In other implementations In scheme, complexing polymer may include being connected to the polyacrylamide of polynucleotides and/or polypeptide.In the reactive mixture It can be used for further hindering movement of the target polynucleotide by reaction mixture including such complexing polymer.

In some embodiments, screening agent may include first with appropriate cross-linking agent or the polymer reacted. For example, screening agent may include the acrylamide reacted with bisacrylamide and/or two (acryloyl) cystamines.

In some embodiments, reaction mixture includes at least one diffusion reduction agent.In some embodiments, Diffusion reduces agent and includes reducing polynucleotides from higher concentration region to any chemical combination of the migration in the region with low concentration Object.In some embodiments, diffusion reduces the migration that agent includes any component (no matter large or small) of reduction nucleic acid amplification reaction Any compound.In some embodiments, the component of nucleic acid amplification reaction includes globule/primer, polynucleotides, recombination Enzyme, polymerase, nucleotide, ATP and/or confactor.

It should be noted that the concept that screening agent and diffusion reduce agent is not certain mutually exclusive；Screening agent can usually have Effect ground reduces diffusion of the target compound by reaction mixture, and diffusion reduction agent can usually have the screening to reactive component Effect.In some embodiments, identical compound or reaction mixture additive can be used as sieving agent and/or diffusion subtracts Few agent.Any screening agent disclosed herein can be linked up in some embodiments reduces agent as diffusion, and vice versa.

In some embodiments, diffusion reduces agent and/or screening agent includes polyacrylamide, agar, agarose or fibre Dimension plain polymer such as hydroxyethyl cellulose (HEC), methylcellulose (MC) or carboxymethyl cellulose (CMC).

In some embodiments, sieve agent and/or diffusion reduce agent at least 1%, 2%, 5%, 10%, 15%, 20%th, 25%, 30%, 40%, 50%, 74%, 90% or 95%w/v (unit volume of weight/reaction mixture of reagent) Concentration include in the reactive mixture.

In some embodiments, reaction mixture includes at least one crowding agent.For example, crowding agent can pass through Generating crowded reaction environment increases the concentration of one or more components in nucleic acid amplification reaction.In some embodiments, instead Mixture is answered to include screening agent and crowding agent.

In some embodiments, include one or more surfaces for the method for nucleic acid amplification.In some embodiments In, surface can be connected with multiple the first primers, it is multiple in the first primer there is common the first primer sequence.

In some embodiments, surface can be outside or uppermost layer or the boundary of object.In some implementations In scheme, surface can be the inside on the boundary of object.

In some embodiments, reaction mixture include multiple and different surfaces, such as reaction mixture may include it is more A globule (such as particle, nano particle, particle etc.) and at least two different polynucleotide templates can be on different surfaces On expand with being cloned, so as to form at least two different surfaces, each of the surface is connected to amplicon.One In a little embodiments, reaction mixture includes single surface (for example, array of the surface of glass slide or reative cell) and extremely Two different regions or position are amplified few two different polynucleotide templates on the surface, and two are connected to so as to be formed The single surface of a or more amplicon.

In some embodiments, surface can be porous, semi-porous or non-porous.In some embodiments In, surface can be plane surface and concave surface, convex surface or its arbitrary combination.In some embodiments, surface can be small Pearl, particle, particle, sphere, filter, flow cell, hole, ditch, tank therefor, gel or capillary inner wall.In some embodiments In, surface includes the inner wall of capillary, slot, hole, ditch, slot, container.In some embodiments, surface may include texture (example Such as, etching, coelosis, aperture, three-dimensional rack or bulge).

In some embodiments, particle can have sphere, hemispheroidal, cylinder, barrel-shaped, annular, bar Shape, plate-like, it is conical, triangle, cube shaped, polygon.Tubulose, linear or irregular shape Shape.

In some embodiments, surface can be prepared from any material, and the material includes glass, Pyrex, silicon Stone, quartz, vitreous silica, mica, polyacrylamide, plastic polystyrene, makrolon, polymethacrylates (PMA), Polymethyl methacrylate (PMMA), dimethyl silicone polymer (PDMS), silicon, germanium, graphite, ceramic, silicon, semiconductor, High refractive index dielectric, crystal, gel, polymer or film (for example, film of gold, silver, aluminium or diamond).

In some embodiments, film can be magnetic or paramagnetic beads (for example, magnetic or paramagnetic nanoparticles Grain or particle).In some embodiments, paramagnetic particles can be connected to the paramagnetic beads of streptavidin (such as from Invitrogen, the Dynabeads of Carlsbad, CA^TMM-270).Particle can have iron core or include water Gel or agarose (such as Sepharose^TM)。

In some embodiments, surface can be connected with multiple the first primers.Surface can be coated with acrylamide, carboxyl Or aminated compounds is to connect nucleic acid (such as the first primer).It in some embodiments, can be by amido modified nucleic acid (example Such as primer) it is connected to the surface coated with carboxylic acid.In some embodiments, amido modified nucleic acid can be with EDC (or EDAC) React the surface to be connected to carboxylic acid coating (with or without NHS).The propylene that the first primer can be fixed on surface Amide compound coating.Particle can be coated with avidin sample compound (such as streptavidin) and be given birth to combining The nucleic acid of object element.

In some embodiments, surface includes the surface of globule.In some embodiments, globule includes polymer Material.For example, globule includes gel, hydrogel or acrylamide polymer.Globule can be porous.Particle can have chamber Or aperture or it may include three-dimensional rack.In some embodiments, particle can be Ion Sphere^TMParticle.

In some embodiments, disclosed method (and relevant composition, system and kit) including by one or It is multiple nucleic acid-templated to be fixed on one or more supports.It can include but not limited to physical absorption by any method, pass through Ion or covalent bond form or combination, by cDNA chip on solid support.Solid support may include polymerization, glass Or metal material.The example of solid support include film, plane surface, microwell plate, globule, filter, test-strips, glass slide, Coverslip and test tube.Mean any solid phase material for synthesizing on it, adhering to, connecting or fix in other ways oligomer.Branch Object is held optionally including " resin ", " phase ", " surface " and " support ".Support can be by organic polymer such as polyphenyl second Alkene, polyethylene, polypropylene, polyvinyl fluoride, polyethyleneoxy (polyethyleneoxy) and polyacrylamide and its copolymer It is formed with graft.Support can also be inorganic, such as glass, silica, controlled pore glass (CPG) or reverse-phase silica. The structure of support can be globule, sphere, particle, particulate, gel or the form on surface.Surface can be plane, base In sheet plane or it is nonplanar.Support can be porous or non-porous, and can have expansion or unexpansive characteristic. Support can be shaped to comprising one or more holes, recess or other reservoirs, container, feature or position.It can be by multiple Hold the different location of object configuration in an array.Support is optionally addressable (for example, the robot for reagent is passed Send) or include passing through the scanning of laser irradiation by detection means and be copolymerized the collection of burnt or deflect light.It can will expand support (such as globule) be placed within another support or on (such as in the hole of the second support).

In embodiments, solid support is " particle ", " globule ", " microballon " etc. (optionally but not necessarily in shape It is spherical on shape), there is 50 microns or smaller, preferably 10 microns or smaller, 3 microns or smaller, about 1 micron or smaller, about 0.5 micron or smaller, for example, about 0.1,0.2,0.3 or 0.4 micron or smaller (for example, less than 1 nanometer, about 1-10 nanometers, About 10-100 nanometers or about 100-500 nanometers) minimum cross-section length (such as diameter).Particle (such as from Dynal, The Dynabead of Oslo, Norway) it can be made of a variety of inorganic or organic material, the material includes but not limited to glass (example Such as controlled pore glass), silica, zirconium, crosslinked polystyrene, polyacrylate, polymethyl methacrylate, titanium dioxide Titanium, latex, polystyrene etc..Magnetization can help reagent (such as polynucleotides or the connection of the connection of particle after amplification Enzyme) collection and concentration, and also assist in other step (such as washing, reagent removal etc.).In certain implementations of the present invention In scheme, the Particle Swarm with different shape size and/or color can be used.It is optionally for example micro- with quantum-dot coding Grain is in order to individually or uniquely particle as identification.

In some embodiments, bead surface can be functionalized to connect multiple the first primers.In some embodiments In, globule can be any size that can be assembled in reative cell.For example, a globule can be assembled in reative cell.At some In embodiment, more than one globule can be assembled in reative cell.In some embodiments, the minimum cross-section of globule is long Degree (such as diameter) can be about 50 microns or smaller or about 10 microns or smaller or about 3 microns or smaller, about 1 micron or Smaller, about 0.5 micron or smaller, for example, about 0.1,0.2,0.3 or 0.4 micron or smaller (for example, less than 1 nanometer, about 1-10 Nanometer, about 10-100 nanometers or about 100-500 nanometers).

In some embodiments, globule can be connected with multiple one or more different primer sequences.In some implementations In scheme, globule can be connected with a kind of multiple primer sequences or can be connected with two or more multiple different primer sequences Row.In some embodiments, globule can be connected at least 1,000 primer or about 1,000-10,000 primer or about 10,000-50,000 primer or about 50,000-75,000 primer or about 75,000-100,000 primer or more Multiple primers.

In some embodiments, reaction mixture includes recombinase.Recombinase may include can inducing recombination event or Increase any reagent of the occurrence frequency of recombination event.Recombination event includes the different polynucleotide chain of two of which and weighs each other Any event of group.Recombination may include homologous recombination.Recombinase can be enzyme or its genetically engineered derivative.Recombinase Optionally (such as combination) is associated with single stranded oligonucleotide (such as the first primer).In some embodiments, catalysis is same The enzyme of source recombination can form nucleoprotein complex by combining single stranded oligonucleotide.In some embodiments, homologous recombination enzyme The analogous parts of double-stranded polynucleotide can be combined as nucleoprotein complex hair part.In some embodiments, polynucleotides Analogous parts can be hybridized at least part of the first primer.In a certain embodiment, the analogous parts of polynucleotides can portion Divide ground or be fully complementary at least part of the first primer.

In some embodiments, homologous recombination enzyme can be by forming nucleoprotein complex and being bound to double-strand multinuclear glycosides The analogous parts of acid are catalyzed chain intrusion (U.S. of Zarling to form the recombination intermediate (D- rings are formed) with triple strand structure State's patent No. 5,223,414, the U.S. Patent number 5,273,881 and 5,670,316 of Sena, and U.S. Patent number 7,270, 981st, 7,399,590,7,435,561,7,666,598,7,763,427,8,017,339,8,030,000,8,062,850 and 8071308).In some embodiments, homologous recombination enzyme includes wild type, mutant, recombinant, fusion or its segment. In some embodiments, homologous recombination enzyme include from it is any biology include Myoviridae (myoviridae) (such as from UvsX, RB69 of Bacteriophage T4 etc.), the enzyme of Escherichia coli (such as recA) or people (such as RAD51).

In some embodiments, include one or more auxilins for the method for nucleic acid amplification.For example, auxiliary Albumen can improve the activity (authorizing United States Patent (USP) 8,071,308 of Piepenburg etc.) of recombinase.In some embodiments In, auxilin can combine the single-stranded of nucleic acid or recombinase can be loaded on nucleic acid.In some embodiments, egg is assisted Include wild type, mutant, recombinant, fusion or its segment in vain.In some embodiments, auxilin can derive from With for carrying out any combinations of the identical or different species of the recombinase of nucleic acid amplification reaction.Auxilin, which can derive from, appoints What bacteriophage includes myovirus bacteriophage.The example of myovirus bacteriophage include T4, T2, T6, Rb69, Aeh1, KVP40, acinetobacter bacteriophage 133, Aeromonas bacteriophage 65, cyanophage P-SSM2, blue-green alge phagocytosis Body PSSM4, cyanophage S-PM2, Rb14, Rb32, Aeromonas bacteriophage 25, vibrio phage nt-1, phi- 1st, Rb16, Rb43, bacteriophage 31, bacteriophage 44RR2.8t, Rb49, bacteriophage Rb3 and bacteriophage LZ2.Auxilin can source Include Escherichia coli, solfataricus genus (such as sulphur ore deposit sulfolobus solfataricus) or Methanococcus (such as Zhan Shi in any bacterial species Methanosarcina).

In some embodiments, it may include single strand binding protein for the method for nucleic acid amplification.Single strand binding protein packet Include myovirus gp32 (such as T4 or RB69), Sso SSB for carrying out bin cure ore deposit sulfolobus solfataricus, the MjA from Methanococcus jannaschii SSB or Escherichia coli SSB albumen.

In some embodiments, the recombinase that may include to increase on nucleic acid for the method for nucleic acid amplification is loaded into Albumen.For example, recombinase, which is loaded into albumen, includes UsvY albumen (United States Patent (USP) 8,071,308 for authorizing Piepenburg).

In some embodiments, the albumen of at least one combination nucleic acid is may include for the method for nucleic acid amplification, including Unlock the albumen (such as unwindase) of double-strandednucleic acid.

In some embodiments, may include for the method for nucleic acid amplification at least one for recombinase or polymerase The confactor of activity.In some embodiments, confactor includes one or more bivalent cations.Bivalent cation Example include magnesium, manganese and calcium.

In some embodiments, premature precincubation nucleic acid amplification reaction under conditions of reacting starting can inhibited.Example Such as, one or more components of nucleic acid amplification reaction can be detained from reaction vessel to prevent premature reaction from originating.In order to start Reaction, can add in bivalent cation (such as magnesium or manganese).In another example, can at a temperature of inhibitory enzyme activity pre-temperature Educate nucleic acid amplification reaction.Precincubation reaction starting can be reacted at about 0-15 DEG C or about 15-25 DEG C to inhibit premature.Then Can at higher temperatures incubation reaction to cause enzymatic activity.

In some embodiments, it may include at least one recombination on nucleic acid for the method for nucleic acid amplification Enzyme assembles or the confactor for homologous nucleic acid pairing.In some embodiments, confactor includes any type of ATP includes ATP and ATP γ S.

In some embodiments, it may include at least one confactor for generating ATP for the method for nucleic acid amplification. Such as confactor includes converting ADP to the enzyme system of ATP.In some embodiments, confactor includes phosphocreatine And creatine kinase.

In some embodiments, any nucleic acid amplification method disclosed herein can be in isothermal or substantially isothermal The step of being carried out under amplification condition or may include carrying out under the described conditions.In some embodiments, isothermal duplication condition packet Include the nucleic acid amplification reaction of temperature change as experience：The temperature change (or entire expands at least certain part of amplification Increasing process) during be confined in limited range, including such as temperature change be equal to or less than about 20 DEG C or about 10 DEG C or About 5 DEG C, or about 1-5 DEG C, or about 0.1-1 DEG C, or less than about 0.1 DEG C.

In some embodiments, can carry out isothermal nucleic acid amplification reaction about 2,5,10,15,20,30,40,50,60 or 120 minutes.

In some embodiments, can at about 15-25 DEG C or about 25-35 DEG C or about 35-40 DEG C or about 40-45 DEG C, Or isothermal nucleic acid amplification reaction is carried out at about 45-50 DEG C, or about 50-55 DEG C, or about 55-60 DEG C.

In some embodiments, include multiple and different polynucleotides for the method for nucleic acid amplification.In some implementations In scheme, multiple and different polynucleotides include the mixture of single-stranded or double-stranded polynucleotides or both.In some embodiments In, multiple and different polynucleotides include the polynucleotides with same or different sequence.In some embodiments, Multiple and different polynucleotides include the polynucleotides with same or different length.In some embodiments, it is more A different polynucleotides include about 2-10, or about 10-50, or about 50-100, or about 100-500, or about 500-1,000, or About 1,000-5,000, or about 10³–10⁶, or about 10⁶ -10¹⁰A or more different polynucleotides.In some embodiments In, multiple and different polynucleotides include the polymer of deoxynucleotide, ribonucleotide and/or its analog.At some In embodiment, multiple and different polynucleotides include it is naturally occurring, synthesis, recombination, clone, amplification, do not expand Increasing or filing (such as preservation) form.In some embodiments, multiple and different polynucleotides include DNA, CDNA, RNA or chimeric rna/dna and nucleic acid analog.

In some embodiments, multiple and different polynucleotide templates, which may include having, is connected with nucleic acid linker sequence One or two end double-stranded polynucleotide library construction body.For example, polynucleotides library construction body may include the first He Second end, wherein first end are connected to the first nucleic acid linker.Polynucleotides library construction body, which may also include, is connected to second The second end of nucleic acid linker.First and second connectors can have same or different sequence.In some embodiments, At least part (part i.e. as polynucleotides library construction body) of first or second nucleic acid linker can be hybridized to first and draw Object.In some embodiments, homologous recombination enzyme can be with connecing as the part of nucleoprotein complex with first or second nucleic acid The polynucleotides library construction body of header sequence combines.

In some embodiments, polynucleotides library construction body can be suitable for any kind of microarray dataset and include Chemical degradation, chain termination, synthesis order-checking, pyrophosphate, large-scale parallel, ion-sensitive and unimolecule platform.

In some embodiments, include dilution with globule (for example, being connected with multiple for the method for nucleic acid amplification The globule of one primer) reaction polynucleotides amount to reduce the percentage of the globule reacted with more than one polynucleotides. In some embodiments, nucleic acid amplification reaction, the polynucleotides can be carried out to globule ratio using such polynucleotides It is selected to optimize the percentage of the globule of monoclonal polynucleotides group attached thereto to globule ratio.It for example, can With with about 1:2 to 1:Any one polynucleotides in the range of 500 carry out nucleic acid amplification reaction to globule ratio.In some realities It applies in scheme, polynucleotides include about 1 to globule ratio:2, or about 1:5, or about 1:10, or about 1:25, or about 1:50, or about 1:75, or about 1:100, or about 1:125, or about 1:150, or about 1:175, or about 1:200, or about 1:225, or about 1:225, Or about 1:250.In some embodiments, nucleic acid amplification reaction can be generated without the globule for being connected to its polynucleotides, Other globules of a type of polynucleotides attached thereto and more than one type attached thereto it is more Other globules of nucleotide.

In some embodiments, reaction mixture includes one or more primers.For example, reaction mixture may include At least the first Oligonucleolide primers.In some embodiments, the first primer may include being hybridized to a chain of polynucleotides At least part of forward direction amplimer.In some embodiments, the first primer is included for the extendible of nucleotide polymerization 3 ' ends.

In some embodiments, include being hybridized to the other primer of template for the method for nucleic acid amplification.For example, Second primer can be at least part of reversed amplimer for a chain for being hybridized to polynucleotides.In some embodiments In, the second primer includes extendible 3 ' end.In some embodiments, the second primer is not attached to surface.

In some embodiments, third primer can be hybridized to polynucleotides a chain it is at least part of just To amplimer.In some embodiments, third primer includes extendible 3 ' end.In some embodiments, third Primer is not attached to surface.In some embodiments, third primer include for Enrichment Amplification nucleic acid binding partners or Affine part (such as biotin).

In some embodiments, primer (such as first, second, and third primer) is including single stranded oligonucleotide.

In some embodiments, at least part of primer can be at least one chain of polynucleotides in reaction mixture Partial hybridization.For example, at least part of primer can be miscellaneous with the nucleic acid linker for one or two end for being connected to polynucleotides It hands over.In some embodiments, at least part of primer can partially or even wholly be complementary to part or the nucleic acid of polynucleotides Connector.In some embodiments, primer can be suitable for any kind of microarray dataset include chemical degradation, chain termination, Synthesis order-checking, pyrophosphate, large-scale parallel, ion-sensitive and unimolecule platform.

In some embodiments, primer (such as first, second or third primer) can have not in reaction mixture 5 ' or the 3 ' of the partial hybridization of at least one chain of polynucleotides are prominent tail portion (magnetic tape trailer primer).In some embodiments, band Tail primer can have any length, include the length of 1-50 or more nucleotide.

In some embodiments, primer includes the polymer of deoxynucleotide, ribonucleotide and/or its analog. In some embodiments, primer includes shape that is naturally occurring, synthesis, recombination, clone, amplification or not expanding Formula.In some embodiments, primer includes DNA, cDNA, RNA or chimeric rna/dna and nucleic acid analog.

In some embodiments, primer can be any length, including about 5-10 nucleotide or about 10-25 core Thuja acid or about 25-40 nucleotide or about 40-55 nucleotide or longer.

In some embodiments, it may include one or more different polymerases for the method for nucleic acid amplification.One In a little embodiments, polymerase include any enzyme that can be catalyzed the polymerization of nucleotide and/or nucleotide analog or its segment or Subunit.In some embodiments, polymerase needs extendible 3 ' end.For example, polymerase needs the end of nucleic acid primer 3 ' OH are polymerize with initiation nucleotide.

Polymerase includes that nucleotide (including its analog) can be catalyzed to any enzyme of the polymerization in nucleic acid chains.Normally But not necessarily such nucleotide polymerization can be occurred with template dependent manner.In some embodiments, polymerase can To be high fidelity polymerase.Such polymerase may include but be not limited to naturally occurring polymerase and its any subunit and Truncate, mutated polymerase, variant polymerase, recombination, fusion or the polymerase being engineered in other ways, chemical modification Polymerase, synthesis molecule or assembly and its remain any analog of the such ability polymerizeing of catalysis, derivative Object or segment.Optionally, polymerase can be the mutated polymerase for including one or more mutation, and the mutation includes one A or multiple amino acid by the insertion or missing or two kinds of other amino acid replacements, one or more amino acid from polymerase or The connection of the part of more kinds of polymerases.As used herein, belong to " polymerase " and its variant also refer to comprising at least two that The fusion protein of the part of this connection, wherein first part include the peptide that can be catalyzed the polymerization in nucleotide to nucleic acid chains and company Second part is connected to, the second part includes the second polypeptide, such as reporter enzyme or processivity promote enzyme.Normally, Polymerase includes one or more active sites, and the catalysis that nucleotide combines and/or nucleotide combines can occur on it. In some embodiments, polymerase includes or lacks other enzymatic activity such as 3 ' to 5 ' exonuclease activities or 5 ' to 3 ' Exonuclease activity.In some embodiments, recombinant DNA technology or chemical synthesis process can be detached or used from cell Generate polymerase.In some embodiments, polymerase can be expressed in protokaryon, eukaryon, virus or phage biocontrol.One In a little embodiments, polymerase can be the albumen or its segment of posttranslational modification.

In some embodiments, polymerase may include the bioactivity piece of any one or more polymerases or polymerase Section, be described in Davidson et al. in U.S. Patent Publication No. 2011/0262903 disclosed in 27 days October in 2011 and/ Or Vander Horn et al. in 2 months 2013 WO of International PCT publication number disclosed in 14 days 2013/023176.

In some embodiments, polymerase can be archaeal dna polymerase and including but not limited to DNA of bacteria polymerase, true Core biology archaeal dna polymerase, archael DNA polymerase.Viral dna polymerase and phage DNA polymerase.

In some embodiments, polymerase can be replicase, DNA dependences polymerase, primase, RNA dependences Polymerase (such as reverse transcriptase of the archaeal dna polymerase including RNA dependences), thermal instability polymerase or thermal stability polymerization Enzyme.In some embodiments, polymerase can be any family A or Type B polymerase.Family A (such as Escherichia coli Pol I), B (such as Escherichia coli Pol II), C (such as Escherichia coli Pol III), D (such as wealthy archeobacteria Pol II), X (such as people Pol β) and Y (such as Escherichia coli UmuC/DinB and eucaryote RAD30/ xeroderma pitmentosums variant) polymerase Many type specifications in Rothwell and Watsman 2005Advances in Protein Chemistry 71: In 401-440.In some embodiments, polymerase can be T3, T5, T7 or SP6 RNA polymerase.

In some embodiments, can using a type or mixture for polymerase, recombinase and/or ligase come Carry out nucleic acid amplification reaction.In some embodiments, nucleic acid expansion can be carried out using low fidelity or high fidelity polymerase Increase reaction.

It in some embodiments, can be by thermostabilization or thermal instability polymerase come catalytic nucleic acid amplified reaction.

In some embodiments, polymerase can lack 5 ' -3 ' exonuclease activities.In some embodiments, gather Synthase can have strand-displacement activity.

In some embodiments, extinct plants and animal archaeal dna polymerase can be but not limited to thermal stability or thermophilic DNA gathers Synthase is for example：Bacillus subtilis (Bacillus subtilis) (Bsu) DNA polymerase i large fragment；Thermus aquaticus (Taq) archaeal dna polymerase；Filamentous Thermus (Thermus filiformis) (Tfi) archaeal dna polymerase；Phi29DNA polymerases； Bacillus stearothermophilus (Bst) archaeal dna polymerase；9 ° of N-7DNA polymerases of hyperthermophilic Gu bacterium (Thermococcus sp.)； Shi Shi bacillus (Bacillus smithii) (Bsm) DNA polymerase Large fragments；Beach is thermophilic coccus (Thermococcus Litoralis) (Tli) DNA polymerases or Vent^TM(exo-) archaeal dna polymerase (coming from New England Biolabs)；Or " Deep Vent " (exo-) archaeal dna polymerase (New England Biolabs).

In some embodiments, the nucleotide of one or more types is may include for the method for nucleic acid amplification.Nucleosides Acid combines polymerase including alternative or can be by any compound of polymerization enzymatic polymerization.It is normally but nonessential, nucleotide With being to pass through polymerizeing in the nucleotide to nucleic acid chains of polymerase after the selective binding of polymerase；However occasionally nucleosides Acid can be from polymerization enzymatic hydrolysis without being incorporated in nucleic acid chains, which is herein referred to as " unproductive " event.It is such Nucleotide not only further includes including naturally occurring nucleotide and alternative combines polymerase or can be by any of polymerization enzymatic polymerization Analog (no matter its structure).Although naturally occurring nucleotide generally comprises base, sugar and phosphate moiety, the disclosure The nucleotide of content may include the compound of any, some or all for lacking such part.In some embodiments In, nucleotide is optionally including phosphorus atoms chain, and it includes 3,4,5,6,7,8,9,10 or more phosphorus atoms.In some realities It applies in scheme, phosphorus chain may be connected to any carbon such as 5 ' carbon of saccharide ring.Using the O or S of insertion by phosphorus chain link to sugar. In one embodiment, one or more of chain phosphorus atoms can be the part of the phosphate group with P and O.Another In a embodiment, using O, NH of insertion, S, methylene, the methylene of substitution, ethylene, the ethylene of substitution, CNH₂、C (O)、C(CH₂)、CH₂CH₂Or C (OH) CH₂Phosphorus atoms in chain are connected to one by R (wherein R can be 4- pyridines or 1- imidazoles) It rises.In one embodiment, the phosphorus atoms in chain, which can have, includes O, BH₃Or the side group of S.In phosphorus chain, have in addition to O Side group phosphorus atoms can be substitution phosphate group.In phosphorus chain, the phosphorus atoms of the atom with the insertion in addition to O can Be substitution phosphate group.Some examples of nucleotide analog are described in the U.S. Patent number 7,405,281 of Xu.

Some examples of nucleotide available for disclosed method and composition include but not limited to ribonucleotide, take off Oxygen ribonucleic acid, the ribonucleotide of modification, the DNA of modification, ribonucleotide polyphosphate, deoxyribose core Sour polyphosphate, the ribonucleotide polyphosphate of modification, the DNA polyphosphate of modification, peptide nucleotide, modification Peptide nucleotide, metal nucleosides, phosphonate ester nucleosides and the phosphate of modification-sugar backbone nucleotide, the class of aforesaid compound Like object, derivative or variant etc..In some embodiments, nucleotide may include substitution bridge joint nucleotide α phosphate and Sugar or α the and β phosphate of nucleotide or any other two phosphate of the β and γ phosphate of nucleotide or nucleotide it Between, or any combination thereof the non-oxygen part such as sulphur-of oxygen part or borine-part.

In some embodiments, nucleotide is unlabelled.In some embodiments, nucleotide includes label simultaneously Herein referred to as " nucleotide of label ".In some embodiments, label can be any portion for being connected to nucleotide Divide (being the phosphate group farthest from sugar including base, sugar or any phosphate group intervened therebetween or terminal phosphate group) The form of fluorescent dye.

In some embodiments, nucleotide (or its analog) is connectable to label.In some embodiments, it marks Note includes optically detectable part.In some embodiments, label includes being generally not present in naturally occurring nucleosides Part in acid.For example, label may include fluorescence, shine or radioactive part.

In some embodiments, it may also include enriching step for the method for nucleic acid amplification.In some embodiments In, for nucleic acid amplification method can generate it is at least one be connected with it is multiple have and the sequence of template polynucleotide complementation The globule of polynucleotides (such as nucleic acid of amplification).At least one of the polynucleotides of globule is connected to be hybridized to life The polynucleotides (such as reversed amplified production with third primer) of object element part.In some embodiments, it is enriched with Step includes passing through the polynucleotides with biotin moiety and the purifying globule for being connected to streptavidin part (such as para-magnetic beads) (such as the MyOne from Dynabeads^TMBead) with reference to forming purifying compound.In some realities It applies in scheme, can will purify compound from reaction mixture separation/removal by using the attraction of magnet.

In some embodiments, the amplicon of the nucleic acid group comprising substantially monoclonal is respectively placed, distributed or determined Different loci in site array.

In some embodiments, disclosed method is included single template molecule (such as the single target multinuclear glycosides of sample Acid) distribution, place or in other ways positioning in reative cell or site (such as in array).It can be by making with multinuclear The reacted room of fluid stream of thuja acid sample distributes single polynucleotides into reative cell from sample.In distribution to reative cell Single polynucleotides can be single-stranded or double-strand.In some embodiments, after the distribution in reative cell or site Middle amplification of nucleic acid.

In some embodiments, different single target polynucleotide can be distributed to arrangement in an array from sample In each of different reative cells.It can be by making to have the reacted room of fluid stream of polynucleotides sample will be different single Polynucleotides are distributed from sample into each of different reative cells.In distribution to each of different reative cells not Same single polynucleotides can be single-stranded or double-strand.

In some embodiments, method includes distributing into reative cell single polynucleotides and in reative cell expanding Increase single polynucleotides, so as to generate monoclonal nucleic acid group in the reaction chamber.

In some embodiments, for single target polynucleotide being distributed into reative cell and is expanded single target multinuclear The method of thuja acid includes sample of nucleic acid.In some embodiments, it can be distributed from the sample of nucleic acid with multiple polynucleotides single A polynucleotides or different single polynucleotides.For example, sample of nucleic acid may include about 2-10 or about 10-50 or about 50- 100, or about 100-500, or about 500-1,000, or about 1,000-5,000, or about 10³-10⁶, or more different multinuclears Thuja acid.Different polynucleotides can have same or different sequence.Different polynucleotides can have identical or different Length.Sample may include the mixture of single-stranded or double-strand polynucleotides or both.

In some embodiments, for single target polynucleotide being distributed into reative cell and is expanded single target multinuclear The method of thuja acid includes distributing single polynucleotides.In some embodiments, single polynucleotides may include single-stranded and double The nucleic acid molecules of chain.In some embodiments, nucleic acid may include deoxynucleotide, ribonucleotide and/or its analog Polymer.In some embodiments, nucleic acid may include it is naturally occurring, synthesis, recombination, clone, amplification, not Amplification or filing (such as preservation) form.In some embodiments, nucleic acid may include DNA, cDNA, RNA or chimeric RNA/DNA and nucleic acid analog.In some embodiments, single polynucleotides may include being included in one or two end It is connected to the nucleic acid library construct of the nucleic acid of oligonucleotide joint.In some embodiments, nucleic acid library construct can Be suitable for any kind of microarray dataset include chemical degradation, chain termination, synthesis order-checking, pyrophosphate, large-scale parallel, Ion-sensitive and unimolecule platform.

In some embodiments, the site of array may include that one or more reative cells (can be on the surface of solids Hole).Reative cell can have the hole for limiting width and depth.The scale of reative cell can be enough to allow the deposition of reagent Or for being reacted.Reative cell can have any shape to include cylindrical, polygon or combination of different shapes.Reative cell Any wall can have smooth or irregular surface.Reative cell may include having plane, concave or convex surface Bottom.The bottom of reative cell and side wall may include same or different material and/or can be coated with chemical group, the chemistry base Group can be with biomolecule such as nucleic acid, albumen or enzyme reaction.

In some embodiments, reative cell can be aligned in one of multiple reative cells in grid or array.Battle array Row may include two or more reative cells.Multiple reative cells randomly can be arranged or be arranged in orderly array.Orderly Array may include with arrange or with row and column two-dimensional grid arrange reative cell.

Array may include any amount of reative cell for depositing reagent and carrying out multiple individual reactions.For example, array It may include at least 256 reative cells or at least 256,000 or at least 1-3 million or at least 3-5 million or at least 5-7 hundred Ten thousand or at least 7-9 million, at least 9-11 million, at least million reative cells of 11-13 or even higher density include 13- 700000000 reative cells or more.Be arranged in reative cell in grid can have less than about 10 microns or less than about 5 microns, Or the centre distance (such as pitch (pitch)) between the adjacent reaction room less than about 1 micron or less than about 0.5 micron.

Array may include the reative cell with any width and depth dimension.For example, reative cell can be single micro- with adapting to The scale of grain (such as microballon) or multiple particles.Reative cell can accommodate the water volume of 0.001-100 picoliters.

In some embodiments, at least one reative cell can be coupled to one or more sensors or can be welded on On one or more sensors.Be coupled to the reative cell of sensor can provide be deposited on the closing of reagent therein so as to come Self-reacting product can be detected by sensor.Sensor can detect the variation of the product from any kind of reaction, described anti- Should include any nucleic acid reaction such as primer extend or or nucleotide be incorporated to reaction.Sensor can detect ion (such as hydrogen Ion), the variation of proton, phosphate group such as pyrophosphate group.In some embodiments, at least one reative cell can It is coupled to one or more ion-sensitive field-effect transistors (ISFET).It is coupled to the battle array of the reative cell of ISFET sensors The example of row is found in U.S. Patent number 7,948,015 and U.S. serial 12/002,781.

In some embodiments, it is (and relevant that amplification method described herein can be carried out in the array of reative cell Composition, system and device), wherein the reative cell of array forms the part of single fluid sexual system.In some embodiments In, the arrays of multiple reative cells may include fluidity interface, the fluidity interface make fluid (such as liquid or gas) with by The laminar flow of control flows through reative cell.In some embodiments, the array of reative cell can include on reative cell for laminar flow Fluid head space.In some embodiments, the array of reative cell can be the part of flow cell or flow chamber, wherein Reative cell is in fluid communication to each other.For example, fluid can be flowed on array so that partially or fully filling the one of array A or multiple reative cells.In some embodiments, fluid can be completely filled with multiple reative cells, and excessive fluid can flood The top of reative cell is in the top of reative cell formation fluid layer.Fluid layer above reative cell can be multiple anti-in array Room is answered to provide fluid communication.In some embodiments, the fluid communication in array between multiple reative cells can be used for multiple The parallel reaction detached in reative cell.For example, it is in fluid communication multiple available for polynucleotides and/or reagent are delivered to Reative cell is to carry out parallel nucleic acid amplification reaction.

In some embodiments, can the sample with multiple and different polynucleotides be applied to flow chamber to incite somebody to action list A polynucleotides distribute each reative cell into array.In some embodiments, other reagent can be applied to flowing Room is to distribute in each reative cell into array.For example, other reaction reagent may include particle, one or more enzymes, enzyme Confactor, primer and/or nucleoside triphosphate.It in some embodiments, can in any order, including continuously or base Polynucleotides and reagent are delivered to the array of reative cell by the combination in sheet simultaneously or both.For example, in some embodiment party In case, it then can distribute the array that polynucleotides are distributed to reative cell to reagent first or opposite sequence can be used or can Essentially simultaneously distribute polynucleotides and reagent.

In some embodiments, any method can be used to be delivered to polynucleotides and/or reagent (including flow chamber) The reative cell of certain percentage in array.For example, the percentage for the reative cell being loaded in array is including about 1-25% or about 25% or about 30% or about 40% or about 50% or about 60% or about 70% or higher percentage.In some implementations In scheme, polynucleotides and/or reagent can be loaded with to increase by carrying out the loading step of two or more bouts The percentage of reative cell.For example, (a) in first leg, polynucleotides and/or reagent can be distributed it is multiple anti-into array Room and (b) is answered polynucleotides and/or reagent can be distributed to identical array in second leg.It can carry out additional loading Bout (such as third, 4th or more bout).In some embodiments, can be appointed between any loading bout The reaction of what type and/or any kind of reaction can be carried out after the completion of multiple loading bouts.It for example, can be in any loading Nucleic acid amplification reaction is carried out between bout or nucleic acid amplification reaction can be carried out after the completion of multiple loading bouts.In some implementations In scheme, after each loading bout, polynucleotides and globule can be prevented to migrate out instead on array compound laying Answer room.For example, after each loading bout, it can be by the solution laying comprising at least one screening agent on array.One In a little embodiments, screening agent includes cellulose derivative.It alternatively, can be by oil reservoir laying in the hole of array or indoor reaction The top of mixture.

In some embodiments, disclosed method (and relevant composition, system and kit) is additionally included in amplification By the nucleic acid-templated site for being connected to array before template.Optionally, site includes primer, and the connection includes to draw Object is hybridized to the primer binding site of template.For example, the site in array may include at least one fixed primer, it is described to draw Object includes the sequence of at least part complementation of the primer binding site of template with being assigned or being placed at site.Primer promotes Template to array connection.In some embodiments, most of site in array includes at least one primer.Different positions The primer of point can be mutually the same.Alternatively, the primer of different loci can be different from each other.In an exemplary reality It applies in scheme, at least two sites respectively include different target specific primers.

Any appropriate method can be used that primer is connected to the site of array.Primer is connected to reative cell by particle The surface of nano-array (for example, ISFET array types for the sequencing based on ion), first in at least some of array It can be useful to react indoor synthesis or prepare three dimensional matrix.In embodiments, can by polymer substrate precursor applications in With the array in the united hole of one or more sensors.Polymer substrate precursor can be polymerize to the battle array to form polymer substrate Row.These polymer substrates can be conjugated in oligonucleotides and include hereditary sequencing technologies available for a variety of analytical technologies.

In some embodiments, by hydrophilic polymer matrix distribute with sensor (such as the sensing of sensor array Device) in united hole.In instances, hydrophilic matrix is such as hydrogel matrix.Hydrophilic matrix can be with one-to-one structure pair It should be in the sensor of sensor array.In other examples, the sensor of sensor array may include field-effect transistor (FET) sensor, such as ion-sensitive field-effect transistor (ISFET).Particularly, host material be in-situ solidifying and It is adapted to the structure of a body opening of sensing device.Void area between hole can there is no polymer substrate. In example, the bonding surface for being for example covalently bonded to hole of host material.In instances, hole has 100 nanometers to 10 microns models Depth or thickness in enclosing.In another example, hole can have the characteristic diameter in 0.1 micron to 2 micron ranges.

It, can be by the way that the aqueous solution comprising polymer precursor be applied in the hole of hole array come shape in illustrative methods Into polymer substrate.The appearance of hydrous material that the immiscible fluid being placed on hole array isolation can be used to be limited by hole array Amount.Can starting soln the capacity through isolation to promote the polymerization of matrix precursor, so as to cause matrix battle array of the distribution in hole Row.In instances, the aqueous solution comprising matrix precursor is distributed to the hole of sensing device by the way that aqueous precursor is made to flow through hole. In another example, it is included in aqueous solution as dispersed phase in emulsion.Dispersed phase can be settled or is excited in hole array Kong Zhong.It can be used and be placed in the polymerization that the initiation factor in water phase or in immiscible fluid carrys out starting substrate precursor.In another reality It, can hot starting polymerization in example.

In another illustrative methods, it can passed by the surface that starting molecule is anchored in the hole of hole array Array of substrates is formed in the hole of induction device.The solution for including matrix precursor can be provided on the hole of hole array.Initiation factor can rise The polymerization of beginning matrix precursor causes to form polymer substrate in the hole of hole array.In other examples, the combination above method Aspect to further enhance the formation of array of substrates.

In special embodiment, sequencing system include wherein be placed in sensor array flow cell, including with sensing The telecommunication circuit of device array electronic communication and the container including being in fluid communication with flow cell and fluid control.In instances, Figure 13 shows the expanded view of flow cell 100 and sectional view and the part for showing flow chamber 106.Reagent flow 108 flows through Kong Zhen The surface of row 102, wherein reagent flow 108 are flowed above the open end in the hole of hole array 102.Hole array 102 and sensing Device array 105 can form the integrated unit for the lower wall (or bottom surface) for forming flow cell 100 together.Reference electrode 104 can be fluidly It is coupled to flow chamber 106.In addition, flow cell lid 130 encapsulates flow chamber 106 so that reagent flow 108 to be included in limited region It is interior.

Figure 14 shows the expanded view of hole 201 and sensor 214 (as shown at the 110 of Figure 13).It can be based on occurring Reaction property and the labelling technique (if any) or reagent, by-product that use come the volume of selecting hole, shape, Length-width ratio (such as bottom width is to hole depth rate) and other size characteristics.Sensor 214 can be chemical field-effect transistor (chemFET), more particularly ion-sensitive FET (ISFET), with floating gate 218, floating gate 218 has optionally Pass through the sensor board 220 detached inside material layer 216 and hole.(do not show in addition, conductive layer can be placed on sensor board 220 Go out).In instances, material layer 216 includes ion-sensitive material layer.Material layer 216 can be ceramic layer, such as except other The nitride of zirconium, hafnium, tantalum, aluminium or titanyl compound or titanium in addition.In instances, material layer 216 can have 5nm to 100nm, Such as the thickness of 10nm to 70nm, 15nm to 65nm or 20nm to 50nm.

Although material layer 216 is shown as extending in except the boundary of the FET components of display, material layer 216 can be along hole 201 bottom and optionally along the wall in hole 201 extend.Sensor 214 can sense (and generating relevant output signal) and be present in biography The amount of charge 224 in the opposite material layer 216 of sensor plate 220.The variation of charge 224 can cause the source electrode 221 of chemFET Curent change between drain electrode 222.And then chemFET can be used directly for providing the output signal based on electric current or utilization Other circuit provides the output signal based on voltage indirectly.Reactant, washing solution and other reagents can pass through diffusion machine System 240 is movable into and out hole.

In embodiments, the reaction carried out in hole 201 can be the characteristic for identifying or measuring purpose analyte Or the analysis reaction of property.Such reaction can either directly or indirectly have an impact the quantity of electric charge near sensor board 220 By-product.If such by-product generate in a small amount decay rapidly or with other component reactions, can simultaneously in hole 201 Multiple copies of middle analysis same analyte, to increase the output signal generated.It in embodiments, can be by the more of analyte A copy is connected to solid support 212 before or after hole 201 is placed in.Solid support 212 can be polymer matrix Matter, such as hydrophilic polymer matrix, such as hydrogel matrix or similar.For simplicity and ease of explanation, solid support 212 are also referred to as polymer substrate herein.

Hole 201 can be defined by wall construction, and the wall construction can be formed by one or more layers material.In instances, wall construction Can have 0.01 micron to 10 microns that upper surface is extended to from the lower surface in hole, such as 0.05 micron to 10 microns, 0.1 is micro- Rice is to 10 microns, 0.3 micron to 10 microns or 0.5 micron to 6 microns of thickness.Particularly, thickness can be at 0.01 micron To in the range of 1 micron, such as in the range of 0.05 micron to 0.5 micron or 0.05 micron to 0.3 micron.Hole 201 can With no more than 5 microns, such as no more than 3.5 microns, no more than 2.0 microns, no more than 1.6 microns, no more than 1.0 microns, Characteristic diameter no more than 0.8 micron or no more than 0.6 micron, the diameter are multiplied by the flat of cross-sectional area (A) divided by Pi by 4 Root (for example, sqrt (4*A/ π)) defines.In instances, hole 201 can have at least 0.01 micron of characteristic diameter.

Although Figure 14 shows single-wall structure and monolayer material layer 216, system may include one or more wall constructions Layer, one or more conducting shells or one or more material layers.For example, wall construction can be formed by one or more layers, the layer Oxide comprising TEOS or silicon or the nitride comprising silicon.

In the specific example shown in fig.15, system 300 includes the hole wall structure 302 for the array for defining hole 304, institute Hole is stated to be placed on the sensing pad of sensor array or be operationally coupled to it.Hole wall structure 302 defines upper surface 306.With The united lower surface 308 in hole is placed on the sensing pad of sensor array.Hole wall structure 302 defines upper surface 306 and lower surface Side wall 310 between 308.As described above, the material layer contacted with the sensing pad of sensor array can be along the array in hole 304 The lower surface 308 in hole extends along at least part of the wall 310 defined by hole wall structure 302.Upper surface 306 can not have material Layer.Particularly, polymer substrate can be placed in the hole of the array in hole 304.Upper surface 306 can there is no polymer Matrix.For example, upper surface 306 may include the area of no polymer substrate, such as at least the 70% of the gross area, the gross area At least 80%, at least the 90% of the gross area or about the 100% of the gross area.

Although the wall surface of Figure 14 is shown as substantially perpendicularly and outwardly extends, wall surface can be in difference Side upwardly extend and can have different shapes.Term " substantially perpendicularly " means on the surface with being defined by sensing pad Extend on orthogonal component direction.For example, as shown in Figure 16, hole wall 402 can vertically extend, with being determined by sensing pad The normal component 412 on the surface of justice is parallel.In another example, wall surface 404 is on the outward direction of sensing pad is left Substantially perpendicularly extend, so as to provide the hole of the following table area bigger than hole opening.As shown in Figure 16, wall surface 404 It is upwardly extended in the 412 parallel vertical component side of normal component with surface 414.In alternative example, wall surface 406 exists Substantially perpendicularly extend in inward direction, so as to provide opening area more smaller than the following table area in hole.Wall surface 406 Extend on the component direction parallel with the normal component 412 on surface 414.

Although by straight line display surface 402,404 or 406, some semiconductors or CMOS manufacturing process can cause to have There is the structure of non-linear shape.Particularly, wall surface such as wall surface 408 and upper surface such as upper surface 410 in shape may be used To be arc or can have various non-linear forms.Although in company with the structure and equipment of display be described as having linear layer, Surface or shape, but can be different in some extent by practical layer, surface or the shape that semiconductor technology generates, it may packet Include the variation of the non-linear and arc of the embodiment of display.

Figure 17 shows the illustrative hole for including ion-sensitive material layer.For example, pore structure 502 can define hole Array, such as exemplary bore 504,506 or 508.Hole (504,506 or 508) can operationally be coupled to the sensor of lower section (not shown) or the sensor for being connected to such lower section.Exemplary bore 504 includes the bottom for defining hole 504 and extends to knot Ion-sensitive material layer 510 in structure 502.Although not shown in Figure 17, conducting shell, such as grid, such as ion-sensitive Property field-effect transistor floating gate, can be located at ion-sensitive material layer 510 lower section.

In another example, as shown by hole 506, ion-sensitive material layer 512 can define the bottom in hole 506 Portion and be not extend in structure 502.In other examples, hole 508 may include along the side wall in hole 508 defined by structure 502 The ion-sensitive layer 514 of 516 at least part extension.Same Shangdi, ion-sensitive material layer 512 or 514 can be underlying On the conducting shell or grid of electronic device.

In fig. 14, host material 212 and pore structure are conformal.Particularly, host material can in-situ solidifying with hole Wall and bottom surface it is conformal.Defining the upper surface in hole may include there is no the area of host material, such as the gross area is at least 70%, at least the 80% of the gross area, at least the 90% of the gross area or about the 100% of the gross area.Depending on the property of pore structure, Polymer substrate can physically be fixed to hole wall structure.In another example, polymer substrate chemically is bound to hole Wall construction.Particularly, polymer substrate can covalently be bound to hole wall structure.In another example, can by hydrogen bond or from Polymer substrate is bound to hole wall structure by sub-key.

Can be from matrix precursor, such as the monomer that can polymerize completely, such as the monomer based on vinyl, form polymer matrix Matter.Specifically, monomer may include hydrophilic monomer, such as acrylamide, vinyl acetate, hydroxy alkyl methacrylate (hydroxyalkylmethacrylate), its variant or derivative, its copolymer or its arbitrary combination.In specific example In, hydrophilic monomer is acrylamide, such as it is functionalized with comprising hydroxyl, amino, carboxyl, halogen or combination acryloyl Amine.In instances, hydrophilic monomer is aminoalkyl acrylamides, (D is shown the functionalized acrylamide of POLYPROPYLENE GLYCOL blocked with amine Be shown in hereinafter), propylene piperazine (C, display below) or combination.In another example, acrylamide can be hydroxyl Alkyl acrylamide, such as hydroxyethyl acrylamide.Specifically, alkylol acrylamides may include N- tri- (methylol) first Base) acrylamide (A, display below), N- (methylol) acrylamide (B, display is below) or combination.Another In one example, comonomer may include the acrylate or acrylamide of halogen modification, such as N- (5- acetyl bromides amidos penta Base) acrylamide (BRAPA, E, display is below).In another example, comonomer may include oligonucleotides-modified Acrylate or acrylamide monomer.In other examples, the mixture of monomer can be used, such as alkylol acrylamides With the mixture or acrylamide of amine-functionalized acrylamide and the mixture of amine-functionalized acrylamide.In instances, may be used With with such alkylol acrylamides:Amine-functionalized acrylamide or acrylamide:Amine-functionalized acrylamide Ratio includes amine-functionalized acrylamide, and the ratio is 100:1 to 1:In the range of 1, such as 100:1 to 2:1、50:1 To 3:1、50:1 to 5:1 or 50:1 to 10:In the range of 1.It in another example, can be with such hydroxyalkyl acryloyl Amine:The functionalized acrylamide of bromine or acrylamide:The ratio of the functionalized acrylamide of bromine includes amine-functionalized acryloyl Amine, the ratio is 100:1 to 1:In the range of 1, such as 100:1 to 2:1、50:1 to 3:1、50:1 to 5:1 or 50:1 to 10:In the range of 1.

In other examples, it may include the functionalized acrylamide of oligonucleotides or acrylate monomer, such as Acrydite^TMOligonucleotides is incorporated in polymer substrate by monomer.

Another exemplary substrates precursor includes crosslinking agent.In instances, with monomer to the 15 of crosslinking agent:1 to 1:2, example Such as 10:1 to 1:1、6:1 to 1:1 or 4:1 to 1:1 mass ratio includes crosslinking agent.Particularly, crosslinking agent can be that divinyl is handed over Join agent.For example, divinyl crosslinking agent may include bisacrylamide, such as N, N '-（Ethane -1,2- diyls）Two（2- ethoxys） Acrylamide, N, N '-（2- hydroxypropyl alkane -1,3- diyls）Bisacrylamide or combination.In another example, divinyl is handed over Join agent and include ethyleneglycol dimethacrylate, divinylbenzene, hexamethylene bisacrylamide, trimethylol propane trimethyl propylene Acid esters, its shielded derivative or combination.

In one aspect, in the range of polymer network is comprising having 3-20%, in the range of more preferably 5-10% The polyacrylamide gel of total monomer percentage.In one embodiment, the crosslinking agent percentage of monomer is in the model of 5-10% In enclosing.In specific embodiments, polymer network includes 10% total acrylamide, wherein 10% is bisacrylamide Crosslinking agent.

It can be by the initiation factor in solution come starting polymerization.For example, initiation factor can be based on water.Another In a example, initiation factor can be hydrophobic initiation factor, be preferably placed in hydrophobic phase.Exemplary initiation factor includes over cure Sour ammonium or TEMED（Tetramethylethylenediamine）.TEMED can accelerate the synthesis speed of the free radical from persulfate, and then be catalyzed Polymerization.Acrylamide monomer is for example converted into free radical by persulfate free radical, with non-activated monomer reaction with rise Beginning Polymerization chain reaction.The polymer chain of extension can be randomly crosslinked, so as to cause with the porous gel of characteristic, institute Porosity is stated depending on polymerizing condition and monomer concentration.Riboflavin（Or riboflavin -5 '-phosphate）It also is used as free radical Source is usually applied in combination with TEMED and ammonium persulfate.In the presence of light and oxygen, riboflavin is converted into it The colorless form of activity with starting polymerization, this is commonly referred to as photochemical polymerization.

In another example, nitrogenous initiation factor can be used for starting polymerization.Illustrative water-soluble nitrogenous starting because Son is shown in table 1, and the illustrative nitrogenous initiation factor of oil-soluble is shown in table 2.Particularly, nitrogenous initiation factor can be with It is azodiisobutyronitrile（AIBN）.

Table 1

Water-soluble nitrogenous initiation factor compound

Table 2

The nitrogenous initiation factor compound of oil-soluble

In other examples, the precursor of polymer substrate may include surfactant additive to enhance and the combination on surface. Exemplary additives include functionalized propylene acid monomers or functionalized acrylamide monomer.For example, acrylic monomers can be through official Energyization is with mating surface material, such as forms the bottom in hole or the ceramic material of side wall.In instances, additive may include propylene Base-phosphonate ester, such as metering system phosphonate ester.In another example, additive may include dimethacrylamide or poly- Dimethacrylamide.In other examples, additive may include by polymerizable group (such as acrylic acid groups) modification Polylysine.

In another example, atom transfer radical polymerization (ATRP) can be used to promote to polymerize.ATRP systems can wrap Include initiation factor, transition metal ions and ligand.Exemplary transition metal ion complex includes the complex compound based on copper.Show Example property ligand includes 2,2 '-two pyridines,-two pyridine of 4,4 '-two -5- nonyls -2,2 ', 4,4 ', 4 "-three (5- nonyls) -2, 2’:6 ', 2 "-terpyridyl, N, N, N ', N ', N "-pentamethyl-diethylenetriamine, three second four of 1,1,4,7,10,10- hexamethyls Amine, three (2- dimethylaminoethyls) amine, N, N- bis- (2- picolyls) octadecylamine, N, N, N ', N '-four [(2- pyridines) methyl] Ethylenediamine, three [2- pyridines) methyl] amine, three (2- aminoethyls) amine, three (2- bis- (3- butoxy -3- oxygen propyl groups) aminoethyl) amine, Three (2- bis- (3- (2- ethyl hexyls oxygen) -3- oxygen propyl groups) aminoethyl) amine, three (2- bis- (ten dioxy -3- oxygen propyl groups of 3-) aminoethyls) Amine, its variant and derivative or combination.Exemplary initiation factor includes 2- bromopropionitriles, ethyl 2- isobutyl bromides ester, ethyl 2 bromopropionic acid ester, methyl 2 bromopropionic acid ester, 1- phenyl bromoethane, paratoluensulfonyl chloride, 1- cyanogen -1- Methylethyls diethyl two Thiocarbamate, 2- (N, N- diethyl-dithio carbamyl)-ethyl isobutyrate, dimethyl 2,6- dibromo pimelic acid Ester, its variant or derivative and its arbitrary combination.Optionally, it can be used as branching agent there are BRAPA monomers during ATRP systems.

In instances, ATRP is originated on surface so that polymer bonds directly are bonded to surface.It for example, can be in transition metal In the presence of ion/ligand complex compound, by acrylic monomers, acrylamide monomer, Acrydite^TMMonomer, succinyl are sub- Amine acrylate, double acrylic acid or bisacrylamide monomer, its derivative or combination are applied to the table of starting in the solution Face.

In another, ATRP systems can be used for using modified phosphonate, sulfonate, silicate or zirconates Polymer is connected to the surface in hole by compound.Specifically, amine or hydroxy-end capped phosphonate ester or its alkoxy can be spread out Biologic applications are originated in surface and using initiation factor.Can application of catalyst complex compound and monomer, with extensional surface chemical combination Object.

In illustrative methods, the aqueous solution of the precursor comprising polymer substrate can be applied to the array in definition hole In the hole of structure.The polymerization of polymer precursor in solution on hole in offer immiscible fluid and starter hole can be provided Carry out the aqueous solution in clearance hole.

For example, Figure 18 shows the exemplary bore structure 602 for defining hole 604.One or more sensors (not shown) can To be operationally coupled or be connected to hole 604.For example, one or more sensors may include and at least bottom surface in hole 604 electricity The gate structure of communication.It is provided on hole and includes the aqueous solution 606 of polymer precursor in addition to other components and will include poly- The solution of polymer precursor is distributed into hole 604.Exemplary polymer precursor includes monomer, crosslinking agent, starting among other things The factor or surfactant, such as described above.Optionally, hydrophilic solution can be used before the deposition, such as comprising Water, the solution of alcohol or its mixture or the solution comprising water and surfactant, to soak hole 604.Exemplary alcohols include different Propyl alcohol.In another example, alcohol includes ethyl alcohol.Although not showing, the side wall in the bottom surface in hole and optionally hole can wrap Include ion-sensitive material.Such ion-sensitive material can overlay on the electronic device such as field-effect transistor of lower section On conducting structure.One of surfactant additive processing hole can be used before application is comprising the solution of polymer precursor Or multiple surfaces.

Distribution in aqueous solution comprising polymer precursor to hole 604 can be by shaking structure (such as by rotation or whirlpool Rotation) further strengthen.In another example, vibration such as sound wave or ultrasonic activation can be used to exist to increase aqueous solution Distribution in hole 604.In other examples, the degasification of vacuum pump device to hole can be used and apply solution under negative gauge pressure.In example In, aqueous solution is distributed to hole at room temperature.In another example, aqueous solution is distributed at a temperature below the room temperature, it is special It is not so assigned when using the initiation factor based on water.Alternatively, aqueous solution is distributed at elevated temperatures.

As shown in Figure 19, immiscible fluid 708 is applied on hole 604, aqueous solution 606 is pushed away into hole Aqueous solution 606 in top and clearance hole 604, as shown in Figure 20.Exemplary immiscible fluid includes mineral oil, silicone oil (such as poly- (dimethyl siloxane)), heptane, carbonic acid ester oil (such as diethylhexyl carbonic ester (Tegosoft)) Or combination.

Initiation factor can be applied in aqueous solution 606.Alternatively, initiation factor can be provided in immiscible fluid 708.It can By changing substrate temperature come starting polymerization.Alternatively, polymerization can occur at room temperature.It specifically, can be molten by polymer precursor Liquid is kept for 10 minutes to 5 at 20 DEG C to 100 DEG C, such as at a temperature of 25 DEG C to 90 DEG C, 25 DEG C to 50 DEG C or 25 DEG C to 45 DEG C Hour, such as 10 minutes to 2 hours or 10 minutes to 1 hour.

Due to polymerization, the array of polymer substrate 912 is formed in the hole 604 defined by pore structure 602, is such as schemed Shown by 21.Optionally, array can be washed with NaOH (such as 1N NaOH) to remove polymerization from the void area between hole Object.

In alternate example, using in immiscible fluid as the breast of the aqueous solution of the polymer precursor of dispersed phase Agent is used for the droplet deposition of aqueous solution in hole.For example, as shown in Figure 22, pore structure 1002 defines hole 1004.Kong Ke To be operationally coupled or be electrically connected to one or more sensors (not shown).As described above, the bottom of the wall in hole 1004 Surface and optionally side surface can be defined by ion-sensitive material layer, and the material layer can overlay on the electronic device of lower section On conducting parts.

Emulsion 1006 may include the aqueous droplet for including polymer precursor disperseed in continuous immiscible fluid 1010 1008.Droplet 1008 can be deposited in hole 1004.Particularly, aqueous droplet 1008 has than 1010 bigger of immiscible fluid Density.Exemplary immiscible fluid include mineral oil, silicone oil (such as poly- (dimethyl siloxane)) heptane, carbonic acid ester oil (such as Diethylhexyl carbonic ester (Tegosoft)) or combination.It in other examples, can be by rotating, being vortexed or surpass Sonication fluid or structure promote the distribution in aqueous droplet to hole 1004.It optionally, can be before the deposition using hydrophilic molten Liquid, such as the solution comprising water, alcohol or its mixture or the solution comprising water and surfactant, to soak hole 604.It is micro- Temperature during dropping to the distribution in hole can carry out at room temperature.Alternatively, it can be allocated at elevated temperatures.

As shown in Figure 23, droplet merges to provide the molten of the isolation for including polymer precursor 1112 in hole 1004 Liquid.Optionally, immiscible fluid 1010 or different immiscible fluid of the immiscible fluid for example without droplet 1008 can be used 1116 replace emulsion 1006.Exemplary immiscible fluid includes mineral oil, silicone oil (such as poly- (dimethyl siloxane)) heptan Alkane, carbonic acid ester oil (such as diethylhexyl carbonic ester (Tegosoft)) or combination.Alternatively, emulsion 1006 can It is held in place during polymerization.After this manner, the solution 1112 in hole 1004 is kept apart with the solution in other holes 1004. Can starting polymerization, lead to the polymer substrate 1214 in hole 1004, as shown in Figure 24.As described above, it hot can originate poly- It closes.In another example, oil phase initiation factor can be used to carry out starting polymerization.Alternatively, water phase initiation factor can be used to rise Begin to polymerize.Particularly, the second emulsion can be applied on hole 1004.Second emulsion may include including the dispersion of water phase initiation factor Water phase.

It can be by changing substrate temperature come starting polymerization.Alternatively, polymerization can occur at room temperature.Specifically, can will gather Polymer precursor solution is kept at 20 DEG C to 100 DEG C, such as at a temperature of 25 DEG C to 90 DEG C, 25 DEG C to 50 DEG C or 25 DEG C to 45 DEG C 10 minutes to 5 hours, such as 10 minutes to 2 hours or 10 minutes to 1 hour.Optionally, NaOH (such as 1N NaOH) can be used Array is washed to remove polymer from the void area between hole.

In another example, the initiation factor on surface for being fixed on hole array can be used to be formed in the hole of hole array The array of host material.For example, as shown in Figure 25, structure 1302 can define hole 1304.Material layer 1306 can define hole 1304 lower surface.It can will be anchored compound 1308, such as the anchoring chemical combination for atom transfer radical polymerization (ATRP) Object is fixed to the material layer 1306 for the bottom surface for defining hole 1304.It is exposed to alternatively, being limited in the hole or layer of structure 1302 Side-wall material in hole 1304 can be anchored the compound that compound is for example used for ATRP as described above.

In such example, can on structure 1302 and hole 1304 in application comprising polymer precursor such as monomer, The solution 1310 of crosslinking agent and optionally surfactant additive.Anchoring compound 1308 can be caused to promote from anchoring chemical combination Polymerization is isolated in hole 1304 and polymer is fixed to hole 1304 by the polymerization of object extension.In instances, it is anchored compound Group is formed with surface active groups and distal end free radical.Surface active groups may include phosphonate, silicate sulfonate, Zirconates, titanate or combination.Distal end free radical, which forms group, may include amine or hydroxyl, and the amine or hydroxyl can undergo example Such as with the transfer of halogenated (such as bromo) compound and subsequently forming for polymer, polymer precursor and by generated polymer It is anchored into the free radical of hole surface.Normally, ATRP systems may be selected with the monomer of statistically average length or quantity to add Polymerization is terminated after adding.In this way, the polymerization sum in controllable drilling 1304.Prolong in other examples, chain can will be influenced It stretches or terminates other actually using and added to aqueous solution 1310.

In another example shown in fig. 26, structure 1402 can define hole 1404.Hole 1404 may include along structure 1402 and hole 1404 side wall 1410 extend material layer 1406.Initiation factor1 408 can be along the bottom in hole 1404 and along side wall 1410 are fixed to material layer 1406.It can be applied on structure 1402 and hole 1404 comprising polymer precursor, crosslinking agent and other The solution 1412 of reagent.

As shown in Figure 27, polymer substrate 1512 is because surface of the starting out of hole 1304 is (such as by material layer 1306 definition surfaces) extension polymerization and formed.

It can be by changing substrate temperature come starting polymerization.Alternatively, polymerization can occur at room temperature.Specifically, can will gather Polymer precursor solution is kept at 20 DEG C to 100 DEG C, such as at a temperature of 25 DEG C to 90 DEG C, 25 DEG C to 50 DEG C or 25 DEG C to 45 DEG C 10 minutes to 5 hours, such as 10 minutes to 2 hours or 10 minutes to 1 hour.

After formation, can activated polymer matrix it is conjugated with target analyte such as polynucleotides to promote.For example, it can increase Functional group in strength polymer matrix is to allow and the combination of target analyte or analyte receptor (such as Oligonucleolide primers). In specific example, available can be the activity that can undergo nucleophilic or parental materials by hydrophilic polymer functional group conversions Partial reagent modifies the functional group of hydrophilic polymer matrix.It for example, can be by using sulfonic acid group or chlorine substituted hydroxy At least partly carry out the hydroxyl in activated polymer matrix.Exemplary sulfonic acid group can derive from three fluoro ethanesulfonyls (tresyl), methylsulfonyl, tolysulfonyl or to fluorobenzene sulphonyl (fosyl) chlorine or its arbitrary combine.Sulphonic acid ester is used to allow parent Core group substituted sulfonic acid ester.Sulphonic acid ester can also react the chlorination to provide the process that can be used for being conjugated matrix with the chlorine being released Functional group.In another example, amido that can be in activated polymer matrix.

For example, target analyte or analyte receptor can be bound to hydrophilic polymer by the nucleophilic displacement of fluorine with sulfonic acid group. In specific example, the target analyte receptor blocked with nucleophilic group such as amine or sulfydryl can undergo nucleophilic displacement of fluorine to replace Sulfonic acid group in polymer substrate.Due to activation, conjugated polymer substrate can be formed.

In another example, the polymer substrate of sulfonation can further with the single or multiple nucleopilic reagent of single or multiple functionality (such as maleimide) reacts, and the connection that the reagent can form with matrix is preserved for the widow for including electrophilic group simultaneously The nucleophilic reactivity of nucleotide.In addition, remaining nucleophilic reactivity can be converted by being connected to the reagent for including more electrophilic groups Electrophilic live sex will then be connected to the oligonucleotides for including nucleophilic group.

In another example, the monomer for including functional group can be added during polymerization.Monomer is included for example comprising carboxylic Acid, ester, halogen or other amine reactive groups acrylamide.Ester group can be before reacting by water with few amine (amine oligo) Solution.

Other conjugation techniques include the use of amine-containing monomer.Amido is can be by amine reactivity difunctionality parents electricity The nucleophilic group that reagent is further modified, the reagent generate mono-functional's electrophilic group after polymer substrate is connected to. Such electrophilic group can be reacted with the oligonucleotides with nucleophilic group (such as amine or sulfydryl), lead to the company of oligonucleotides It connects (by the electrophilic precursor reactant with vacating).

If preparing polymer substrate from the combination of amino-and hydroxy-acrylamide, polymer substrate includes nucleophilic Amino and the combination with Neutral hydroxy.It can be repaiied using the double electrophilic moiety such as diisocyanate of difunctionality or two-NHS esters Amino is adornd, so as to generate the hydrophilic polymer matrix that there is reactivity to nucleophilic group.Exemplary two-NHS esters include two-amber Amber acid imide acyl C2-C12 alkyl esters, such as two-succinimide acyl suberate or two-succinimide acyl glutarate.

Other activating chemicals include mixing multiple steps so that specific functional group conversions are specific desired to adapt to Connection.For example, the hydroxyl that sulphonic acid ester is modified can be transformed into nucleophilic group by several method.In instances, sulphonic acid ester with The reaction of azide anion generates the hydrophilic polymer of azide substitution.Azide can be used directly for by can " CLICK " chemical action carried out in the case of with or without copper catalysis is conjugated to the biomolecule of acetylene substitution. Optionally, azide can be converted to amine by using the catalysis reduction of hydrogen or using the reduction of organic phosphine.Then may be used The amine of gained is converted to electrophilic base using plurality of reagents such as diisocyanate, two-NHS esters, cyanuric chloride or combination Group.In instances, urea bond (urea linkage) is generated between polymer and connector using diisocyanate, this causes to remain Remaining isocyanate groups, the biomolecule that can replace with amino react to generate urea between connector and biomolecule Key.In another example, amido bond and remaining NHS ester groups between polymer and connector are produced using two-NHS esters, The biomolecule that the NHS ester groups can replace with amino is reacted to be connect with generating amide between connector and biomolecule. In other examples, amino-triazines key and two remaining chlorine three between polymer and connector are produced using cyanuric chloride Piperazine group, one of the chlorotriazine group biomolecule that can replace with amino react with connector and biomolecule it Between generate amino-triazines key.It can be activated by sulfonic acid acyl and other nucleophilic groups are incorporated in matrix.For example, sulfonic acid acyl group matter with Sulfydryl is incorporated in matrix by the hydrolysis of the Thiobenzoate of reaction and the generation of thiobenzoate anion, the sulfydryl with The biomolecule that can replace afterwards with maleimide is reacted to be connect with generating with sulphur-succinimide of biomolecule.

Sulfydryl can also be reacted with bromo- acetyl group or bromo- amide groups (bromo-amidyl group).In specific example In, it, can be by being formed for gathering when being comonomer comprising n- (5- acetyl bromide amidos amyl) acrylamide (BRAPA) Thiobenzamide-oligonucleotide compound of the reaction of the bromo- acetyl group on object is closed to be incorporated to oligonucleotides, such as following aobvious Show.

It can be by following dithiobenzoic acid-NHS compounds being made to be reacted with the oligonucleotides that amine blocks and to activate two thio Benzamide-oligonucleotide compound is thio to be formed to form the thiobenzamide-oligonucleotide compound being shown above Benzamide-oligonucleotide compound.

Alternatively, acrydite oligonucleotides can be incorporated to oligonucleotides during polymerization.Exemplary acrydite is few Nucleotide may include the oligonucleotides of ion exchange.

It can divide by using on the electrophilic moiety or substrate being coupled on substrate with the nucleophilic moiety in biomolecule with biology The nucleophilic of electrophilic connection coupling on son connects to be served as a contrast to generate biomolecule in refractory material (refractory) or polymeric material Covalent linkage on bottom.Due to the hydrophilic nmature of most of general object biomolecule, being coupled for these for selection is molten Agent be water or water comprising a certain water-miscible organic solvent with by biomolecule dispersion on substrate.Particularly, usually in water Polynucleotides are coupled to substrate in system, because they have polyanion property.Since water is by the way that electrophilic body is hydrolyzed Come to compete electrophilic body with nucleophilic group in conjugated inactive part in pairs, thus aqueous system can lead to the coupling of low-yield Product, wherein yield are based on couplet electrophilic moiety.It is high when the high yield of the electrophilic moiety of expected response couplet The nucleophilic group driving of concentration reacts and slows down hydrolysis, leads to the invalid use of nucleophilic group.In the feelings of Polynucleotide Under condition, biomolecule can be dissolved in polarity, nothing by phosphatic alkaline counter-ion by lipophile balance ionic compartmentation with help In the nonaqueous solvents of reactivity.These solvents may include amide or urea such as formamide, N,N-dimethylformamide, acetyl Amine, DMAC N,N' dimethyl acetamide, hexamethyl-phosphoramide, pyrrolidones, N-Methyl pyrrolidone, N, N, N ', N '-tetramethyl Urea, N, N '-dimethyl-N, N '-trimethyleneurea or combination；Carbonate such as dimethyl carbonate, propene carbonate or its group It closes；Ester such as tetrahydrofuran；Sulfoxide and sulfone such as dimethyl sulfoxide (DMSO), dimethyl sulfone or combination；Hinder alcohol (hindered Alcohol) such as tertiary butyl ethyl alcohol；Or combination.Lipophilic cation may include tetra-allkylammonium or four aryl ammonium cations for example Tetramethyl-ammonium, tetraethyl ammonium, tetrapropyl ammonium, tetrabutylammonium, four pentyl ammonium, tetrahexyl ammonium, four heptyl ammoniums, four octyl group ammoniums and its Alkyl and aryl mixture, four aryl phosphine cation such as tetraphenylphosphoniums, tetraalkyl arsine or four aryl arsines such as four phenylarsines and Trialkylsulfonium cation such as trimethylsulfonium, or combination.Can Polynucleotide be carried out by multiple standards cation exchange technology To the conversion of organic solvent soluble material (by the way that metal cation is exchanged with lipophilic cation).

In specific embodiments, polymer substrate is exposed to target polynucleotide, the target polynucleotide have with It is conjugated in the section of the oligonucleotides complementation of polymer substrate.Make polynucleotides experience amplification, such as pass through polymerase chain reaction Answer (PCR) or recombinase polymeric enzymatic amplification (RPA).For example, target polynucleotide is provided with low concentration, so as to single polynucleotides It is likely located in the single polymer substrate of the array of polymer substrate.Polymer substrate can be exposed to enzyme, nucleotide, salt Or other components for being enough to promote the duplication of target polynucleotide.

In specific embodiments, enzyme such as polymerase is present in, is connected to or close proximity to polymer substrate. Multiple nucleic acids polymerase can be used for method described herein.In an exemplary embodiment, polymerase may include to be catalyzed more The enzyme of the duplication of nucleotide, its segment or subunit.In another embodiment, polymerase can be naturally occurring polymerization Enzyme, recombination polymerase, mutated polymerase, variant polymerase, fusion or the polymerase being engineered in other ways, chemistry are repaiied The polymerases of decorations, synthetic molecules or its analog, derivative or segment.

In some embodiments, for distributing to reative cell and expand single target multinuclear glycosides single target polynucleotide The method of acid includes nucleic acid amplification reaction.In some embodiments, any kind of nucleic acid amplification reaction can be carried out to include gathering Polymerase chain reaction (PCR) (United States Patent (USP) 4,683,195 and 4,683,202 for authorizing Mullis), ligase chain reaction (LCR)(Barany 1991Proceedings National Academy of Science USA 88:189-193； Barnes 1994Proceedings National Academy of Science USA91:2216-2220), unwindase according to Rely property amplification (HDA) or isothermal Autonomous maintenance serial response (Kwoh 1989Proceedings National Academy of Science USA 86:1173-1177；WO 1988/10315；With United States Patent (USP) 5,409,818,5,399,491 and 5,194, 370)。

In some embodiments, amplified reaction includes recombinase polymeric enzymatic amplification (RPA).(see, for example, Zarling U.S. Patent number 5,223,414, the U.S. Patent number 5,273,881 and 5,670,316 of Sena, and U.S. Patent number 7, 270,981、7,399,590、 7,435,561、7,666,598、7,763,427、8,017,339、8,030,000、8,062, 850 and 8071308).

In some embodiments, for distributing to reative cell and expand single target multinuclear glycosides single target polynucleotide The method of acid includes isothermal duplication condition.In some embodiments, nucleic acid amplification reaction can be carried out under isothermal conditions. In some embodiments, isothermal duplication condition includes the nucleic acid amplification reaction of temperature change as experience：The temperature change It is confined in limited range during at least certain part of amplification, including such as temperature change at about 20 DEG C or about 10 DEG C, or about 5 DEG C, or within about 1-5 DEG C, or about 0.1-1 DEG C, or less than about 0.1 DEG C.It in some embodiments, can be in isothermal Or nucleic acid amplification reaction is carried out under thermal cycle conditions.

In some embodiments, the nucleic acid of content amplification can be used for any nucleic acid sequencing workflow according to this teaching, Including sequencing (such as the SOLiD from Life Technologies for being connected and being detected by oligonucleotide probe^TM, WO 2006/084131), probe-anchoring connection sequencing (such as Complete Genomics^TMOr Polonator^TM), synthesis order-checking (such as Genetic Analyzer and HiSeq from Illumina^TM), pyrophosphate sequencing is (such as from 454Life The Genome Sequencer FLX of Sciences), ion-sensitive sequencing (such as from Ion Torrent Systems, Inc. Personal Genome Machine (PGM^TM) and Ion Proton^TMSequencer) and single-molecule sequencing is put down Platform (such as from Helicos^TMHeliScope^TM)。

In some embodiments, can the nucleic acid that content expands according to this teaching, institute be sequenced by any sequencing approach Sequencing approach is stated to include synthesis order-checking, include the use of field-effect transistor (such as FET and ISFET) detection sequencing by-product Sequencing based on ion, chemical degradation sequencing, the sequencing based on connection, sequencing by hybridization, pyrophosphate detection sequencing, capillary Electrophoresis, gel electrophoresis, the next generation, large-scale parallel microarray dataset, detection hydrogen ion or other sequencings that by-product is sequenced are put down Platform and single-molecule sequencing platform.In some embodiments, at least one times for being hybridized to polynucleotide constructs can be used The sequencing primer of what part (including nucleic acid linker or target polynucleotide) carries out sequencing reaction.

In some embodiments, the method for one or more by-products that detection nucleotide is incorporated to can be used to be sequenced The nucleic acid of content amplification according to this teaching.It can by the polymerase extension detection for the physical chemistry by-product for detecting extension Including pyrophosphate, hydrogen ion, electric charge transfer, heat etc., such as e.g., as disclosed in the U.S. Patent number of Rothberg et al. 7,948, In the U.S. Patent Publication No. 2009/0026082 (being incorporated herein by reference in their entirety) of 015 and Rothberg et al..Inspection The other examples for surveying the method for the extension based on polymerase are found in such as Pourmand et al., Proc.Natl.Acad.Sci.,103:6466-6470(2006)；Purushothaman et al., IEEE ISCAS, IV-169- 172；Anderson et al., Sensors and Actuators B Chem., 129:79-86(2008)；Sakata et al., Angew.Chem.118:2283-2286(2006)；Esfandyapour et al., U.S. Patent Publication No. 2008/01666727； With Sakurai et al., Anal.Chem.64:In 1996-1997 (1992).

The reaction of the generation and the detection that involve ion is widely carried out.It is monitored using direct ion detection method The process of such reaction can simplify many existing bioassay.For example, it is generated as by detection by polymerase catalysed The hydrogen ion for the natural by-product that nucleotide is incorporated to can be monitored to be synthesized by polymerase-mediated template-dependent nucleic acid.Ion is quick Ionic byproducts such as hydrogen ion is utilized in perception sequencing (also referred to as " based on pH's " or " based on ion " nucleic acid sequencing) The direct detection of (being generated as the by-product that nucleotide is incorporated to).In the exemplary system that one is used for the sequencing based on ion In, it can be by trapping nucleic acids to be sequenced in micropore, and nucleotide is made to flow through one at a time under the conditions of nucleotide is incorporated to Hole.Appropriate nucleotide is incorporated to the chain of growth by polymerase, and the pH of solution can be changed in the hydrogen ion being released, can by with The ion transducer of hole coupling detects.The technology does not need to the label of nucleotide or the optical module of costliness, and allows to survey Sort run is much completed faster.The example of such method for nucleic acid sequencing and platform based on ion includes Ion Torrent PGM^TMOr Proton^TMSequenator (Ion Torrent^TM Systems,Life Technologies Corporation)。

In some embodiments, the target polynucleotide generated using the method for this teachings, system and kit can As the substrate reacted for biological or chemical, the reaction includes field-effect transistor (FET) to detect by sensor And/or monitoring.FET is chemFET or ISFET in different implementation scenarios." chemFET " or chemical field-effect transistor It is used as the field-effect transistor types of chemical sensor.It is the analogue of mosfet transistor, wherein passing through Process applies the charge on gate electrode." ISFET " or ion-sensitive field-effect transistor be used for measure in solution from Sub- concentration；When ion concentration (such as H+) changes, will correspondingly be changed by the electric current of transistor.Operate the detailed of ISFET Theory sees " Thirty years of ISFETOLOGY:what happened in the past 30years and what may happen in the next 30years,”P.Bergveld,Sens. Actuators,88(2003), In pp.1-20.

In some embodiments, FET can be FET array.As used herein, " array " is that element for example senses Device or the planar alignment in hole.Array can be one-dimensional or two-dimentional.One-dimensional array can have a row (or row) in the first dimension Element and in arrays of second dimension with multiple row (or row).In the first and the second dimension the number of row (or row) can with or not Can be identical.FET or array may include 102,103,104,105,106,107 or more FET.

In some embodiments, one or more microfluidic structures can be welded on FET sensor array to carry For the receiving and/or closing of biological or chemical reaction.For example, in an instrument, microfluidic structures can be configured to be placed in battle array On the one or more sensors of row one or more holes (or micropore or reative cell or reacting hole, it is interchangeable herein Ground uses these terms), to be analyzed in given bore one or more sensors detection placed on it and measurement given bore Presence, level and/or the concentration of object.In some embodiments, the correspondence of FET sensor and reacting hole can be 1: 1。

Micropore or reative cell are typically hole or hole with clearly defined shape and volume, can be machined to substrate In and conventional microbonding connection technology can be used to weld, such as disclosed in following references：Doering and Nishi,Editors,Handbook of Semiconductor Manufacturing Technology,Second Edition(CRC Press,2007)；Saliterman,Fundamentals of BioMEMS and Medical Microdevices(SPIE Publications,2006)；Elwenspoek et al,Silicon Micromachining (Cambridge University Press,2004)；It is and similar.The structure of micropore or reative cell (such as spacing, shape Shape and volume) example be disclosed in the U.S. Patent Publication 2009/0127589 of Rothberg et al.；The English of Rothberg et al. In state patent application GB24611127.

In some embodiments, can contacted with FET such as chemFET or ISFET, operationally coupling or capacitance Property coupling solution or reative cell in carry out biological or chemical reaction.FET (or chemFET or ISFET) and/or reative cell can It is the array of FET or reative cell respectively.

In some embodiments, biological or chemical reaction can be carried out in the two-dimensional array of reative cell, wherein each anti- Answer room that can be coupled to FET, and each reative cell is no more than 10 μm on capacity³(i.e. 1pL).In some embodiments, often A reative cell is no more than 0.34pL, 0.096pL or 0.012pL on capacity.Reative cell is optionally in the cross-sectional area at top It is upper to be no more than 2,5,10,15,22,32,42,52,62,72,82,92 or 102 square microns.Preferably, array has at least 10²、10³、10⁴、10⁵、10⁶、10⁷、10⁸、10⁹Or more reative cell.In some embodiments, reative cell is at least one Operationally it is coupled at least one of FET.

It can be conventional use of according to the CMOS welding techniques of conventional cmos welding technique and improvement and in CMOS welding Other semiconductor welding technologies other than technology, to weld for the FET battle arrays according to various embodiments in the present disclosure Row.In addition, various lithographics can be used as the part of array welding procedure.

The exemplary FET array of method disclosed in being suitable for and micropore and fluid and the method for preparing it are disclosed in Such as U.S. Patent Publication No. 20100301398；U.S. Patent Publication No. 20100300895；U.S. Patent Publication No. 20100300559；U.S. Patent Publication No. 20100197507, U.S. Patent Publication No. 20100137143；U.S. Patent Publication Numbers 20090127589；In U.S. Patent Publication No. 20090026082, above-mentioned reference is integrally incorporated this by reference Text.

In one aspect, disclosed method, composition, system, device and kit can be used for carrying out unmarked nucleic acid survey Sequence is based particularly on the nucleic acid sequencing of ion.The concept for the markless detection that nucleotide is incorporated to is had been described in document, including Following document (it is incorporated herein by reference)：The U.S. Patent Publication 2009/0026082 of Rothberg et al.； Anderson et al., Sensors and Actuators B Chem., 129:79-86(2008)；With Pourmand et al., Proc.Natl.Acad.Sci.,103:6466-6470(2006).Briefly, in nucleic acid sequencing application, it is polymerize by measuring The natural by-product of the extension of enzymatic includes hydrogen ion, polyphosphate, PPi and Pi (such as there are pyrophosphatases When) be incorporated to measure nucleotide.The example of such method for nucleic acid sequencing and platform based on ion includes Ion Torrent PGM^TMOr Proton^TMSequenator (Ion Torrent^TM Systems,Life Technologies Corporation)。

In some embodiments, present disclosure generally relates to sequencing by introduction provided herein Hold the method for nucleic acid expanded.In an exemplary embodiment, present disclosure is generally related to from multinuclear glycosides The method that acid obtains sequence information, including：(a) amplification of nucleic acid；(b) by the nucleic acid of amplification generated during step (a) It is at least one to carry out template-dependent nucleic acid synthesis as template.Optionally according to any amplification side described herein Method is expanded.

In some embodiments, Template Dependent synthesis is included one or more nucleotide with Template Dependent side Formula is incorporated to newly synthesized nucleic acid chains.

Optionally, method may also include generate as one or more ionic byproducts for being incorporated to of nucleotide.

In some embodiments, method may also include being incorporated in the one or more nucleotide to sequencing primer of detection. Optionally, detection may include detecting hydrionic release.

In another embodiment, the method that present disclosure generally relates to sequencing nucleic acid, including：(a) basis The methods disclosed herein amplification of nucleic acid；(b) nucleic acid of amplification is placed in multiple reative cells, wherein the one of reative cell or It is multiple to be contacted with field-effect transistor (FET).Optionally, method further includes the core for the amplification being placed in one of reative cell Acid is contacted with polymerase, so as to synthesize new nucleic acid by the way that one or more nucleotide are continuously incorporated to nucleic acid molecules Chain.Optionally, method, which further includes, generates the by-product that one or more hydrogen ions are incorporated to as such nucleotide.Optionally, Method is further included detects being incorporated to for one or more nucleotide by using the one or more hydrionic generations of FET detections.

In some embodiments, detection includes detecting the voltage and/or current-responsive in array at least one FET The variation of one or more hydrionic generations.

In some embodiments, FET may be selected from：Ion-sensitive FET (isFET) and chemosensitivity FET (chemFET)。

One exemplary system of the sequencing of ionic byproducts including being incorporated to by detecting nucleotide is Ion Torrent PGM^TMOr Proton^TMSequenator (Life Technologies) is to be produced as nucleotide by detection to be incorporated to The hydrogen ion of by-product carry out the sequencing system based on ion of sequencing nucleic acid template.Normally, hydrogen ion be released as by Existing nucleotide is incorporated to by-product during polymerase-mediated template-dependent nucleic acid synthesis.Ion Torrent PGM^TMOr Proton^TMSequenator is incorporated to by detecting the hydrogen ion by-product that nucleotide is incorporated to detect nucleotide.Ion Torrent PGM^TMOr Proton^TMSequenator may include it is multiple to be sequenced nucleic acid-templated, wherein each template is placed in array respectively Sequencing reaction hole in.The hole of array can respectively be coupled at least one ion transducer, and the sensor can detect as core The H that the by-product that thuja acid is incorporated to generates⁺The release of ion or the variation of pH value of solution.Ion transducer includes being coupled to and can sensing H⁺The field-effect transistor (FET) of the ion-sensitive detection layers of the presence of ion or the variation of pH value of solution.Ion transducer can The output signal that is incorporated to of instruction nucleotide is provided, is represented by voltage change, the magnitude of the voltage change and respective hole or H in reative cell⁺Ion concentration is related.Different nucleotide types can be continuously flowed into reative cell, and can pass through polymerization Enzyme is incorporated to extension primer (or polymerization site) with the sequence determined by the sequence of template.Each nucleotide is incorporated to can be along with H in reacting hole⁺The release of ion and the parallel variation of local pH.H⁺The release of ion can be recorded by the FET of sensor, production The signal that nucleotide is incorporated to occurs for raw instruction.The nucleotide not being incorporated into specific nucleotide stream can not generate signal. The wave amplitude of signal from FET can also be related to the quantity for the certain types of nucleotide for being incorporated to extension nucleic acid molecules, so as to permit Perhaps parsing homopolymer region.Therefore, during the operation of sequenator, into multiple nucleotide streams of reative cell and a large amount of The permissible equipment of monitoring that is incorporated to of hole or reacting chamber space simultaneously parses many nucleic acid-templated sequences.About Ion Torrent PGM^TMOr Proton^TMComposition, design and the other details of operation of sequenator are found in such as U.S. Patent Application Serial 12/002781, now it is disclosed as U.S. Patent Publication No. 2009/0026082；U.S. Patent Application Serial 12/474897, it is existing It is disclosed as U.S. Patent Publication No. 2010/0137143；With U.S. Patent Application Serial 12/492844, it is now disclosed as the U.S. In patent publication No. 2010/0282617, above-mentioned reference is incorporated herein by reference in their entirety.

Figure 28 shows the block diagram of the component of the system for the nucleic acid sequencing according to exemplary implementation.Component Flow cell 101, reference electrode 108, multiple reagents 114, the valve body for sequencing being included on IC apparatus 100 116th, solution 110, valve 112, fluid control 118, circuit 120/122/126, channel 104/109/111, waste material container are washed 106th, array control unit 124 and user interface 128.IC apparatus 100 includes covering micropore battle array on an array of sensors Row 107, the sensor array include chemical sensor as described in this article.Flow cell 101 includes entrance 102, outlet 103 and the flow chamber 105 of reagent flow path that is defined on microwell array 107.

Reference electrode 108 can have any appropriate type or shape, including with flow channel or insertion channel 111 Chamber in conducting wire coaxial clyinder.Can reagent 114 be driven by flowing road by pump, air pressure or other appropriate methods Diameter, valve and flow cell 101, and can be discharged it in waste material container 106 behind the outlet 103 for leaving flow cell 101.Fluid Operation of the appropriate software control for the driving force of reagent 114, valve 112 and valve body 116 can be used in controller 118.

Microwell array 107 includes the array of reaction zone as described in this article, herein also referred to as micropore, can grasp Make ground chemical sensor joint corresponding with sensor array.For example, each reaction zone, which can be coupled to, is suitable for detection instead Answer the chemical sensor of the purpose analyte or reaction property in area.Microwell array 107 can be integrated into IC apparatus In 100, so that microwell array 107 and sensor array are the parts of single device or chip.

Flow cell 101 can control path and flow rate of the reagent 114 on microwell array 107 with a variety of Structure.Array control unit 124 provides bias voltage and timing and control signal for reading for IC apparatus 100 The chemical sensor of sensor array.Array control unit 124 is also provided with reference to bias voltage for reference electrode 108 with to flowing through 114 biasing of reagent of microwell array 107.

During the experiment, what the chemical sensor from sensor array was collected and processed to array control unit 124 passes through collection Output signal into the output port on circuit device 100 Jing Guo bus 127.Array control unit 124 can be computer or its Its calculating instrument.Array control unit 124 may include memory for storing data and application software, for accessing data With the processor and the component of promotion and the communication of the various assemblies of the system in Figure 28 for performing application.

The value of the output signal of chemical sensor indicates one occurred in the correspondence reaction zone in microwell array 107 Or the physically and/or chemically parameter of multiple reactions.For example, it in an exemplary embodiment, can be used public in following reference The value of technical finesse output signal opened：The U.S. Patent Application No. 13/ that Rearick et al. was submitted on December 29th, 2011 339,846, the U.S. Provisional Patent Application No. 61/428,743 submitted based on December 30th, 2010 and on January 3rd, 2011 The U.S. Provisional Patent Application No. 61/429,328 and Hubbell of submission are in the United States Patent (USP) submitted on December 29th, 2011 Application number 13/339,753, the U.S. Provisional Patent Application No. 61/428,097 submitted based on December 29th, 2010.

User interface 128 can be shown about flow cell 101 and received from the sensor array on IC apparatus 100 In chemical sensor output signal information.User interface 128 may also display equipment and be set and controlled and allow user defeated Enter or equipment is set to be set and controlled.

In an exemplary embodiment, fluid control 118 can be by individual reagent 114 to flow cell 101 during the experiment Delivering with IC apparatus 100 is controlled in predetermined order, predetermined duration, predetermined stream Speed.Array control unit 124 then can collect and analyze the chemistry of chemical reaction that instruction occurs in response to the delivering of reagent 114 The output signal of sensor.

During the experiment, the temperature of IC apparatus 100 can be also monitored and controlled in system, so as to known advance It reacts and measures at determining temperature.

Configurable system is so that single fluid or reagent contact reference electrode in entire multistep reaction during operation 108.Valve 112 can be closed to prevent any washing solution 110 when reagent 114 flows in flow channel 109.Although washing is molten The flowing of liquid can be prevented from, but continuous stream can be still had between reference electrode 108, channel 109 and microwell array 107 Body and telecommunication.The distance between tie point between reference electrode 108 and channel 109 and 111 may be selected, so as to no or pole A small amount of flowing in channel 109 and the reagent possibly diffused in channel 111 reach reference electrode 108.In exemplary reality It applies in scheme, washing 110 selected as of solution can be continuously contacted with reference electrode 108, this can be particularly used for using frequent The multistep reaction of washing step.

Figure 29 shows the sectional view and expanded view of the part of IC apparatus 100 and flow cell 101.During operation, The flow chamber 105 of flow cell 101 closes the reagent through delivering for the open end for flowing through the reaction zone in microwell array 107 Reagent flow 208.Property that can be based on the reaction of generation and the labelling technique (if any) or reagent, by-product used To select the volume of reaction zone, shape, length-width ratio (such as bottom width is to hole depth rate) and other size characteristics.

Relevant reaction in chemical sensor sensing (and generating output signal) microwell array 107 of sensor array 205 Chemical reaction in area is analyzed with testing goal or or reaction property.Can be for example of chemical sensor of sensor array 205 Learn sensibility field-effect transistor (chemFET), such as ion-sensitive field-effect transistor (ISFET).Available for implementing The chemical sensor of scheme and the example of array structure are described in U.S. Patent Application Publication No. 2010/0300559,2010/ 0197507th, 2010/0301398,2010/0300895,2010/0137143 and 2009/0026082 and U.S. Patent number 7, In 575,865, each above-mentioned reference is incorporated herein by reference.

Figure 30 is shown according to two of exemplary implementation representative chemical sensors and its corresponding reaction zone Sectional view.In fig. 30, it is shown that two chemical sensors 350,351 represent the biography that may include millions of chemical sensors The sub-fraction of sensor array.In some embodiments, sensor array may include at least 1,000,000 chemical sensors and Optionally at least 1,000,000 corresponding reaction zones, at least 1,000 ten thousand chemical sensors are corresponding with optionally at least 1,000 ten thousand Reaction zone, at least 100,000,000 chemical sensors and optionally at least 100,000,000 corresponding reaction zones, at least 500,000,000 chemical sensitisations Device and optionally at least 500,000,000 corresponding reaction zones or at least 1,000,000,000 chemical sensors are corresponding with optionally at least 1,000,000,000 Reaction zone.

Chemical sensor 350 is coupled to corresponding reaction zone 301, and chemical sensor 351 is coupled to corresponding reaction zone 302.Chemical sensor in 350 representative sensor array of chemical sensor.In the example of display, chemical sensor 350 is Ion-sensitive field-effect transistor.Chemical sensor 350 includes floating gate structure 318, and the floating gate structure 318 has The floating gate conductor (herein referred to as sensor board 320) detached by sensing material 316 with reaction zone 301.Such as figure It is shown in 30, the uppermost figure of the conductive material in floating gate structure 318 of the sensor board 320 below reaction zone 301 Pattern layer.

In the example of display, floating gate structure 318 is included in the more of the conductive material in the layer of dielectric substance 319 A pattern layer.As described in greater detail below, the upper surface of sensing material 316 is used as the sensing surface of chemical sensor 350 317。

In the embodiment of display, sensing material 316 is ion-sensitive material, so as to ion or other charge kinds Presence in solution of the class in reaction zone 301 changes the surface potential of sensing surface 317.The variation of surface potential is because molten Caused by the protonation or deprotonation of surface charge group present in liquid caused by ion at sensing surface.Sensing Material 316 can be placed in by using multiple technologies or manufacture works for forming the one or more of chemical sensor 350 During skill natural feature into.In some embodiments, sensing material 316 is metal oxide, for example, silicon, tantalum, aluminium, lanthanum, The oxide of titanium, zirconium, hafnium, tungsten, palladium, iridium etc..

In some embodiments, sensing material 316 is the oxide on the upper strata of the conductive material of sensor board 320.Example Such as, the upper strata of sensor board 320 can be titanium nitride, and sensing material 316 may include titanium oxide or titanium oxynitrides.More generally, Sensing material 316 may include the one or more of a variety of different materials to promote the sensibility to specific ion.For example, nitridation Silicon or silicon oxynitride and metal oxide such as silica, aluminium oxide or tantalum oxide are usually provided to hydrionic sensitivity Property, however the sensing material comprising the polyvinyl chloride with valinomycins provides the sensibility to potassium ion.Depending on instrument, The material to other ions (such as sodium, silver, iron, bromine, iodine, calcium and nitrate) sensitivity also can be used.

Chemical sensor 350 further includes source area 321 and drain region 322 in Semiconductor substrate 354.321 He of source area Drain region 322 includes the semi-conducting material of doping, has the conductivity type different from the conductivity type of substrate 354.Example Such as, source area 321 and drain region 322 may include the P- type of semiconductor material of doping, and substrate may include the N- types half of doping Conductor material.

Slot area 323 detaches source area 321 and drain region 322.Floating gate structure 318 is overlayed on slot area 323, and is led to Gate-dielectric 352 is crossed to detach with substrate 354.Gate-dielectric 352 can be for example silica.Alternatively, other dielectrics Available for gate-dielectric 352.

As shown in Figure 30, reaction zone 301 extends through the packing material 310 on dielectric substance 319.Fill material Material 310 can be for example comprising one or more layers dielectric substance such as silica or silicon nitride.

The scale (such as width and depth) and its pitch (centre-to-centre spacing between adjacent reaction area of reaction zone 301,302 From) can between instrument and instrument it is different.In some embodiments, reaction zone, which can have, is no more than 5 microns, such as do not surpass 3.5 microns are crossed, no more than 2.0 microns, no more than 1.6 microns, no more than 1.0 microns, no more than 0.8 micron, no more than 0.6 Micron, no more than 0.4 micron, the characteristic diameter no more than 0.2 micron or no more than 0.1 micron, the diameter is multiplied by flat by 4 The square root of face figure cross-sectional area (A) divided by Pi (for example, sqrt (4*A/ π)) defines.

In some embodiments, the pitch between adjacent reaction area is no more than 10 microns, no more than 5 microns, is no more than 2 microns, no more than 1 micron or no more than 0.5 micron.

In the embodiment of display, reaction zone 301,302 is detached with being equal to the distance of its width.It is alternatively, adjacent anti- The separating distance between area is answered to be smaller than its width.For example, separating distance can be used to be used for forming reaction zone 301,302 Technique (such as litho technique) minimum feature size.In this case, separation directly may significantly be less than that a The width in precursor reactant area.

Sensor board 320, sensing material 316 and reaction zone 301 can be for example with circular cross sections.Alternatively, these can be with It is non-circular.For example, cross section can be square, rectangle, hexagon or irregular shape.

Depending on wherein realizing the device and array structure of chemical sensor described herein, the device in Figure 30 is also It may include other element such as accessing in the array line of chemical sensor (such as wordline, bit line), substrate 354 Other doped region and other circuits (such as access circuit, biasing circuit etc.) for operating chemical sensor.In some realities It applies in scheme, it can be for example using U.S. Patent Application Publication No. 2010/0300559,2010/0197507,2010/ 0301398th, 2010/0300895,2010/0137143 and 2009/0026082 and U.S. Patent number 7,575,865 (it is every One is incorporated herein by reference) described in technology process units.

In operation, reactant, washing solution and other reagents can be moveable into and out reaction zone by flooding mechanism 340 301.Chemical sensor 350 senses (and generating relevant output signal) and is present in the sensing material opposite with sensor board 320 The amount of charge 324 on 316.The variation of charge 324 causes the voltage change on floating gate structure 318, and then causes crystal The variation of the threshold voltage of pipe.It can be surveyed by measuring the electric current in the slot area 323 between source area 321 and drain region 322 Measure the variation of the threshold voltage.Therefore, chemical sensor 350, which can be used directly for providing, is connected to source area 321 or drain region Output signal based on electric current on 322 array line is provided using other circuit based on the defeated of voltage indirectly Go out signal.

In embodiments, the reaction carried out in reaction zone 301 can identify or measure purpose analyte The analysis reaction of characteristic or property.Such reaction can either directly or indirectly have an impact the charge near sensor board 320 The by-product of amount.If such by-product generate in a small amount decay rapidly or with other component reactions, can simultaneously anti- Multiple copies that same analyte is analyzed in area 301 are answered, to increase the output signal generated.In embodiments, can will divide Multiple copies of analysis object are connected to solid support 312 before or after being placed in reaction zone 301.Solid support 312 can To be particle, nano particle, globule, solid or porous including gel etc..It is supported for simplicity with ease of explanation, solid phase Object 312 is also referred to as particle herein.For nucleic acid analyte, the copies of multiple connections can by rolling circle amplification (RCA), Index RCA, recombinase polymeric enzymatic amplification (RPA), PCR amplification (PCR), emulsion PCR amplification or similar skill Prepared by art, to generate amplicon without solid support.

In multiple exemplary implementations, method described herein, system and computer-readable medium can be advantageous Ground is used to handling and/or analyzing the data and signal obtained from the nucleic acid sequencing based on electronics or charge.Based on electronics or charge Sequencing (for example, sequencing based on pH) in, can the day of polymerase catalysed nucleotide extension be generated as by detection The ion (such as hydrogen ion) of right by-product measures nucleotide and incoming event.This can be used for sequencing sample or template nucleic acid, It can be the segment of such as purpose nucleic acid sequence, and it either directly or indirectly can be connected to solid branch as clone group Object is held such as particle, particle, globule.Sample or template nucleic acid can operationally associate simultaneously can be through to primer and polymer (it can be referred to " nucleotide stream " herein, should " nucleotide for the nucleotide addition for going through repetition and the cycle washed or " stream " Stream " can cause nucleotide to be incorporated to).Primer can be annealed to sample or template so as to whenever addition and next base in template 3 ' ends of primer can be extended by polymerase during the nucleotide of complementation.It sequence subsequently, based on known nucleotide stream and is based on The output signal of ion concentration during each nucleotide stream of instruction of the chemical sensor of measurement, can measure and be present in idol The information of the type of nucleotide, sequence and quantity that the sample nucleic acid being coupled in the reaction zone of chemical sensor associates.

It, can be by detecting by polymerase catalysed extension in the embodiment of the nucleic acid sequencing typically based on ion It reacts the hydrionic presence generated and/or concentration is incorporated to detect nucleotide.It in one embodiment, can will optionally It is loaded with the template that sequencing primer and/or polymerase are combined in advance to reative cell (such as in Rothberg cited herein et al. Disclosed micropore) in, followed by the nucleotide addition repeated and the cycle of washing.In some embodiments, in this way Template solid support such as particle, globule or similar can be connected to as clone group, and the clone group is loaded into anti- It answers in room.

In another embodiment, it will be allocated in, be placed in or be positioned at array optionally in combination with the template to polymerase Different loci.The site of array includes primer and method may include being hybridized to different templates in different sites Primer.

In each addition step of cycle, only when next base in template is the complementation of the nucleotide of addition During object, polymerase can be by being incorporated to the nucleotide of addition come extension primer.If there is a complementary base, then there are one It is incorporated to, if there are two, there are two to be incorporated to, if there are three, it is incorporated to, etc. there are three.For each in this way Be incorporated to, there are the hydrogen ion of release, and jointly release hydrogen ions template group change reative cell local pH.Hydrogen from The quantity of continuous complementary base (and participates in the template point of extension with primer and polymer in the generation of son and template The total quantity of son) it is monotonically correlated.Therefore, when in template there are during many consecutive identical complementary base (i.e. homopolymer regions), The hydrionic quantity of generation and the thus magnitude of caused local pH variation, can be with the quantity of consecutive identical complementary base It is proportional.If the next base and the nucleotide of addition in template be not complementary, then is incorporated to and does not occur and do not discharge hydrogen Ion.In some embodiments, after the step of each addition nucleotide, additional step can be carried out, wherein will be Under predetermined pH without buffering washing solution be used for remove the nucleotide of previous steps with prevent Posterior circle in Mistake mixes.In some embodiments, after the step of each addition nucleotide, additional step can be carried out, wherein With nucleotide destroy reagent such as apyrase process chamber with remove be retained in it is indoor any remaining Nucleotide, these nucleotide can lead to false extension in subsequent cycle.

In an exemplary embodiment, different types of nucleotide is continuously added to reative cell, so as to each Reaction can be exposed to different nucleotide one at a time.For example, nucleotide can be added by following sequence：dATP,dCTP, DGTP, dTTP, dATP, dCTP, dGTP, dTTP, etc.；Washing step is carried out after exposing each time.Depending on desired sequence The length of column information repeats cycle 50 times, 100 times, 200 times, 300 times, 400 times, 500 times, 750 times or more times.

It in some embodiments, can be according to PGM^TMOr Proton^TMThe user's manual that sequenator provides is sequenced. Embodiment 3 provides to use Ion Torrent PGM^TMSequenator (Ion Torrent^TM Systems,Life Technologies, CA) the sequencing based on ion an exemplary arrangement.

In some embodiments, the method that present disclosure generally relates to sequencing template polynucleotides group, packet It includes：(a) multiple amplicons are generated by expanding multiple template polynucleotides with cloning on multiple surfaces, wherein reacting Carried out in the single continuous phase of mixture the amplification and wherein generated amplicon at least 10%, 20%, 30%, 40%th, 50%, 60%, 70%, 80%, 90% or 95% inherently substantially monoclonal.In some embodiments In, the amplicon of sufficient amount of substantially monoclonal is generated in single amplified reaction in Ion Torrent PGM^TM314th, at least 100MB, 200MB, 300MB, 400MB, 500MB, 750MB, 1GB or 2GB are generated on 316 or 318 sequenators AQ20 sequencing reading.As used herein, term " AQ20 " and its variant refer in Ion Torrent PGM^TMIn sequenator Measure the ad hoc approach of sequencing accuracy.Accuracy of measurement, the Phred- samples Q it can be divided in a manner of Phred- sample Q scores Number accuracy of measurement in logarithmic scale：Q10=90%, Q20=99%, Q30=99.9%, Q40=99.99% and Q50= 99.999%.For example, in specific sequencing reaction, by prediction algorithm or the reality with known reference gene group can be passed through It compares and carrys out accuracy in computation measurement.The mass fraction (" Q scores ") of prediction can derive from such algorithm：It considers input letter Number intrinsic property and give whether single base by alignment fairly accurate comment for being included in sequencing " reading " Valency.In some embodiments, the mass fraction of such prediction can be used for filtering and removal low-quality before downstream compares Measure reading.In some embodiments, reporting accuracy, the Phred- samples Q can be divided in a manner of Phred- sample Q scores Number accuracy of measurement in logarithmic scale：Q10=90%, Q17=98%, Q20=99%, Q30=99.9%, Q40= 99.99% and Q50=99.999%.In some embodiments, can will be obtained from the data filtering of given polymeric enzyme reaction with Only measurement measure " N " a nucleotide or it is longer and be more than certain threshold value such as Q10, Q17, Q100 (herein referred to as " NQ17 " score) Q scores polymerase reading.For example, 100Q20 scores may indicate that is in length obtained from given reaction The quantity of at least 100 nucleotide and the reading with Q20 (99%) or higher Q scores.Similarly, 200Q20 scores The quantity for being at least 200 nucleotide and the reading with Q20 (99%) or higher Q scores is may indicate that in length.

In some embodiments, can also based on the appropriate of reference gene group sequence is used to compare come accuracy in computation, Herein referred to as " original " accuracy.This is single-pass accuracy (single pass accuracy), including with it is single The measurement of the relevant "true" per base mistake of reading, this is with shared accuracy on the contrary, shared accuracy measurement comes from shared sequence The error rate of row is the result of multiple readings.It can be reported in a manner of " AQ " (writing a Chinese character in simplified form for " quality of comparison ") score Original accuracy measurement.In some embodiments, the data filtering of given polymeric enzyme reaction can be will be obtained from only to measure measurement " N " a nucleotide or it is longer and be more than certain threshold value such as AQ10, AQ17, AQ100 (herein referred to as " NAQ17 " Score) AQ scores polymerase reading.For example, 100AQ20 scores may indicate that obtained from given polymeric enzyme reaction in length Upper is the quantity of at least 100 nucleotide and the reading with AQ20 (99%) or higher AQ scores.Similarly, 200AQ20 scores may indicate that in length to be at least 200 nucleotide and with AQ20 (99%) or higher AQ scores The quantity of reading.

In some embodiments, this teachings provides the system for nucleic acid amplification, and the system includes following Any combinations：It is connected with globule, the second primer, third primer, polynucleotides, recombinase, the recombinase of multiple the first primers It is loaded into albumen, single strand binding protein (SSB), polymerase, nucleotide, ATP, phosphocreatine, creatine kinase, hybridization solution and/or washes Wash solution.System may include all or some of these components.In some embodiments, for the system of nucleic acid amplification also It may include any combinations of buffer solution and/or cation (such as bivalent cation).

In some embodiments, this teachings provides the kit for nucleic acid amplification.In some embodiments In, kit includes any reagent available for nucleic acid amplification.In some embodiments, kit may include following appoint What is combined：Globule, the second primer, third primer, polynucleotides, recombinase, the recombinase for being connected with multiple the first primers are loaded into Albumen, single strand binding protein (SSB), polymerase, nucleotide, ATP, phosphocreatine, creatine kinase, hybridization solution, washing solution, Buffer solution and/or cation (such as bivalent cation).Kit may include all or some of these components.

In some embodiments, present disclosure generally relates to parallel in multiple divided reaction capacities Ground expands method, composition, the system of different nucleic acid-templated (such as opposite with the amplification in single Continuous Liquid Phase).Example Such as, nucleic acid-templated distribution extremely or can be placed in the array of reative cell or the array of reaction capacity, so as at least two in array A such room or capacity respectively receive single nucleic acid-templated.In some embodiments, the reaction of multiple separation is formd Capacity.Closed reaction chamber's (or reaction capacity) optionally before amplification.It in another embodiment, can be mixed by reaction Object is closed to divide or detach to being dispersed in multiple microreactors in the continuous phase of emulsion.Divided or separation reaction capacity It is not optionally mixed with each other or connects or can not be mixed with each other or connect.In some embodiments, at least some reactions Room (or reaction capacity) includes recombinase and optionally polymerase.Polymerase can be strand displacement polymerase.

In some embodiments, present disclosure generally relate to nucleic acid synthesis and/or amplification comprising emulsion Composition, system, method, apparatus and kit.As used herein, term " emulsion " is including including the first liquid and second Any composition of the mixture of liquid, wherein the first and second liquid are substantially immiscible each other.Normally, liquid One kind be hydrophilic and other liquid be hydrophobic.Normally, emulsion includes dispersed phase and continuous phase.For example, First liquid can form the dispersed phase being dispersed in second liquid, and the second liquid forms continuous phase.The optional landlord of dispersed phase It to be made of the first liquid.Continuous phase is optionally mainly made of second liquid.In various embodiments, identical two kinds Liquid can form different types of emulsion.For example, the mixture comprising oil and water can be initially formed oil-in-water emulsion, wherein Oil is dispersed phase, and water is decentralized medium.Secondly, they can form water-in-oil emulsion, and wherein water is dispersed phase, and oil is foreign minister. Multiple emulsion is also possible, including " W/O/W " emulsion and " Water-In-Oil packet oil " emulsion.In some embodiments, Dispersed phase includes one or more wherein nucleic acid-templated microreactors that can be amplified eachly.One or more microreactors The divided reaction capacity for the amplified reaction that can wherein detach can be formed.For the appropriate medium of nucleic acid amplification An example include water-in-oil emulsion, wherein based on water mutually comprising it is several be dispersed in it is aqueous micro- in the oil phase of emulsion Reactor.In some embodiments, emulsion also may include emulsifier or surfactant.Emulsifier or surfactant can For the stable emulsion under nucleic acid synthesizing conditions.

In some embodiments, present disclosure is usually directed to the composition comprising emulsion, and the emulsion includes reaction Mixture.Emulsion may include water phase.Water phase is dispersed in the continuous phase of emulsion.Water phase may include one or more micro- reactions Device.In some embodiments, reaction mixture is included in multiple liquid phase microreactors in the phase of emulsion.Optionally, instead Mixture is answered to include recombinase.Optionally, reaction mixture includes multiple and different polynucleotides.Optionally, reaction mixing Object includes multiple supports.Optionally, reaction mixture includes recombinase, multiple and different polynucleotides and/or multiple supports Any combinations of object.Optionally, at least one support is connectable to the nucleic acid group of substantially monoclonal.

In some embodiments, present disclosure is usually directed to the composition comprising reaction mixture, and the reaction is mixed It closes object and includes (i) multiple supports, (ii) multiple and different polynucleotides and (iii) recombinase, reaction mixture is included in breast In multiple liquid phase microreactors in agent.

In some embodiments, present disclosure is usually directed to the composition comprising reaction mixture, and the reaction is mixed It closes object and includes (i) recombinase and (ii) multiple supports, at least one support is connectable to the nucleic acid of substantially monoclonal Group, wherein reaction mixture are included in multiple liquid phase microreactors in emulsion.

Optionally, emulsion includes aqueous favoring.

Optionally, emulsion includes the aqueous favoring being dispersed in hydrophobic phase.For example, emulsion may include water-in-oil emulsion.

In some embodiments, aqueous favoring includes multiple microreactors.

Optionally, reaction mixture is included in single reaction vessel.

Optionally, the sequence of multiple and different polynucleotides can be identical or different.

Optionally, at least one of multiple supports is connected to multiple the first primers (such as positive amplimer).

Optionally, reaction mixture also includes multiple second primers (such as reversed amplimer).

In some embodiments, at least one of multiple supports also includes multiple second primers.

In some embodiments, at least one of multiple supports includes multiple first and second primers.

In some embodiments, the first and second primers include identical sequence.In some embodiments, first Different sequences is included with the second primer.

In some embodiments, support includes the inner wall of globule, particle, plane surface or slot or pipe.

In some embodiments, reaction mixture also includes polymerase and multiple nucleotide.

In some embodiments, present disclosure is usually directed to the composition comprising emulsion.Optionally, emulsion includes Aqueous favoring and hydrophobic phase.Optionally, emulsion includes the aqueous favoring being dispersed in hydrophobic phase.Optionally, aqueous favoring may include multiple Any combinations of polynucleotide template, multiple supports and/or recombinase.Optionally, aqueous favoring may include multiple polynucleotides Template.Optionally, aqueous favoring may include multiple supports.Selection of land, aqueous favoring may include recombinase.

In some embodiments, composition includes emulsion, and the emulsion includes aqueous favoring and hydrophobic phase, wherein hydrophilic Mutually include multiple polynucleotide templates, multiple supports and recombinase.

In some embodiments, present disclosure is usually directed to the composition comprising emulsion, and the emulsion includes dispersion Aqueous favoring in hydrophobic phase.Optionally, aqueous favoring includes multiple microreactors.Optionally, at least two in multiple are micro- anti- Device is answered to include different polynucleotide templates.Optionally, the sequence of different polynucleotide templates is identical or different.Appoint Selection of land, the first microreactor includes the first polynucleotide template and the second microreactor includes the second polynucleotide template. Optionally, the first and second polynucleotide templates include identical or different sequence.Optionally, at least two in multiple are micro- Reactor includes recombinase.

In some embodiments, composition includes emulsion, and the emulsion includes the aqueous favoring being dispersed in hydrophobic phase, Wherein aqueous favoring include multiple microreactors, it is multiple at least two microreactors include different polynucleotide templates and Recombinase.

In some embodiments, aqueous favoring includes multiple aqueous microreactors, and at least two of microreactor respectively wraps Containing different polynucleotide templates, support and recombinase.

Optionally, the first microreactor includes the first polynucleotide template and the second microreactor includes the second multinuclear Thuja acid template.Optionally, the first and second polynucleotide templates include identical or different sequence.

In some embodiments, the first and second primers include identical sequence.

In some embodiments, the first and second primers include different sequences.In some embodiments, it is hydrophilic Mutually also include polymerase.

In some embodiments, polymerase includes strand displacement polymerase.In some embodiments, aqueous favoring includes Nucleotide.

In some embodiments, present disclosure generally relates to nucleic acid synthetic method (and relevant composition And system), including：(a) reaction mixture is formed；(b) reaction mixture is made to undergo amplification condition.Optionally, reaction mixing Object is included in the aqueous favoring of emulsion.Optionally, emulsion includes aqueous favoring and hydrophobic phase.Optionally, emulsion is thin comprising being dispersed in Aqueous favoring in water phase.Optionally, reaction mixture includes multiple supports, multiple and different polynucleotides and/or recombination Any combinations of enzyme.Optionally, reaction mixture includes multiple supports.Optionally, reaction mixture includes multiple and different Polynucleotides.Optionally, the sequence of different polynucleotide templates is identical or different.Optionally, the first microreactor packet The second polynucleotide template is included containing the first polynucleotide template and the second microreactor.Optionally, more than first and second Oligonucleotide template includes identical or different sequence.Optionally, reaction mixture includes recombinase.Optionally, amplification condition packet Include isothermal or thermal cycling temperature condition.Optionally, method further include to be formed at least two supports make emulsion undergo amplification condition Multiple supports are resulted in, wherein at least two of support are connected to the nucleic acid group of substantially monoclonal each independently.

In some embodiments, present disclosure generally relates to nucleic acid synthetic method (and relevant composition And system), including：(a) reaction mixture for including multiple supports, multiple and different polynucleotides and recombinase is formed, instead Mixture is answered to be included in the aqueous favoring of emulsion；(b) make the emulsion experience isothermal duplication condition comprising reaction mixture, from And it generates multiple supports and is connected to the nucleic acid group of its substantially monoclonal.

In some embodiments, emulsion includes water-in-oil emulsion.In some embodiments, liquid phase microreactor Include aqueous favoring.In some embodiments, emulsion includes the aqueous favoring being dispersed in hydrophobic phase.In some embodiments, Reaction mixture is formed in single reaction vessel.Optionally, the sequence of multiple and different polynucleotide templates is identical or not With.Optionally, the first polynucleotide template includes First ray and the second polynucleotide template includes the second sequence.Appoint Selection of land, the first and second polynucleotide template sequences are identical or different.Optionally, at least one company of multiple supports It is connected to multiple the first primers (such as positive amplimer).Optionally, reaction mixture also comprising multiple second primers (such as Reversed amplimer).In some embodiments, at least one of multiple supports also includes multiple second primers.One In a little embodiments, at least one of multiple supports includes multiple first and second primers.In some embodiments, One and second primer include identical sequence.In some embodiments, the first and second primers include different sequences. In some embodiments, nucleic acid synthesis methods further include from reaction mixture recycling is at least some and are connected to substantially nucleic acid list Clone the support of group.In some embodiments, nucleic acid synthesis methods are further included is connected at least some substantially singly The support of the nucleic acid group of clone is placed on the surface.In some embodiments, nucleic acid synthesis methods are further included by near The support of some few nucleic acid groups for being connected to substantially monoclonal is placed on surface to form array.In some embodiment party In case, nucleic acid synthesis methods further include the nucleic acid group that at least one substantially monoclonal for being connected to support is sequenced.At some In embodiment, support includes the inner wall of globule, particle, plane surface or slot or pipe.In some embodiments, it reacts Mixture also includes polymerase and multiple nucleotide.In some embodiments, polymerase includes strand displacement polymerase.

In some embodiments, include forming emulsion for nucleic acid synthetic method.Optionally, emulsion includes hydrophilic Phase and hydrophobic phase.Optionally, emulsion includes the aqueous favoring being dispersed in hydrophobic phase.Optionally, aqueous favoring includes multiple micro- reactions Device.Optionally, at least two microreactors in multiple include individual polynucleotide template.Optionally, at least two in multiple A microreactor includes different polynucleotide templates.Optionally, the first microreactor includes the first polynucleotide template, and And second microreactor include the second polynucleotide template.Optionally, the first and second polynucleotide templates have identical or not Same sequence.Optionally, at least two microreactors in multiple include recombinase.

In some embodiments, present disclosure generally relates to nucleic acid synthetic method (and relevant composition And system), including：The emulsion for including the aqueous favoring being dispersed in hydrophobic phase is formed, the aqueous favoring includes multiple micro- reactions Device, it is multiple at least two microreactors include different polynucleotide template and recombinase.

In some embodiments, emulsion includes water-in-oil emulsion.In some embodiments, aqueous favoring also includes Polymerase.In some embodiments, polymerase is strand displacement polymerase.In some embodiments, aqueous favoring includes nucleosides Acid.In some embodiments, emulsion is formed in single reaction vessel.Optionally, the sequence of different polynucleotide templates It is identical or different.Optionally, the first microreactor includes the first polynucleotide template and the second microreactor includes Second polynucleotide template.Optionally, the first and second polynucleotide templates include identical or different sequence.In some realities Apply in scheme, it is multiple at least two microreactors include multiple supports.Optionally, multiple supports is at least one It is connected to multiple the first primers (such as positive amplimer).Optionally, reaction mixture also includes multiple second primer (examples Such as reversed amplimer).In some embodiments, at least one of multiple supports also includes multiple second primers.One In a little embodiments, at least one of multiple supports includes multiple first and second primers.In some embodiments, One and second primer include identical sequence.In some embodiments, the first and second primers include different sequences. In some embodiments, aqueous favoring includes reaction mixture.In some embodiments, reaction mixture includes multiple multinuclears Thuja acid template, multiple supports and recombinase.In some embodiments, being further included for nucleic acid synthetic method makes emulsion (such as including reaction mixture) undergoes isothermal duplication condition, so as to generate the nucleic acid group of multiple substantially monoclonals.At some In embodiment, the nucleic acid group of multiple substantially monoclonals is connected to multiple supports.In some embodiments, nucleic acid closes It is further included into method from reaction mixture and recycles at least some supports for being connected to substantially nucleic acid monoclonal group.At some In embodiment, nucleic acid synthesis methods, which further include, puts at least some supports of nucleic acid group for being connected to substantially monoclonal It puts on the surface.In some embodiments, nucleic acid synthesis methods are further included by being connected at least some substantially singly The support of the nucleic acid group of clone is placed on surface to form array.In some embodiments, nucleic acid synthesis methods also wrap Include the nucleic acid group that at least one substantially monoclonal for being connected to support is sequenced.In some embodiments, support includes The inner wall of globule, particle, plane surface or slot or pipe.In some embodiments, reaction mixture is also comprising polymerase and more A nucleotide.In some embodiments, polymerase includes strand displacement polymerase.

Paragraph heading used herein, which is only used for organizational goal, should not be construed as limitation description Theme.

All documents and similar material quoted in the application, including but not limited to patent, patent application, article, book Nationality, paper and internet page are integrally incorporated herein explicitly by reference for any purpose.When the reference money being incorporated to It, should be to be provided in this teachings when the definition of term in material seems different from the definition provided in this teachings Definition subject to.

It will be understood that there is implicit " about " before the temperature discussed in this teachings, concentration, number etc., therefore light Micro- and unsubstantiality difference is in the range of this teachings.

Unless the context otherwise requires, the term comprising plural number and plural number will be included odd number by singular term.

Term "comprising", " comprising ", " containing ", " having ", " carrying " and " having " are not intended to be restricted.

It will be understood that foregoing general description and detailed description below are only exemplary and explanatory and do not limit The present invention.

Unless otherwise defined, the scientific and technical terms relatively used with described herein teachings will have Have by the normally understood meaning of those skilled in the art.Normally, with cell and tissue culture, molecular biology and albumen The nomenclature and its technology relatively used with widow-or more-nucleotide chemistry and hybridization is known in the art and usually used 's.Standard technique is for example for nucleic acid purification and preparation, chemical analysis, recombinant nucleic acid and oligonucleotide synthesis.According to manufacture The specification of quotient carries out as usually realized in this field or as described in this article enzymatic reaction and purification technique.Usually According to conventional method as known in the art and such as reference in entire this specification and the various generality discussed and more specifically Technology and methods described herein are carried out described in reference.See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual(Third ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.2000).Nomenclature used in connection with and laboratory method described herein and skill Art is known in the art and usually used.

As according to used in exemplary implementation provided herein, following term, should be by except as otherwise indicating It is interpreted as that there is following meaning：

As used herein, term " amplification " and its variant include at least certain part for generating polynucleotides Any process of multiple copies or complement, the polynucleotides are commonly referred to as " template ".Template polynucleotide can be single Chain or double-strand.It can lead to the generation of polynucleotide amplification product group, the polynucleotide amplification product to the amplification of solid plate Group is collectively referred to as " amplicon ".The polynucleotides of amplicon can be single-stranded or double-stranded or two kinds mixtures.Usually Ground, template will include target sequence, and generated amplicon will be included with substantially the same with target sequence or substantially mutual The polynucleotides of the sequence of benefit.In some embodiments, the polynucleotides of specific amplicon are substantially the same or base each other Complementation in sheet；Alternatively, in some embodiments, the polynucleotides in given amplicon can have nucleosides different from each other Acid sequence.Amplification can be carried out in a manner of linear or index, and may include the repetition to solid plate and continuous duplication To form two or more amplified productions.Some typical amplified reactions include based on template nucleic acid synthesis it is continuous and Repetitive cycling, leads to the formation of multiple sub- polynucleotides, the sub- polynucleotides include the nucleotide sequence of template at least certain A part and the nucleotide sequence homology (or complementary) that at least a certain degree is enjoyed with template.In some embodiments In, each nucleic acid synthesizes (it can be referred to " cycle " of amplification) including primer annealing and primer extension procedures；Optionally, It may also include the other denaturing step that wherein template is partially or completely denaturalized.In some embodiments, one expands back Close the given number of repetition for including single amplification cycles.For example, amplification bout may include particular cycle 5,10,15,20, 25th, 30,35,40,50,75,100 or more repetitions.In an exemplary embodiment, amplification includes wherein specific more Oligonucleotide template undergoes any reaction of two continuous nucleic acid synthesis cycles.Synthesis may include that template-dependent nucleic acid synthesizes. Each cycle of nucleic acid synthesis optionally includes single primer annealing step and single extension step.In some embodiments In, amplification includes isothermal duplication.

As used herein, term " contact " and its variant, when for any group of component in use, including wherein will The component for waiting to be contacted mixes into identical mixture any mistake of (for example, being added into identical compartment or solution) Journey, and the actual physics contact being not necessarily required between the component.It can in any order or any combinations (or subgroup Close) the contact component, and may include such situation：Wherein then by one of the component or some are from mixture Removal, optionally before the other components of addition.For example, " contacting A with B and C " is including under arbitrary and whole Row situation：(i) A with C is mixed, B is then added to mixture；(ii) A and B are mixed into mixture；By B from mixing Object removes and C then is added to mixture；A is added to the mixture of B and C by (iii)." by template with reacting mixing Object contacts " including arbitrary or whole following state：(i) template is contacted to generate with the first component of reaction mixture Mixture；Then by other components of reaction mixture in any order or combination be added to mixture；(ii) with template Before mixing, reaction mixture is formed completely.

As used herein, term " support " and its variant include on it can fixating reagent such as nucleic acid it is any The article of solid or semisolid.

As used herein, term " isothermal " and its variant are used when about any reaction condition of finger, process or method When, it is included in condition, process and the method carried out under conditions of substantially isothermal.Substantially the condition of isothermal includes its medium temperature Degree is confined to any condition in limited range.In an exemplary embodiment, the variation of temperature is no more than 20 DEG C, usually No more than 10 DEG C, 5 DEG C or 2 DEG C.Isothermal duplication includes carrying out at least two wherein under conditions of substantially isothermal continuously Any amplified reaction of nucleic acid synthesis cycle, and including wherein at least two continuous nucleic acid synthesis recycle it is lasting when Between during temperature variation no more than 20 DEG C, 10 DEG C, 5 DEG C or 2 DEG C of amplified reaction, although wherein in the other of amplification procedure During part, including during other nucleic acid synthesis cycle, the variation of temperature can be more than 20 DEG C.Optionally, it is anti-in isothermal Should (including isothermal duplication) in, by temperature or about 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C or 70 DEG C keep at least about 10,15,20, 30th, 45,60 or 120 minutes.Optionally, in one or more amplification cycles (for example, 1,5,10,20 or all carried out Amplification cycles) during, any temperature variation is no more than 20 DEG C, optionally within 10 DEG C, such as within 5 DEG C or 2 DEG C. In some embodiments, isothermal duplication may include thermal cycle, and wherein temperature change is within the scope of isothermal.In instances, it is denaturalized Temperature change between step and another step are for example annealed and/or extended is restricted.In instances, for one or Multiple amplification cycles, the difference between denaturation temperature and annealing or elongating temperature is no more than 20 DEG C, optionally within 10 DEG C, Such as within 5 DEG C or 2 DEG C.Optionally, temperature at least 5,10,15,20,30, the 35 or substantially all cycles in amplification Variation is limited.

As used herein, term " sequencing " and its variant are included commonly by least one in measure nucleic acid molecules The information of a little nucleotide (including its nucleobase component) obtains sequence information from nucleic acid chains.And in some embodiments, The given area of " sequencing " nucleic acid molecules includes each nucleotide in the region that identification is sequenced, and " sequencing " may also include The information of wherein one or more nucleotide is determined and the information of some nucleotide is still undetermined or improperly Determining method.

As used herein, term " homogeneity " and " identical " and its variant, when for two or more nucleic acid sequences Row are in use, refer to the sequence similarity of two or more sequences (such as nucleotide or peptide sequence).At two or more Under the background of a homologous sequence, the percentage identity or homology of sequence or its subsequence represent all identical monomer lists First (such as nucleotide or amino acid) ratio (i.e. about 70% homogeneity, preferably 75%, 80%, 85%, 90%, 95% Or 99% homogeneity).When compare and compare comparison window or specified region on such as lacked by using with described below The maximum for saving BLAST the or BLAST2.0 sequence comparison algorithms of parameter or being measured by manually comparing and estimating detection is corresponding During property, percentage identity can be the percentage identity on specific region.When in amino acid levels or in nucleotide level During the homogeneity of upper presence at least 85%, then it is assumed that sequence is " substantially the same ".Preferably, at least about in length 25th, there are homogeneity on the region of 50 or 100 residues or in the whole length of at least one sequence compared.With It is BLAST and BLAST2.0 algorithms in the typical algorithm of measure Percent sequence identity and sequence similarity, is described in Altschul et al., Nuc.Acids Res.25:In 3389-3402 (1977).Other methods include Smith＆ Waterman,Adv.Appl.Math. 2:482 (1981) and Needleman＆Wunsch, J.Mol.Biol.48:443(1970) Deng algorithm.It is two molecules or its complement in stingent hybridization that two nucleic acid sequences, which are another substantially the same instructions, Under the conditions of hybridize each other.

As used in herein with respect to two or more polynucleotides, term " complementary " and its variant refer to wrap (such as hybridizing containing accumulation base pairing can be undergone on antiparallel direction in two or more individual corresponding sites Duplex in) any nucleic acid sequence polynucleotides.Optionally, it may be present between the first and second nucleic acid sequences " complete " or " entire " is complementary, wherein each nucleotide in the first nucleic acid sequence can in second nucleotide sequence The stable base pairing of polynucleotides experience interacts (however, term " complementary " itself in corresponding antiparallel position It may include nucleic acid sequence non-fully complementary over the entire length)；" part " is complementary to describe its more control sequences at least 20% but the nucleic acid sequence of the residue complementation in residue and other nucleic acid sequences less than 100%.In some embodiments, At least 50% but the residue less than 100% and the residue in other nucleic acid sequences of nucleic acid sequence are complementary.In some embodiments In, at least 70%, 80%, 90% or 95% of nucleic acid sequence but the residue being less than in 100% residue and other nucleic acid sequences It is complementary.When a nucleic acid sequence residue at least 85% with the residue mutual added time in other nucleic acid sequences, then it is assumed that sequence is " being substantially complementary "." incomplementarity " is described in the residue and other nucleic acid sequences less than 20% of its more control sequences The nucleic acid sequence of residue complementation.It is not at complementary any position that " mispairing ", which is contained therein two opposite nucleotide,.It is complementary Nucleotide include the core that is effectively incorporated to relative to one another by archaeal dna polymerase during DNA replication dna in physiological conditions Thuja acid.In a typical implementation, complementary nucleotide can form base-pair each other, such as by reversely with each other parallel Position on nucleotide and/or polynucleotides nucleobase between the hydrogen bondings of specific Watson-Crick types formed A-T/U and G-C base-pairs.The complementary of other artificial bases couple can be based on other types of hydrogen bonding and/or base Shape complementary between hydrophobic phase and/or base.

As used in herein with respect to any polynucleotides or nucleic acid molecules, term " double-strand " and its variant refer to With one or more chain and including the region comprising the nucleotide residue with nucleotide residue base pairing (such as such as nucleic acid In duplex) any polynucleotides or nucleic acid molecules.Optionally, the polynucleotides (or nucleic acid molecules) of double-strand can be " fully " or " entirely " double-strand, in this way, each nucleotide residue in polynucleotides (or nucleic acid molecules) with it is another A corresponding nucleotide residue base pairing.In some embodiments, double-stranded polynucleotide includes one or more comprising not With the single stranded zone of the nucleotide residue of any other nucleotide residue base pairing.In some embodiments, double-strand multinuclear glycosides The nucleotide residue of at least 51%, 75%, 85%, 95%, 97% or 99% in sour (or nucleic acid molecules) and other nucleosides Sour residue base pairing.In some embodiments, double-stranded polynucleotide (or nucleic acid molecules) does not connect covalently each other comprising two The chain connect；Alternatively, double-stranded polynucleotide (or nucleic acid molecules) is matched at least certain part of its length with own bases To the single-stranded of (such as in hairpin oligonucleotide).When polynucleotides nucleotide residue at least 85% with corresponding core During thuja acid residue base pairing, then it is assumed that polynucleotides are " substantially double-strands ".When a nucleic acid sequence residue with it is another In a nucleic acid sequence during corresponding residue base pairing, then it is assumed that the two nucleic acid sequences are " double-strands ".In some embodiment party In case, base pairing can occur, such as by parallel nucleotide reversely with each other according to a certain conventional pairing pattern And/or the A-T/U and G-C that the hydrogen bonding of the specific Watson-Crick types between the nucleobase of polynucleotides position is formed Base-pair；In other embodiments, base pairing can be occurred by any other pattern, and wherein base pairing is according to Establishing and predictable rule carries out.

As used herein, term " single-stranded " and its variant, when for any polynucleotides or nucleic acid molecules use When, refer to include comprising not with any polynucleotides in the region of the nucleotide residue of any nucleotide residue base pairing or Nucleic acid molecules.Optionally, single-stranded polynucleotides (or nucleic acid molecules) can be " fully " or " entirely " single-stranded, such as This, each nucleotide residue in polynucleotides (or nucleic acid molecules) not with any other nucleotide residue base pairing. In some embodiments, single stranded polynucleotide includes one or more nucleotide comprising with nucleotide residue base pairing The double stranded region of residue.In some embodiments, in single stranded polynucleotide (or nucleic acid molecules) at least 51%, 75%, 85%th, 95%, 97% or 99% nucleotide residue not with other nucleotide residue base pairings.When the nucleosides of polynucleotides When at least the 85% of sour residue is not with nucleotide residue base pairing, then it is assumed that polynucleotides are " substantially single-stranded ".

As used herein, term " denaturation " and its variant, when for any double-stranded polynucleotide molecule or double-strand multinuclear Nucleotide sequence is in use, base pairing quilt between nucleotide in the opposite chain including wherein duplex molecule or double-stranded sequence Any process destroyed.Normally, denaturation is included so that at least a certain portion of double-stranded polynucleotide molecule or two chains of sequence Divide or region becomes single-stranded.In some embodiments, denaturation includes two by double-stranded polynucleotide molecule or sequence At least certain part or the region of chain are separated from each other.Normally, the region of denaturation or part are then able to be hybridized to another more Nucleic acid molecule or sequence.Optionally, " complete " of double-stranded polynucleotide molecule or sequence may be present or " entirely " become Property.Complete Denaturing be can for example cause a large amount of chains signal portion (such as more than 10%, 20%, 30%, 40% or 50%) with its extend and/or overall length the condition that is kept completely separate of complement.Normally, complete or entire denaturation destroys two All base pairings of the nucleotide of chain to each other.Similarly, optionally when the individual molecular of sample more than 80% or During 90% any double-strand of shortage (or lacking any hybridization to complementary strand), then it is assumed that sample of nucleic acid is denaturalized completely.

Alternatively, double-stranded polynucleotide molecule or sequence part or can be non-fully denaturalized.When at least one of nucleic acid The part holding of chain and hybridizing for complementary strand, and another part is in non-hybridized state (even if in situation existing for complementary strand Under) when, it is believed that given nucleic acid molecules are partial denaturations.Non-hybridized part optionally have at least 5,7,8,10, 12nd, the length of 15,17,20 or 50 nucleotide.The part of hybridization optionally has at least 5,7,8,10,12,15,17,20 Or the length of 50 nucleotide.Partial denaturation includes wherein some of a chain or the nucleotide of sequence but not all and double-strand The situation of some nucleotide bases pairing of other chains or sequence in polynucleotides.In some embodiments, partial denaturation Polynucleotides (or sequence) a chain nucleotide residue at least 20% but less than 100% not with the core in opposite chain Thuja acid residue base pairing.Under exemplary condition, the nucleosides in double-stranded polynucleotide molecule (or double-stranded polynucleotide sequence) At least the 50% of sour residue is single-stranded (or non-hybridized) form, but the residue less than 20% or 10% is double-strand.

Optionally, when the substantial portions of the individual nucleic acid molecules of sample (for example, more than 20%, 30%, 50% or 70%) when in partial hybridization state, it is believed that sample of nucleic acid is partial denaturation.Optionally, substantial amount is less than in sample Individual nucleic acid molecules be denaturalized completely, such as the nucleic acid point in sample no more than 5%, 10%, 20%, 30% or 50% Son.Under exemplary condition, at least the 50% of the nucleic acid molecule of sample is partial denaturation, but is less than 20% or 10% It is denaturalized completely.In other situations, at least the 30% of the nucleic acid molecule of sample is partial denaturation, but less than 10% or 5% is denaturalized completely.Similarly, when individual nucleic acid molecules a small number of in sample be partially or completely be denaturalized when, it is believed that Sample of nucleic acid is non denatured.

In embodiments, part Denaturing is realized by the way that duplex is maintained at appropriate temperature range.Example Such as, nucleic acid is maintained at fully raising to realize some thermal denaturations (such as higher than 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C or 70 DEG C) but be not high enough to realize the temperature of complete thermal denaturation (for example, being less than 95 DEG C or 90 DEG C or 85 DEG C or 80 DEG C or 75 DEG C). In embodiments, using the condition part denaturing nucleic acid of substantially isothermal.

Can also by other methods, such as the chemical denaturant such as urea or formamide that are suitably adjusted using concentration or Using the chemical method of high or low pH (such as pH between 4-6 or 8-9), to realize partial denaturation.In embodiments, make Partial denaturation and amplification are realized with recombinase-polymeric enzymatic amplification (RPA).Exemplary RPA methods description is in this article.

In some embodiments, double-stranded polynucleotide sequence to be denatured is handled by using appropriate denaturant To realize complete or partial denaturation.For example, it can be made by raising the temperature to the point for the denaturation for wherein realizing aspiration level double Chain polynucleotides experience thermal denaturation (also by interchangeably referenced as heat denatured).In some embodiments, double-stranded polynucleotide Complete thermal denaturation, temperature-adjustable to realize being kept completely separate for the two of polynucleotides chains, in this way, at least the 90% of chain It is single stranded form in its whole length.In some embodiments, by the way that polynucleotide molecule (or sequence) is exposed to height In at least 5 DEG C of the melting temperature (Tm) be computed or prediction of polynucleotide molecule or sequence, 10 DEG C, 15 DEG C, 20 DEG C, 25, 30 DEG C, 50 DEG C or 100 DEG C of temperature realizes the complete thermal denaturation of polynucleotide molecule (or polynucleotide sequence).

It alternatively, can be by the way that double-stranded polynucleotide to be denatured be contacted to realize chemistry with appropriate chemical denaturant Denaturation, the chemical denaturant are, for example, highly basic, strong acid, chaotropic agent etc. and may include such as NaOH, urea or the change containing guanidine Close object.In some embodiments, by being exposed to the chemical denaturant such as urea or formamide or make that concentration suitably adjusts It is partially or completely denaturalized with high or low pH (such as pH between 4-6 or 8-9) to realize.In embodiments, using recombination Enzyme-polymeric enzymatic amplification (RPA) realizes partial denaturation and amplification.Exemplary RPA methods description is in this article.

When about given polynucleotides (or given target sequence in polynucleotides) in use, term " melting temperature ", " Tm " or " T_m" and its variant typically refer to such temperature, the multinuclear glycosides given under determining condition group at such a temperature The 50% of acid (or given target sequence) exists with double-stranded form and 50% is single-stranded.In some embodiments, it determines Condition group may include indicating the determining parameter of aqueous reaction condition ionic strength and/or pH.Determining condition can lead to The concentration, temperature, pH, buffer solution and/or formamide of change salt (such as sodium) are crossed to adjust.Normally, the pyrolysis chain being computed Temperature can be T_mUnder about 5-30 DEG C or T_mUnder about 5-25 DEG C or T_mUnder about 5-20 DEG C or T_mUnder about 5-15 DEG C or T_mIt Lower about 5-10 DEG C.For calculating T_mMethod be well known and be found in Sambrook (1989 in " Molecular Cloning:A Laboratory Manual”,2^ndedition,volumes 1-3；Wetmur 1966,J.Mol.Biol., 31:349-370；Wetmur 1991Critical Reviews in Biochemistry and Molecular Biology, 26:In 227-259).For calculating the T for hybridization or denaturing nucleic acid_mOther sources (come from including OligoAnalyze Integrated DNA Technologies) and Primer3 (by the Whitehead Institute for Biomedical Research are issued).In some embodiments, term " melting temperature ", " Tm " and " T_m" and its variant Practical Tm (as empirically measured using determining condition) or pre- including given polynucleotides (or target sequence) Tm surveying or being computed.It in some embodiments, can be by assuming that template includes 4 kinds of common nucleotide acid (A, C, G and T) Certain proportion and come in the sequence without using template with certain length (or in the case of template group, average length) In the case of prediction or calculation template Tm.For example, it may be assumed that on gel hangover the template group that migrates include each 25% A, C, G or T, and the average length with 200,300,400 base-pairs.

As used in herein with respect to chemical part, term " label " and its variant are included comprising optically or non-light Any composition of detectable part on, wherein detectable part by be chemically treated artificially add, connect or It is attached to not labeled second part.Normally, user (or supplied upstream person) is in order to enhance the detectable of second part The addition that the purpose of property is marked.It is present in the optically or non-of the composition in the naturally occurring form of composition Optically detectable component is (for example, be present in the typical DNA molecular in n cell, RNA molecule or nucleotide Hydrogen ion and amino acid) it is not intended to the label of purpose in the present disclosure.Some typical labels include fluorescence part and dye Material.

If nucleic acid by least during the condition selected (such as during amplified reaction) for substantially stablize in a manner of connect Support is connected to, then it is believed that nucleic acid is fixed.Any mechanism, including but not limited to non-covalent bonding, ion phase can be passed through Interaction, covalent linkage, to be attached.If the first nucleic acid is hybridized to fixed the second nucleic acid on the support, if expanded Increasing condition is so so that any or all time during amplification of the first and second nucleic acid of substantial amount is that This association or connection, then it is also contemplated that the first nucleic acid is affixed to support during amplification.Such as first and second core Acid can be associated together by including the hybridization of Watson-Crick base pairings or hydrogen bonding.In instances, the amplification of selection The holding of at least 50%, 80%, 90%, 95% or 99% of the first nucleic acid of conditions permit and hybridizing for the second nucleic acid, or vice versa It is as the same.If nucleic acid does not either directly or indirectly connect or associates with support, it is believed that nucleic acid is loose or non-solid Fixed.

If medium is temporarily, at least liquid mediums object under selected conditions, the liquid mediums object is not substantive Property or fully inhibit or hinder the transfer or movement of loose molecule, then it is believed that medium under selected conditions It is flowable.Loose molecule itself be not fixed to solid support or surface or not with another fixed molecule Association.In embodiments, loose molecule is the solute (such as nucleic acid) by flowable medium.Illustratively Transfer or movement in medium can be by diffusion, convection current, turbulent flow, stirring, Brownian movement, advection, electric current or liquid Other molecular motions from any first point in continuous phase to fluid communication or identical continuous phase in any other point Transfer or movement.For example, in flowable medium, the loose nucleic acid of significant quantity is shifted from a fixed site Another fixation site in the identical continuous phase of flowable medium or that fix site fluid communication with first.Appoint Selection of land, the rate of transfer or the movement of medium amplifying nucleic acid and the rate of transfer or the movement of water amplifying nucleic acid are comparable.One In a little examples, the condition of selection is the condition that medium is undergone during amplification.The condition of selection may or may not allow flow Dynamic medium keeps substantially stationary.Condition may or may not make the effective mixing of flowable medium experience, stirring Or it shakes.Medium is temporarily, at least flowable optionally during amplification.Such as medium is in at least one of selection It is flowable under pre- amplification and/or amplification condition.Optionally, flowable medium do not prevent not substantively it is different not The transfer of the mixing of fixed nucleic acid or loose nucleic acid between the different zones of the continuous phase of flowable medium. The movement or transfer of nucleic acid can for example be caused by diffusion or convection current.If loose nucleic acid is after amplification in different fixations It cannot propagate or move between site or in entire continuous phase, then it is not flowable optionally to think medium.Generally Ground, flowable medium does not limit loose nucleic acid (such as template or amplicon) substantially during amplification period In the effective coverage of reaction capacity or it is limited in fixed position.Optionally, by a variety of methods or change can be passed through The condition of flowable medium is not flowable to become.Optionally, if medium is liquid or is not half solid Body, then it is flowable.If the mobility of medium is comparable with pure water, it is believed that it is flowable. In other embodiments, if medium be there is no polymer fluid or if its viscosity coefficient and pure water it is viscous Degree coefficient is similar, then it is believed that the medium is flowable.

As it will appreciated by a person of ordinary skill, referring to for template, template polynucleotide, extension probes, primer etc. can refer to It is the group of substantially the same nucleic acid molecules or library rather than individual molecule in relevant part.For example, " template " can refer to it is multiple Substantially the same template molecule；" probe " can refer to multiple substantially the same probe molecules etc..It is one or more positions On be degeneracy probe in the case of, it will be understood that the sequence of the probe molecule comprising particular probe will be on degeneracy position not Together, that is, the sequence for forming the probe molecule of particular probe can be substantially the same only on nondegenerate position.In the application These terms are intended to provide support to group or molecule.When being intended to refer to single nucleic acid molecules (i.e. a molecule), term can be used " template molecule ", " probe molecule ", " primer molecule " etc. are replaced.It in some instances, will be it is manifestly intended that substantially phase The plural property of same nucleic acid molecules group.

" template ", " oligonucleotides ", " probe ", " primer ", " template ", " nucleic acid " etc. are intended to interchangeable herein Term.These terms refer to polynucleotides, are not necessarily limited to any length or function.Identical nucleic acid can basis Background is taken as " template ", " probe " or " primer ", and can be converted between these roles at any time." polynucleotides " also by Referred to as " nucleic acid " is the linear polymer or its variant of the two or more nucleotide connected by covalent internucleoside linkage Or functional fragment.In these naturally occurring example, internucleoside linkage is typically phosphodiester bond.It is however, other real Example is optionally comprising other internucleoside linkage such as mercaptan phosphate (phosphorothiolate) keys, and may or may not wrapping Phosphorous acid groups.Polynucleotides include double-strand and single-stranded DNA and double-strand and single-stranded RNA, DNA:RNA heterozygosis The heterozygote of body, peptide nucleic acid (PNA) between PNA and DNA or RNA, and may also include the modification of known type.Multinuclear glycosides Acid optionally by 5 ' or 3 ' ends be connected to one or more non-nucleotide moieties for example mark and other small molecules, Macromolecular such as albumen, fat, sugar and solid or semisolid support.Label includes the detection method detection of selection can be used Any part, and so that the nucleotide or polynucleotides of connection can similarly be detected by using the detection method of selection. Optionally, label transmitting is optically detectable or visible electromagnetic radiation.In some cases, nucleotide or polynucleotides It is not connected to mark, and directly detects the presence of nucleotide or polynucleotides." nucleotide " refer to nucleotide, nucleosides or its Analog.Optionally, optionally, nucleotide is N- the or C- glucosides of purine or pyrimidine bases.(such as include 2- deoxidation-D- cores The dezyribonucleoside of sugar or the ribonucleotide comprising D-ribose).The example of other analogs includes but not limited to D2EHDTPA Ester, phosphoramidate, methyl phosphonate, chiral-methyl phosphonates, 2-O- methyl ribonucleotides.Pass through these terms Any one refers to that nucleic acid should not be interpreted to mean that nucleic acid has the function of any specific activity or property.It is for example, single Word " template " does not indicate that " template " passes through polymerase duplication or template and can not be used as " primer " or " probe ".

It will be understood that in some instances, it can be larger such as template, probe, primer to participate in the nucleic acid reagent of amplification The part of nucleic acid molecules, the larger nucleic acid molecule also include another part without identical function.Optionally, this its It does not partly have any template, probe or primer function.In some instances, substantially it is hybridized to optionally fixed draw The nucleic acid of object (such as on fixed site) is considered as " template ".Before or during amplification ginseng can be generated from other nucleic acid With any one or more nucleic acid reagents (template, fixed chain, fixed or revocable primer etc.) of template walking.Optionally Ground carries out one or more modifications to be generated from input nucleic acid by the nucleic acid walked in medium to being initially introduced into template (and not needing to be same) nucleic acid reagent.Input nucleic acid experience restrictive digestion, connection, one or more can for example be made Amplification cycles, denaturation, mutation etc. are used as the nucleic acid of template, primer etc. to generate during amplification or further amplification.For example, Can be by the input nucleic acid denaturation of double-strand to generate the first single-chain nucleic acid, first single-chain nucleic acid is optionally for generation second Complementary strand.If it is desire in this way, then it is believed that the first single-chain nucleic acid our purpose " template " in this article.Alternatively, from The second complementary strand that first single-chain nucleic acid generates can be considered as " template " with our purpose in this article.In another reality In example, template source is in input nucleic acid and not necessarily identical with input nucleic acid.For example, template may include being not present in inputting Other sequence in nucleic acid.In embodiments, template can be with not complementary with input nucleic acid using one or more 5 ' jags primer from input nucleic acid generate amplicon.

As used in herein with respect to two or more polynucleotides, term " hybridization " and its variant refer to wherein Any one or more nucleic acid sequences (each sequence includes the section of continuous nucleotide residue) in the polynucleotides are at two Or more individual corresponding position experience base pairing (such as hybridization nucleic acid duplex in) any process.Optionally " complete " or " entirely " hybridization may be present, wherein in the first nucleic acid sequence in ground between the first and second nucleic acid sequences Each nucleotide residue can corresponding with the antiparallel position in second nucleotide sequence nucleotide experience base pairing phase Interaction.In some embodiments, hybridization may include two or more not fully complementary over the whole length or non-alkali Base pairing between the nucleic acid sequence of basigamy pair.For example, when two nucleic acid sequences undergo base pairing, one of nucleic acid During residue base pairing at least 20% but residue and other nucleic acid sequences less than 100% of sequence, " part " hybridization Occur.In some embodiments, hybridization includes the base pairing between two nucleic acid sequences, one of nucleic acid sequence At least 50% but less than 100% residue residue base pairing corresponding with other nucleic acid sequences.In some embodiments In, at least 70%, 80%, 90% or 95% of a nucleic acid sequence but residue and other nucleic acid sequences less than 100% Corresponding residue base pairing.When at least 85% residue alkali corresponding with other nucleic acid sequences of the residue of a nucleic acid sequence During basigamy pair, then it is assumed that two nucleic acid sequences are " substantially hybridizing ".A nucleic acid molecules are than another substantially wherein In the situation of longer (or two of which nucleic acid molecules are included and are substantially complementary and substantially not complementary region), even if when one When a or two nucleic acid molecules parts can keep non-hybridized, two nucleic acid molecules can be still described as " hybridization ".It is " not miscellaneous Hand over " residue less than 20% of one of nucleic acid sequence and the core of the residue base pairing in other nucleic acid sequences are described Acid sequence.In some embodiments, base pairing can occur, such as according to a certain conventional pairing pattern by that The hydrogen bond of specific Watson-Crick types between the nucleobase of this antiparallel nucleotide and/or polynucleotides position Close A-T/U the and G-C base-pairs formed；In other embodiments, base pairing can be occurred by any other pattern, Wherein base pairing is carried out according to established and predictable rule.

The hybridization of two or more polynucleotides can be whenever the two or more polynucleotides are appropriate miscellaneous When being contacted under the conditions of friendship.Hybridization conditions include any condition for being suitable for nucleic acid hybridization；The method hybridized It is well known in the present art with for the felicity condition of hybridization.The stringency of hybridization can be influenced by multiple parameters, miscellaneous including treating The degree of homogeneity and/or complementarity between the polynucleotides (or any target sequence in polynucleotides) of friendship；It waits to hybridize Polynucleotides and/or target sequence melting temperature, be called " T_m”；Parameter such as salt, buffer solution, pH, temperature, more The GC% contents and/or time of nucleotide and primer.Normally, hybridize in relatively low temperature and/or raised salinity and It is advantageous under the organic solvent concentration of reduction.High stringency hybridization conditions will usually need relatively elevation between two target sequences Degree it is complementary to hybridize, even and if low strict hybridization conditions shown in two polynucleotides to be hybridized it is low-level It will also promote to hybridize when complementary.It can be in hybridization step or optional and successive washing step or hybridization and optional purge step Hybridization conditions are applied during rapid.

The example of high stringency hybridization conditions includes any one or more following：Salt (the example of about 0.0165 to about 0.0330 Such as NaCl) concentration；Melting temperature (the T of target sequence (or polynucleotides) to be hybridized_m) under about 5 DEG C to about 10 DEG C of temperature； And/or about 50% or higher concentration of forma.Normally, high stringency hybridization conditions allow with high homology for example >= Combination between 95% homogeneity or the sequence of complementarity.In an exemplary implementation of high stringency hybridization conditions In, including 25mM KPO at about 42 DEG C₄At the ultrasound that (pH 7.4), 5 × SSC, 5 × Denhardt solution, 50 μ g/mL are denaturalized The salmon sperm DNA of reason, 50% formamide, 10% dextran sulfate and 1-15ng/mL double-stranded polynucleotides (or double stranded target sequence) Hybridized in hybridization solution, and the about 65 DEG C of washing solution of utilization comprising 0.2 × SSC and 0.1% sodium dodecyl sulfate into Row washing.

The example of moderate stringency hybridization condition includes any one or more following：Salt (the example of about 0.165 to about 0.330 Such as NaCl) concentration；Melting temperature (the T of target sequence to be hybridized_m) under about 20 DEG C to about 29 DEG C of temperature；And/or about 35% Or lower concentration of forma.Normally, such moderate stringency hybridization conditions permit has high or medium homology example Such as the combination between >=80% homogeneity or the sequence of complementarity.In an exemplary implementation of moderate stringency hybridization condition In scheme, 25mM KPO are being included at about 42 DEG C₄(pH 7.4), 5 × SSC, 5 × Denhart solution, 50 μ g/mL are denaturalized super The salmon sperm DNA of sonication, 50% formamide, 10% dextran sulfate and 1-15ng/mL double-stranded polynucleotides (or double-strand target sequence Row) hybridization solution in hybridized, it is and molten in the about 50 DEG C of washings of utilization comprising 2 × SSC and 0.1% sodium dodecyl sulfate Liquid is washed.

The example of low strict hybridization conditions includes any one or more following：About 0.330 to about 0.825 salt (such as NaCl) concentration；Melting temperature (the T of target sequence to be hybridized_m) under about 40 DEG C to about 48 DEG C of temperature；And/or about 25% or Lower concentration of forma.Normally, such low stringency condition allow to have low homology homogeneity for example >=50% or Combination between complementary sequence.

Some exemplary conditions for being suitable for hybridization are included in sodium salt such as NaCl, sodium citrate and/or phosphoric acid Polynucleotides to be hybridized are incubated in the solution of sodium.In some embodiments, hybridize or wash solution and can include about 10- The sodium dodecyl sulfate (SDS) of 75% formamide and/or about 0.01-0.7%.In some embodiments, hybridization solution can To be stingent hybridization solution, it may include 50% formamide, 5x SSC (0.75M NaCl, 0.075M sodium citrates), 50mM Sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt solution, 0.1%SDS and/or 10% dextran sulfate are appointed What is combined.In some embodiments, hybridizing or washing solution may include BSA (can be in bovine serum albumin(BSA)).In some implementations It, can at about 20-25 DEG C or about 25-30 DEG C or about 30-35 DEG C or about 35-40 DEG C or about 40-45 DEG C or about in scheme Hybridized or washed under 45-50 DEG C, or about 50-55 DEG C or higher temperature range.

In some embodiments, can hybridization or washing be subjected to about 1-10 minutes or about 10-20 minutes or about 20- 30 minutes, or about 30-40 minutes, or about 40-50 minutes, or the time range of about 50-60 minutes or longer.

It in some embodiments, can be in the pH ranges of about 5-10 or about pH6-9 or about pH6.5-8 or about pH6.5- Hybridized or washed under 7.

In some embodiments, term " monoclonal " and its variant are used to describe the substance of wherein group member Partly (for example, at least about 50%, typically at least 75%, 80%, 85%, 90%, 95% or 99%) nucleotide sequence level The polynucleotides group of upper shared at least 80% homogeneity.Normally, at least about the 90% of group, normally at least about 95%, More generally at least about 99%, 99.5% or 99.9%) answered by the amplification or Template Dependent of specific polynucleotide sequence It makes to generate, the specific polynucleotide sequence is present among the substantial portions of the member of monoclonal polynucleotides group.It is single All members of clone group need not be identical from one another or complementary.For example, the different piece of polynucleotide template can quilt Amplification or replicate with generate gained monoclonal group member；Similarly, a certain number of " mistakes " and/or not exclusively extension It can occur during the amplification of primary template, it can display sequence otherness among themselves so as to generate its individual member Monoclonal group.In some embodiments, at least the 50% of the member of monoclonal group with reference to nucleic acid sequence (based on i.e. The nucleic acid with determining sequence compared for sequence) it is at least 80% same.In some embodiments, group At least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more member includes and reference Sequence of the nucleic acid sequence at least 80%, 85%, 90%, 95%, 97% or 99% homogeneity (or complementary).One In a little embodiments, nucleic acid expansion that the horizontal mixing of the low or unsubstantialities of non-homogeneous polynucleotides can be described herein Occur during increasing reaction, thus substantially monoclonal group may include a small number of different polynucleotides (such as less than 30%, it is few In 20%, less than 10%, less than 5%, less than 1%, less than 0.5%, the different multinuclears less than 0.1% or less than 0.001% Thuja acid).As used herein, phrase " substantially monoclonal " and its variant, make when for one or more polynucleotides groups Used time refers to one or more multinuclear glycosides for including and having at least polynucleotides of 80% homogeneity with original single template Sour group, the original single template are used as basis and are used for clonal expansion to generate the group of substantially monoclonal.

In some embodiments, at least the 80% of the member of amplicon, normally the member of amplicon is at least 90%, more generally at least 95%, more generally at least 99% homogeneity more than 90% will be shared with polynucleotide template, Normally it is more than 95% homogeneity, more often over 97% and more often over 99% homogeneity.Alternatively, amplification The member of son can have the complementarity more than 90% with primary template, normally the complementarity more than 95%, more generally greatly In 97% complementarity, more often over 99% complementarity.In some embodiments, the substantially nucleic acid of monoclonal The member of group can hybridize each other under stringent hybridization conditions.

In some embodiments, if amplicon includes polyclonal pollutant few enough with by template sequence Detectable signal is generated in any method of the foranalysis of nucleic acids of influence, then is referred to as amplicon " monoclonal " or " basic Upper monoclonal ".For example, " monoclonal " group of polynucleotides may include generating the letter that specific sequencing system can be used to detect Any group of number (such as sequencing signal, nucleotide are incorporated to signal etc.).It is optional, then can signal Analysis correctly to survey Surely it is present in the sequence of any one or more nucleotide in any polynucleotides of group and/or base information.For detecting And/or the example of the appropriate sequencing system of signal as analysis includes Ion Torrent sequencing systems, such as Ion Torrent PGM^TMSequencing system, including 314,316 and 318 systems and Ion Torrent Proton^TMSequencing system, Including Proton I, Proton II and Proton III (Life Technologies, Carlsbad, CA).In some implementations In scheme, monoclonal amplicon allows accurate survey of at least five continuous nucleotide residue on Ion Torrent sequencing systems Sequence.

As used herein, term " clonal expansion " and its variant refer to wherein produce by the amplification of polynucleotide template Any process of the raw substantially polynucleotides group of monoclonal.In some embodiments of clonal expansion, amplification two or more Multiple polynucleotide templates are to generate the polynucleotides group of at least two substantially monoclonals.

As used herein, term " connector " including comprising DNA, RNA, chimeric rna/dna molecule, its analog it is more Nucleotide or oligonucleotides and normally mean to connect or be bound to during operating process purpose target polynucleotide (such as Template) addition or external sequence.The connection of connector to template optionally occurs before or after template amplification. In some embodiments, connector may include primer knot that is substantially the same with the sequence in corresponding primer or being substantially complementary Close sequence.In some embodiments, the first connector comprising the first primer binding site is connected to the one of linear double-stranded template A end, and the second connector comprising the second primer binding site is connected to another end.

As used herein, term " binding partners " includes two molecules or part thereof, has for mutual specific Binding affinity, and normally by prior to and other molecules combination and be bonded to each other.Normally but not necessarily, it is special Some or all structures of one member of fixed combination pair are complementary with some or all structures possessed by another member , two of which member can be especially by the bonding between complementary structure (optional passes through multiple non-covalent attractions) Mode is combined together.

In some embodiments, the molecule as binding partners includes：Biotin (and its derivative) companion in connection Companion's avidin part, streptavidin part (and its derivative)；With reference to the His- labels of nickel, cobalt or copper；Knot Close cysteine, histidine or the histidine section (histidine patch) of Ni-NTA；With reference to maltose-binding protein (MBP) Maltose；Agglutinin-carbohydrate binding partners；Calcium-calbindin (CBP)；Acetylcholine and receptor-acetyl courage Alkali；Albumin A and the anti-FLAG antibody of binding partners；GST and binding partners glutathione；Uracil dna glycosylase (UDG) with Ugi (uracil-DNA glycosylase inhibitor) albumen；The antigen or epitope tag of binding antibody or antibody fragment, particularly It is antigen such as digoxigenin, fluorescein, dinitrophenol dinitrophenolate or bromodeoxyribouridine and its respective antibody；Mouse immune ball egg In vain with goat anti mouse immunoglobulin；With reference to IgG and albumin A；Receptor-receptor agonist or receptor antagonist；Enzyme-enzyme Confactor；Enzyme-enzyme inhibition factor；With thyroxine-cortisol.Another binding partners of biotin can come from chicken Biotin-binding protein (Hytonen et al., BMC Structural Biology 7:8).

Avidin part may include the biotin-binding part of avidin and avidin Any derivative, analog and other unnatural forms.The other forms of avidin part include natural sum The avidin and streptavidin and derivative molecule of recombination, such as nonglycosylated avidin, N- acyl groups avidin and truncated streptavidin.For example, avidin part includes antibiotin The deglycosylation form of albumen, streptomyces (Streptomyces) (such as avidin streptomycete (Streptomyces Avidinii)) generate bacterium streptavidin, truncated streptavidin, the avidin of recombination and Streptavidin and natural, deglycosylated and recombination avidin and natural, recombination and truncate Streptavidin derivative, such as N- acyl group avidins, for example, N- acetyl group, N- phthalyls and N- succinyls avidin and commercial product ExtrAvidin^TM、 Captavidin^TM、Neutravidin^TMWith Neutralite Avidin^TM。

Embodiment

According to the following example it will be further understood that the embodiment of this teachings, these embodiments should not be by It is construed to limit the range of this teachings in any way.

Embodiment 1

Progress nucleic acid amplification is anti-in the single Continuous Liquid Phase of the total reaction volume of~220 μ L in single reaction vessel It should.

It will about 420x10 with water in 1.5mL manages (pipe 1)⁶Globule washing 1 time (vortex/rotation), then in buffer solution Middle washing 1 time (vortex/rotation).

It recombinates enzyme source and comes from TwistAmp^TMBasic kits (come from TwistDx, Cambridge, Great Britain).The sediment through dehydration in kit includes usvX recombinases, usvY recombinases are loaded into albumen, gp32 albumen, Bsu archaeal dna polymerases, dNTP, ATP, phosphocreatine and creatine kinase.It is buffered in the rehydration provided by kit of 120 μ L Rehydration comes from TwistAmp in liquid^TMFour sediments (pipe 2) of Basic kits.Recombination enzyme solutions are vortexed and rotated, It is then ice-cold.Two heat blocks are prepared, one is arranged on about 68-70 DEG C, and one is arranged on 40 DEG C.

Supernatant from globule sediment (pipe 1) is removed, the liquid of about~20 μ L is left in bottom.

By reverse primer (100 μM of stostes of 2 μ L) added to globule pipe (pipe 1), then it is vortexed and rotates.Reverse primer Sequence：5’-ATCCCTGCGTGTCTCCGAC-3.

By biotinylated reverse primer (10 μM of stostes of 2 μ L) added to globule pipe (pipe 1), then it is vortexed and rotates. Biotinylated reverse primer sequences： 5’Bio-ATCCCTGCGTGTCTCCGAC-3’.

By the polynucleotides library (various concentration) of 1 μ L added to globule pipe (pipe 1), vortex/rotation is placed in ice On.Library concentration is according to desired 1:50、1:75、1:200 DNA changes globule ratio.

The recombination enzymatic mixture (pipe 2 is rebuild in 120 μ L rehydration buffer solutions) of rehydration (is managed added to globule pipe 1) it, is vortexed, rotates；It is placed on ice.

The exemplary screening agent in the present disclosure of 65 μ L is added to globule pipe, is vortexed and rotates, be placed on ice.

The ice-cold 280mM Mg- acetate of 11 μ L is added to globule pipe (in centre), then the whirlpool in the case where maximum is set It revolves 3 seconds and puts back to 10 seconds on ice, and incubated 20 minutes in 40 DEG C on heat block.

In heat block heat inactivation will be reacted in 68 DEG C -70 DEG C 10 minutes.

Reaction tube is filled it up with TE buffer solutions, is vortexed and rotates 3 minutes in the case where maximum sets (~20KG), removed from globule Solution leaves~100 μ L.Wash repeatedly step twice.

It is primary that globule is washed with recycling solution.

Reaction tube is filled it up with washing buffer, is vortexed and rotates 3 minutes in the case where maximum sets (~20KG), removed from globule Solution is removed, leaves~100 μ L.Wash repeatedly step twice.

After last rotation, solution is reduced to 100 μ L (washing solution).

By the way that biotinylated polynucleotides (are come from the para-magnetic beads of streptavidin have been conjugated The MyOne of Dynabeads^TMBead) with reference to being enriched with globule.

The globule of enrichment is loaded into Ion Torrent ion-sensitive chips to go forward side by side the quasi- sequencing reaction of rower.Enrichment The signal portion of globule is measured as the group of the substantially monoclonal of the polynucleotides comprising amplification, such as passes through Ion Torrent PGM^TMThe observation of the detectable sequencing signal from such globule is confirmed on sequenator.Signal is sequenced to survey in analysis Surely the sequence being present in the amplicon of each such globule.

Embodiment 2

By about 240x10⁶Globule (combining forward primer) (comes from Ion in 2mL pipes in annealing buffer Sequencing kits, such as PN 4482006) in washed once.It removes (in addition to~50 μ L) and abandons supernatant. Globule is resuspended in 100 μ L annealing buffers.

There to be 300bp or 400bp Insert Fragments (about 120-240x10⁶Copy) bar code DNA library with it is washed Globule prehybridization.Library is connected to the connector for being hybridized to forward primer included in an end and is connected in another end It is hybridized to the insetion sequence of the connector of reverse primer.Detected template/globule ratio includes 1:1、0.75:1 and 0.5:1.With Annealing buffer adjusts final volume to 200 μ L.By being vortexed and rotating mixing tube.Pipe is incubated at 95-100 DEG C 3 minutes, and It is incubated 5 minutes at 37 DEG C.1mL annealing buffers are added, is being vortexed higher than 16,000xG and is rotating pipe 3.5 minutes, abandoning supernatant Liquid.The 10mM potassium acetates of 1mL are added, is being vortexed higher than 16,000xG and is rotating pipe 3.5 minutes, abandoning supernatant.Repeat second Sour potassium washed once.Globule (pipe 1) is resuspended in 480 μ L potassium acetates.

It recombinates enzyme source and comes from TwistAmp^TMBasic kits (come from TwistDx, Cambridge, Great Britain).Component list in the sediment through dehydration from Basic kits can be found in embodiments above 1. In 15mL pipes (pipe 2), rehydration comes from TwistAmp in the rehydration buffer solution provided by kit of 2.88mL^TM 96 sediments of Basic kits.

By 100 μM of reverse primers (revocable primer) of 48 μ L added to the globule (pipe 1) of washed/prehybridization.It will 10 μM of biotinylated reverse primers (revocable primer) of 48 μ L are added to the globule of washed/prehybridization, and vortex tube (pipe 1).By the content of pipe 1 (including library, globule and reverse primer) added to pipe 2 (sediment for including rehydration), Pipe 2 is vortexed 5 seconds and is placed on ice.144 μ L T4gp32 albumen (15 μ g/ μ L) are added, is vortexed and is returned on ice.Addition The exemplary screening agent in the present disclosure of 1.56mL, vortex tube are simultaneously returned on ice.5 points are remained above on ice in reaction After clock, 264 μ L magnesium acetates of addition, vortex tube 3 times, 3 seconds every time.In 96 orifice plates that 50 μ L samples deciles to ice are pre-chilled. 96 orifice plates are incubated 25 minutes at 40 DEG C on thermal cycler (temperature maintains 40 DEG C).

In order to terminate reaction, the 100mM EDTA of 150 μ L are added to each hole.By all reactions together and It is centrifuged 3.5 minutes higher than 16,000xG.Abandon supernatant.Add the Tris/1%SDS of 1mL, vortex tube.In 1mL Globule is washed in OneTouch washing solution twice.Globule is resuspended in 100 μ L.

By with the paramagnetic streptavidin globule (MyOne from Dynabeads^TMGlobule) it is connected with reference to be enriched with The globule of the copy in library.The globule of enrichment is loaded into Ion Torrent PGM ion-sensitive chips.

According to Ion PGM^TMThe explanation of manufacturer in 400 Kit of Sequencing (User Guide PN4474246B) Book carries out standard sequencing reaction.The signal portion of the globule for the enrichment being loaded on chip is measured as comprising the more of amplification The group of the substantially monoclonal of nucleotide such as passes through Ion Torrent PGM^TMIt is detectable from these globules on sequenator The observation of sequencing signal confirmed.Sequencing signal is analyzed by Torrent Suite Software to measure to be present in Sequence in the amplicon of these globules.

Sequencing data generates the average read length (Fig. 9) of 305bp, the mass measurement of comparison be 1.16G (AQ17) and 1.07G(AQ20)。

Embodiment 3

By about 250x10⁶Globule (combining forward primer) (comes from Ion Sequencing in 1.5mL annealing buffers Kit, such as PN 4482006) in washed once, be vortexed and rotate 6 minutes in 15,000xG.Supernatant is abandoned, is stayed in pipe Lower about 50 μ L.

There to be 140bp Insert Fragments (about 50x10⁶Copy) library and washed globule prehybridization.Library includes It is connected to an end and is hybridized to the connector of forward primer and is connected to the connector for being hybridized to reverse primer in another end Insetion sequence.Library (the 62M stostes of 0.81 μ L) and 0.1mL annealing buffers are added to washed globule and passed through Upper and lower imbibition mixing.Globule/template ratios are about 5:1.Pipe is incubated at 92-95 DEG C 7 minutes, and incubate 10 minutes at 37 DEG C. 1mL annealing buffers are added, is being vortexed higher than 15,000xG and is rotating pipe 6 minutes, abandoning supernatant.Add the 10mM second of 1mL Sour potassium is being vortexed higher than 15,000xG and is rotating pipe 6 minutes, abandoning supernatant.Potassium acetate is repeated to washed once and put pipe In on ice.The liquid of about 60 μ L is retained in pipe (pipe 1).

It recombinates enzyme source and comes from TwistAmp^TMBasic kits (come from TwistDx, Cambridge, Great Britain).Component list in the sediment through dehydration from Basic kits can be found in embodiments above 1.About Rehydration comes from TwistAmp in the rehydration buffer solution provided by kit of 240 μ L^TM8 precipitations of Basic kits Object.Sediment and rehydration buffer solution are vortexed, rotation and ice-cold.

10 μM of biotinylated reverse primers of 100 μM of reverse primers (revocable primer) of 4 μ L and 1 μ L are (non-solid Fixed primer) added to the globule (pipe 1) of washed/prehybridization, and be vortexed and rotate and manage (pipe 1).By the content (packet of pipe 1 Containing library, globule and reverse primer) added to pipe 2 (sediment for including rehydration), the vortex of pipe 2 is placed on ice.Add Add the exemplary screening agent in the present disclosure of 130 μ L, vortex tube is simultaneously returned on ice.Add the ice-cold 280mM acetic acid of 24 μ L Magnesium, is vortexed and rotation is managed.Total reaction volume is about 332 μ L.Pipe is incubated 60 minutes at 40 DEG C.

The 100mM EDTA of 1ml are added to terminate reaction.Pipe is vortexed and rotated 6 minutes in 15,000xG.Abandon supernatant Liquid simultaneously leaves about 20 μ L in pipe.It repeats EDTA and terminates reaction step, vortex and spin step.Add the Tris/1% of 1mL Pipe is vortexed and rotated 6 minutes in 15,000 xG by SDS.It abandons supernatant and about 50 μ L is left in pipe.In 1mL By being vortexed and rotating washing globule in OneTouch washing solution, about 100 μ L are left in pipe.By all reactions together Come and rotated 6 minutes in 15,000xG, abandon supernatant, about 100 μ L are left in pipe.

By with the paramagnetic streptavidin globule (MyOne from Dynabeads^TMGlobule) it is connected with reference to be enriched with The globule of the copy in library.The globule of enrichment is loaded into Ion Torrent Proton I^TMIon-sensitive chip.

According to Ion PI^TMManufacturer says in Sequencing 200Kit (User Guide PN MAN0007491) Bright book carries out standard sequencing reaction.The signal portion of the globule for the enrichment being loaded on chip is measured as comprising amplification The group of the substantially monoclonal of polynucleotides such as passes through Ion Torrent Proton^TMOn sequenator from these globules can What the observation of the sequencing signal of detection was confirmed.Sequencing signal is analyzed by Torrent Suite Software to deposit to measure The sequence being in the amplicon of these globules.

Carry out sequencing operation twice.Sequencing data runs the average read length (Figure 10) for generating 96bp in first time, Second of operation generates the reading length (Figure 11) of 94bp.The mass measurement of comparison first time operation be 1.76G (AQ17) and 1.43G (AQ20) is 1.48G (AQ17) and 1.17G (AQ20) in second of operation.

Embodiment 4：

By globule (combining forward primer) (103x10 of 20 μ L⁶/ μ L) and the 10mM potassium acetates of 440 μ L and the 1M of 3 μ L Tris (pH 8) is mixed.Globule is mixed by being vortexed and rotating.

By mixed with the NaOH of 2 μ L be denaturalized 16 μ L have 200bp Insert Fragments (about 199x10⁶Copy) text Library is vortexed and rotates, and allows to stand 1 minute.By add 440 μ L 10mM potassium acetates and 3 μ L 1M Tris pH 8 come Neutralization reaction.Library is connected to the connector for being hybridized to forward primer included in an end and is connected in another end miscellaneous It hands over to the insetion sequence of the connector of reverse primer.

Globule is added to the library of denaturation.Globule/template ratios are about 10:1 (200,000,000,000 globules:200000000 libraries).Whirlpool Coil, and allow be stored at room temperature 5 minutes (pipe 1).

It recombinates enzyme source and comes from TwistAmp^TMBasic kits (come from TwistDx, Cambridge, Great Britain).Component list in the sediment through dehydration from Basic kits can be found in embodiments above 1. In 15mL pipes (pipe 2), rehydration comes from TwistAmp in the rehydration buffer solution provided by kit of 3mL^TMBasic is tried 96 sediments (pipe 2) of agent box.

100 μM of biotinylated reverse primers of 100 μM of reverse primers (revocable primer) of 24 μ L and 2 μ L are (non- Fixed primer) added to the globule of washed/prehybridization, vortex tube (pipe 1) and ice-cold tube.By the present disclosure of 1.6mL Exemplary screening agent be added to pipe 2, by pipe be vortexed 5 seconds, manually overturn/rotate 10 seconds, be vortexed 5 seconds, be placed on ice. 260 μ L 280mM magnesium acetates are added to pipe 2, pipe is vortexed 5 seconds, manually overturn/rotate (vortex and overturning/rotation in 10 seconds 3 times), it is placed on ice.In 96 orifice plates that 50 μ L samples deciles to ice are pre-chilled.96 orifice plates are incubated 60 minutes at 40 DEG C.

The 200mM EDTA for adding 100 μ L are reacted to each hole with terminating.All reactions are gathered and in most high speed Centrifugation 7 minutes.Abandon supernatant.Sediment is resuspended in the 1mL recycling buffer solutions with 1%SDS, is vortexed 30 seconds, and most High speed rotation 6 minutes.After each rotation, pipe is reduced by half by the content for merging two pipes.With 1%SDS 1mL recycling buffer solutions in be resuspended sediment, be vortexed 30 seconds, and rotated 7 minutes in 1550rpm.

By with the paramagnetic streptavidin globule (MyOne from Dynabeads^TMGlobule) it is connected with reference to be enriched with The globule of the copy in library.During enriching step, ES- washing buffers are replaced with the recycling buffer solution with 0.1%SDS. Finally globule is resuspended in 1mL water and is reduced to 100 μ L.The globule of enrichment is loaded into Ion Torrent Proton I^TM Ion-sensitive chip.

Sequencing data generates the average read length (Figure 12) of 144bp, and the mass measurement of comparison is 4G (AQ17).

Embodiment 5

In dH₂By being vortexed and rotating washing 375x10 in O⁶Globule (combining forward primer).Removal supernatant (removes Outside~50 μ L).By DNA library (about 75x10⁶A molecule) added to washed globule.By reverse primer (the non-life of about 4 μ L Object element) and 0.4 μ L biotinylated reverse primer be added to globule.The fusion forward primer of about 0.8 μ L is added To globule.The magnesium acetate of 40 μ L is added to globule (final concentration 14mM).By dH₂It is final total to 320 μ L that O is added to globule Volume.

It recombinates enzyme source and comes from TwistAmp^TMBasic kits (come from TwistDx, Cambridge, Great Britain).Component list in the sediment through dehydration from Basic kits can be found in embodiments above 1.In list In only pipe, rehydration comes from TwistAmp in the rehydration buffer solution provided by kit of 488 μ L^TMBasic reagents 16 sediments of box.For 400bp libraries, the T4gp32 albumen (final concentration of 0.2mg) of 0.25mg/ml is added, for The T4gp32 albumen (final concentration of 0.4 mg) of 0.5mg/ml is added in 600bp libraries.Vortex tube is to mix and rotate.

The content of bead mixture is added to recombinase pipe, be vortexed and is rotated.By globule/recombination enzymatic mixture with The oil of precooling is transferred to pipe, and in Ion Torrent OneTouch^TMIt is received on 10 microns of Sterlitech filters on device Collection.It generates according to the manufacturer's instructions and breaks emulsion.

By with the paramagnetic streptavidin globule (MyOne from Dynabeads^TMGlobule) it is connected with reference to be enriched with The globule (or omitting enriching step) of the copy in library.The globule of enrichment is loaded into Ion Torrent ion-sensitive cores Piece is gone forward side by side the quasi- sequencing reaction of rower.The signal portion of the globule of enrichment is measured as the polynucleotides comprising amplification substantially The group of monoclonal such as passes through Ion Torrent PGM^TMThe sight of detectable sequencing signal from these globules on sequenator Examine what is confirmed.Signal is sequenced to measure the sequence being present in the amplicon of such globule in analysis.

Embodiment 6

It will about 120x10 with annealing buffer⁶Globule (combining forward primer) washed once.Removal supernatant (except~ Outside 50 μ L).Washed globule is resuspended in 100 μ L annealing buffers.By the about 60x10 of DNA library⁶Molecule is added to small Pearl.Final volume is adjusted to 200 μ L with annealing buffer.By being vortexed and rotating mixing globule and library.By globule/library Mixture is heated to 95-100 DEG C and is kept for 3 minutes, is then incubated 5 minutes at 37 DEG C.

The 10mM potassium acetates of 1mL are added to globule/library mixture, is then vortexed and rotates.Abandon supernatant.Weight Multiple potassium acetate washed once.Globule/DNA is resuspended in 120 μ L potassium acetates.

It recombinates enzyme source and comes from TwistAmp^TMBasic kits (come from TwistDx, Cambridge, Great Britain).Component list in the sediment through dehydration from Basic kits can be found in embodiments above 1.In list In only pipe, rehydration comes from TwistAmp in the rehydration buffer solution provided by kit of 720 μ L^TMBasic reagents 24 sediments of box.By the dNTP mixtures (each dNTP for including 10mM) of 54 other μ L added to TwistAmp^TM Mixture.

In third pipe, by the streptavidin (500 μM) of 3 μ L and the 12 biotinylated reverse primers of μ L (100 μM) mixing are subsequently transferred to globule/library pipe.Recombination enzymatic mixture is added to globule/library pipe, be then vortexed with It mixes and ice-cold.By 27 μ L T4 gp32 albumen (15/ μ g/ μ L) added to globule/library pipe, it is vortexed to mix.By 390 μ L Exemplary screening agent be added to globule/library pipe, reverse to manage and be simultaneously vortexed to mix, ice-cold at least 5 minutes.By 80 μ L acetic acid Magnesium is added to globule/library pipe, and pipe is vortexed, is rotated and at least 10 seconds ice-cold.Globule/library pipe 40 is incubated at 40 DEG C to divide Clock.Reaction is terminated by the EDTA (250mM) for adding 500 μ L.Pipe is rotated 3 minutes higher than under 18,000xG.In discarding Sediment is resuspended in the TE with 1%SDS of 1mL in clear liquid.Globule is washed in 1mL OneTouch wash solution twice. Globule is resuspended in 100 μ L.Sediment is resuspended, and pass through and add 2mL Ion Torrent by upper and lower imbibition OneTouch^TMSolution is washed to wash.It is primary to wash repeatedly step.In 300 μ L melt-ofves solution (melt-off solution) Middle resuspension sediment, and incubated 5 minutes under shake.

Embodiment 7

Nucleic acid amplification is carried out on Ion sequence testing chips.Template walking amplified reaction is carried out first on chip, with laggard The amplified reaction of row recombinase-mediated.

Prepare Ion Torrent PGM^TMSequence testing chip is to include low T_MSingle-stranded primer, the primer is in its 5 ' end It is connected to the bottom in hole.Fixed primer includes polyA (30) sequence.

Double-stranded DNA template includes the single stranded end with polyT (30) sequence and protrudes sequence.

With polymer treatment Ion Torrent PGM^TMSequence testing chip is in the bottom in hole generation matrix.Primer is captured to connect It is connected to matrix.

Ion Torrent PGM are pre-washed with the buffer solution containing TE^TMSequence testing chip is primary, and is dried in vacuo.

40 microlitres of solution is mixed and is loaded on chip.The final concentration of solution includes：From New England 1X isothermals buffer solution, the 1.6mM MgSO of Biolabs₄, 3mM dNTP, 1U/uL Bst polymerases (come from New England Biolabs), the water of 0.1nM templates and nuclease free is to 40uL volumes.Chip is centrifuged 5 minutes and incubates 30 points at 37 DEG C Clock.It is dried in vacuo chip.

Template walking amplification：40 microlitres of template walking solution is loaded on chip.The final concentration of template walking solution Comprising：1X isothermals buffer solution, 3.6mM MgSO from New England Biolabs₄, 5mM dNTP, the soluble lists of 2uM The water of strand primer, 6U/uL Bst polymerases (come from New England Biolabs) and nuclease free is to 40uL volumes.It will Chip is centrifuged and is incubated 30 minutes at 60 DEG C.Chip is washed with buffer solutions of the 1X containing TE once and is dried in vacuo.

The amplification of recombinase-mediated：The recombination enzyme source of the present embodiment comes from TwistAmp^TMBasic kits (come from TwistDx,Cambridge,Great Britain).Component list in the sediment through dehydration from Basic kits It can be found in embodiments above 1.50 microlitres of amplification reaction mixtures (including recombinase) are loaded on chip.Amplification is anti- Mixture is answered to include：From TwistAmp^TMBasic kits (come from TwistDx, Cambridge, Great Britain) A sediment, from TwistAmp^TMThe 30uL rehydration buffer solution of Basic kits, a connector with DNA profiling The soluble primer of the 2uM of hybridization and the water of nuclease free are to 50uL total volumes.Chip is centrifuged 2 minutes.By 2 microlitres of vinegar Sour magnesium (280mM stostes) is added to chip.Chip is centrifuged 2 minutes, is then incubated 1 hour at 40 DEG C.

Continuously chip is washed, and washed with washing solution with 0.5M EDTA (pH 8), the buffer solution containing TE, 1%SDS 2X。

The comparison collection of illustrative plates encoded using the color of chip determines the basic of the polynucleotides that most hole includes amplification The group of upper monoclonal.

According to Ion PI^TMManufacturer says in Sequencing 200Kit (User Guide PN MAN0007491) Bright book carries out standard sequencing reaction.Sequencing signal is analyzed by Torrent Suite Software and is present in these to measure Sequence in the amplicon of globule.Sequencing data generates the average read length of 151bp.

Embodiment 8

Directly in Ion Torrent PGM in the presence of recombinase^TMOn sequence testing chip under isothermal conditions into Row nucleic acid amplification.The hydrogel of polymerization is placed in PGM^TMIn the hole of chip.

The recombination enzyme source of the present embodiment comes from TwistAmp^TMBasic kits (TwistDx, Cambridge are come from, Great Britain).Component list in the sediment through dehydration from Basic kits can be found in embodiments above 1。

Using thermal denaturation method or recombination enzyme method (as described above) denatured polynucleotide template, nucleic acid expansion is then carried out Increase step.

(A) thermal denaturation method：Polynucleotide template library is diluted to the final volume of 60 μ L in annealing buffer.It is dilute It releases and is intended to the template merging Ion Torrent Proton of about 5 copies^TMSequence testing chip (about 600-650x10⁶A hole) it is every In a hole.It washs that chip is primary with annealing buffer, is placed in being arranged on 40 DEG C of thermal cycler.By 1:1 annealing buffer The equal portions of 100 μ L of the ratio of water are mixed and in 95 DEG C of preheatings.By template library by being placed on chip and at 95 DEG C 2 minutes are incubated to be denaturalized.Buffer solution/aqueous mixtures (being preheated to 95 DEG C) are drawn in flow cell.Chip is transferred to 40 DEG C Thermal cycler simultaneously incubates 5 minutes.Chip is transferred to experimental bench (about 25 DEG C).Chip is washed with the annealing buffer of 100 μ L. Chip is placed on 4 DEG C of thermal cyclers.Following step is optional：20 μ L water and 30 μ L by kit provide Rehydration comes from TwistAmp in rehydration buffer solution^TM1 sediment of Basic kits.Violent vortex precipitation mixture To dissolve sediment.The precipitation mixture of 50 μ L is loaded on chip.In incubation at room temperature chip at least 1 minute to allow weight Group enzyme combines the primer being preloaded into the hole of chip.

(B) recombinase denaturation method：With annealing/aqueous mixtures of 150 μ L (1 of annealing buffer and water:1 ratio) Wash Ion Torrent Proton^TMSequence testing chip (about 600-650x10⁶A hole).Chip is placed on 40 DEG C of thermal cyclers. Rehydration comes from TwistAmp in the rehydration buffer solution provided by kit of 60 μ L^TM2 precipitations of Basic kits Object.Polynucleotide template library is diluted to the final volume of 50 μ L in annealing buffer.Dilution is intended to copy about 5 Template merging Ion Torrent Proton^TMIn each hole of sequence testing chip.The diluted template of total volume is added to water again The precipitation mixture of change.Volume is adjusted to 100 μ L with water.Vortex precipitation/template is to mix and rotate.It will be by precipitation/template Mixture is loaded into Ion Torrent Proton^TMSequence testing chip (being placed on 40 DEG C of thermal cyclers) simultaneously incubates 20 minutes.It will Chip is removed from thermal cycler and is stood at room temperature.

It is following to carry out nucleic acid amplification：All reagents are kept on ice.Delay in the rehydration provided by kit of 30 μ L Rehydration comes from TwistAmp in 100 μM of reversed amplimers of fliud flushing, the nuclease-free water of 16 μ L and 1 μ L^TMBasic reagents 1 sediment of box.By being vortexed and rotating dissolving sediment.Before chip is loaded into immediately, by 280 μM of 3 μ L Magnesium acetate is added to precipitation mixture.Entire precipitation mixture is loaded on chip and is incubated 1 hour at 40 DEG C.

Chip is washed by using 0.1M EDTA (pH 8) to terminate amplified reaction.Solution washing chip is washed with chip. Chip is washed with 1%SDS.Solution washing chip is washed with TEX twice.

Chip is prepared for sequencing：Chip is washed using melt-off solution.3 juxtapositions of chip are washed with annealing buffer In on 40 DEG C of thermal cyclers.Sequencing primer is prepared in individual pipe：50% annealing buffer of 80 μ L and 50% sequencing are drawn Object mixes, and is then preheated to 95 DEG C.Prepare the 1 of annealing buffer and water:1 mixture is simultaneously preheated to 95 DEG C.Use pre-add The annealing of heat/water buffer solution washing chip.The pre-warmed primer mixture of 80 μ L is loaded into chip.Chip is incubated at 40 DEG C 5 minutes.It is primary with annealing/water buffer solution washs chip.In individual pipe, by the sequencing polymerase of 6 μ L and moving back for 57 μ L Fire/aqueous mixtures mixing, and be loaded on chip.

Standard sequencing reaction is carried out according to the manufacturer's instructions.

Claims

1. the method for the non-diagnostic purpose for nucleic acid amplification, including:

(a) at least two polynucleotides are distributed in multiple sites to support, the support, which includes first and fixes, to be drawn Object is assigned wherein the first polynucleotides are assigned to the first site and the second polynucleotides to the second site, wherein described At least two polynucleotides have different sequences, wherein at least two polynucleotides respectively contain the first primer engaging portion Position and the second primer binding site, and wherein the first primer binding site can be hybridized to the first immobilized primer, the second primer knot Close the second primer that position can be hybridized in solution；

(b) it is more at least two site by being expanded in the presence of the second primer in the first immobilized primer and solution Nucleotide forms the nucleic acid group of at least two substantially monoclonals, wherein at least two site is flowed each other during amplification Body connects, wherein the amplification betides Continuous Liquid Phase, wherein the amplification includes：By at least two polynucleotides and list A reaction mixture contact, the single reaction mixture include：Reagent, recombinase and one or more for nucleic acid synthesis Auxilin, wherein the reagent for nucleic acid synthesis includes polymerase and a variety of nucleotide, one or more auxiliary Albumen includes recombinase load factor and/or single strand binding protein；With

(c) also comprising screening agent, the screening agent limits or slows down polynucleotide amplification product leads to wherein described reaction mixture Cross reaction mixture migration.

2. the method for claim 1 wherein during amplification in the case where being not exclusively denaturalized at least two polynucleotides Carry out the amplification.

3. the method for claim 1 wherein first site and the second site are operationally coupled to sensor, and wherein The sensor energy detection site inner nucleotide is incorporated to the presence of by-product.

4. the method for claim 3, wherein it is indoor hydrophilic poly- comprising the reaction is conformally placed in react indoor each site Polymer matrix.

5. the method for claim 4, wherein the hydrophilic polymer matrix matter includes aquogel polymer matrix or wherein described Hydrophilic polymer matrix is the polymer substrate of in-situ solidifying.

6. the method for claim 4, wherein the hydrophilic polymer matrix matter includes the copolymerization of polyacrylamide, polyacrylamide Object, the derivative of polyacrylamide or combination thereof.

7. the method for claim 6, wherein the polyacrylamide is conjugated in first immobilized primer.

8. the method for claim 3, wherein the sensor includes ion-sensitive field-effect transistor.

9. the method for claim 8 further includes and detects one or more cores using the ion-sensitive field-effect transistor Thuja acid is incorporated to presence of the by-product on site, wherein the detection includes the use of the ion-sensitive field-effect transistor inspection Survey the pH variations occurred at least one reative cell.

10. the method for claim 9, further includes nucleotide introducing site；With detection from sensor by nucleotide extremely Output signal caused by being incorporated to of sequencing primer, wherein the output signal facing based on ion-sensitive field-effect transistor Starting voltage.

11. the method for claim 1 wherein the multiple sites on flow cell.

12. the method for claim 1 wherein the method for cloning expands multiple multinuclear glycosides that the multiple site includes Acid.

13. the method for claim 1 wherein the multiple sites on multiple globules.

14. the method for claim 1 wherein the recombinase load factor includes the uvsY from T4 bacteriophages and the list Chain binding protein includes the gp32 albumen from T4 bacteriophages or the modified gp32 albumen from T4 bacteriophages.

15. the method for any one of claim 1-14, wherein the reaction mixture include a concentration of 0.1 to 20% weight/ The screening agent of volume.

16. the method for any one of claim 1-14, wherein the screening agent is shown in and is dissolved in water with 2 weight percent 5 centipoises measured at 25 DEG C when middle to 15,000 centipoises average viscosity range.

17. the method for any one of claim 1-14, wherein the screening agent shows what is measured at 25 DEG C with 2% aqueous solution The average viscosity range of 10 centipoises to 10,000 centipoises.

18. the method for any one of claim 1-14, wherein the screening agent shows what is measured at 25 DEG C with 2% aqueous solution The average viscosity range of 15 centipoises to 5,000 centipoises.

19. the method for any one of claim 1-14, wherein the screening agent includes polysaccharide polymer, the polysaccharide polymer One or more polymer including being selected from cellulose, glucan, starch, glycogen, agar, chitin, pectin or agarose.

20. the method for any one of claim 1-14, wherein the screening agent includes cellulose derivative.

21. the method for claim 20, wherein the cellulose derivative is sodium carboxymethylcellulose, carboxymethyl 2- ethoxys fibre The plain sodium of dimension, methylcellulose, hydroxyethyl cellulose, 2- hydroxypropyl celluloses, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxyl second Ylmethyl cellulose, hydroxy butyl methyl cellulose, hydroxypropyl methyl cellulose or hydroxyethyl ethylcellulose.