CN109486902B

CN109486902B - Nucleic acid amplification

Info

Publication number: CN109486902B
Application number: CN201810576963.4A
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 Corp
Current assignee: Life Technologies Corp
Priority date: 2012-04-19
Filing date: 2013-03-15
Publication date: 2023-02-28
Anticipated expiration: 2033-03-15
Also published as: WO2013158313A1; CN116064734A; CN109486902A; SG11201406717RA; CN104471075A; CN114854832A; SG10201802883UA; CN104471075B

Abstract

In some embodiments, the present teachings provide methods for nucleic acid amplification comprising forming a reaction mixture, and subjecting the reaction mixture to conditions suitable for nucleic acid amplification. In some embodiments, a method for nucleic acid amplification comprises subjecting a nucleic acid to be amplified to partially denaturing conditions. In some embodiments, the method for nucleic acid amplification comprises amplification without completely denaturing the amplified nucleic acid. In some embodiments, the methods for nucleic acid amplification use an enzyme that catalyzes homologous recombination and a polymerase. In some embodiments, the method for nucleic acid amplification may be performed in a single reaction vessel. In some embodiments, the methods for nucleic acid amplification can be performed in a single continuous liquid phase of a reaction mixture without compartmentalization of the reaction mixture or immobilization of reaction components. In some embodiments, a method for nucleic acid amplification comprises amplifying at least one polynucleotide on a surface under isothermal amplification conditions, optionally in the presence of a polymer. The polymer may include a sieving agent and/or a diffusion reducing agent.

Description

Nucleic acid amplification

Background

The application is a divisional application with the application number of 201380031868.1.

Nucleic acid amplification is very useful in molecular biology and has wide applicability in virtually every aspect of biology, therapeutics, diagnostics, forensics and research. Typically, one or more primers are used to generate an amplicon from a starting template, wherein the amplicon corresponds to or is complementary to the template from which the amplicon was generated. Multiplex amplification also simplifies the process and reduces costs. The application relates to methods and reagents for nucleic acid amplification and/or analysis.

Summary of The Invention

Provided herein are methods, reagents, and products of nucleic acid amplification and/or analysis. Amplification may utilize immobilized and/or solubilized primers. A single set of primers can be mixed with different templates, or a single template can be contacted with multiple different primers, or multiple different templates can be contacted with multiple different primers. The amplicons produced from the methods provided herein are suitable substrates for further analysis, e.g., sequencing.

In some embodiments, the present teachings provide compositions, systems, methods, devices, and kits for nucleic acid amplification.

Detailed description of the drawings

FIG. 1 provides a schematic diagram showing an embodiment of template walking. In an alternative embodiment, the immobilized primer comprises the sequence designated (A) _n The adenosine-rich sequence of (A), for example ₃₀ And the primer binding site on the template for the immobilized primer comprises a complementary T-rich sequence, e.g. (T) ₃₀ 。

FIG. 2 depicts an overview of amplification walking through the template on beads and stacking of beads on a planar array for sequencing.

FIG. 3 depicts some alternative embodiments of semiconductor-based detection using sequencing-by-synthesis. The template walking can be used forClonal amplicon populations are generated on beads or on the base or bottom of a reaction chamber. In an alternative embodiment, the immobilized primer comprises the sequence designated (A) _n An adenosine-rich sequence of (A), e.g. (A) ₃₀ And the primer binding site on the template for the immobilized primer comprises a complementary T-rich sequence, e.g. (T) ₃₀ 。

Fig. 4 depicts some alternative embodiments of the fixing sites on a planar substrate in the form of primer lawn (primer lawn). An array of discrete fixation sites may be used or a single continuous lawn of primers may be considered a random array of fixation sites. Optionally, the position of one or more fixation sites in the continuous lawn of primers may not have been determined, wherein the position is determined at the time of initial template attachment prior to walking, or by the space occupied by the amplified clusters. In an alternative embodiment, the immobilized primer comprises the sequence designated (A) _n An adenosine-rich sequence of (A), e.g. (A) ₃₀ And the primer binding site on the template for the immobilized primer comprises a complementary T-rich sequence, e.g. (T) ₃₀ 。

FIG. 5 shows the effect of temperature on the walking response of the template. Delta Ct before and after template walking amplification was calculated and plotted against reaction temperature.

Figure 6 provides a table of Ct values for 96 dual TaqMan qPCR reactions.

FIG. 7 depicts data showing about 100,000-fold amplification by template walking on beads. The Δ Ct before and after the template walking reaction and the amplification fold before and after the template walking reaction were calculated and plotted for the reaction time.

Fig. 8 provides a schematic depiction of an exemplary chain flipping and walking strategy. (A) template walking, (B) strand inversion to produce an inverted strand, (C) addition of a new primer binding sequence Pg' on the final inverted strand.

FIG. 9 depicts Ion Torrent from polynucleotide templates amplified using recombinase-mediated amplification reactions ^TM Exemplary read length histograms for PGM sequencing runs.

FIG. 10 depictsIon Torrent from polynucleotide templates amplified using recombinase-mediated amplification reactions ^TM Exemplary read length histograms for Proton sequencing runs.

FIG. 11 depicts Ion Torrent from polynucleotide templates amplified using recombinase-mediated amplification reactions ^TM Exemplary read length histograms for Proton sequencing runs.

FIG. 12 depicts Ion Torrent from polynucleotide templates amplified using recombinase-mediated amplification reactions ^TM Exemplary read length histograms for Proton sequencing runs.

Fig. 13 includes an illustration of an exemplary measurement system.

Fig. 14 includes an illustration of an exemplary measurement assembly.

FIG. 15 includes an illustration of an array of exemplary measurement assemblies.

Fig. 16 includes an illustration of an exemplary pore structure.

Fig. 17 includes an illustration of an exemplary aperture and sensor configuration.

Fig. 18, 19, 20, and 21 include illustrations of the work-piece during processing by an exemplary method.

Fig. 22, 23 and 24 include illustrations of the work-piece during processing by the exemplary method.

FIGS. 25, 26, and 27 include illustrations of the work-piece during processing by the exemplary method.

FIG. 28 shows an exemplary block diagram of components of a system for nucleic acid sequencing according to an exemplary embodiment.

Fig. 29 shows an exemplary cross-sectional view of a portion of an integrated circuit device and a flow cell according to an exemplary embodiment.

FIG. 30 shows an exemplary cross-sectional view of a representative chemical sensor and corresponding reaction zone according to an exemplary embodiment.

Detailed Description

Conventional amplification of a nucleic acid template typically involves repeated replication of the template (and/or its progeny) using an appropriate synthesis system. In such conventional methods, each instance of replication is typically initiated by denaturing the template to be amplified using extreme denaturing conditions, thereby rendering the template substantially single-stranded. Some common and widely used examples of extreme denaturing conditions for conventional amplification include thermal denaturation using temperatures well above the melting temperature of the nucleic acid template to be amplified (e.g., conventional PCR involves thermal cycling using denaturation temperatures well above 90 ℃, typically about 94-95 ℃), or exposure of the template to powerful denaturing agents such as NaOH, guanidinium reagents, and the like. Such methods typically require specialized equipment (e.g., thermocyclers) and require additional operations during the amplification process (e.g., annealing steps for conventional PCR; washing steps for removing chemical denaturants, etc.), thereby increasing the cost, effort, and time associated with such amplifications, as well as limiting the yields that can ultimately be obtained using such methods. Furthermore, such extreme denaturing conditions often render the template to be amplified substantially single-stranded, thereby presenting challenges for a large number of applications involving multiple clonal amplifications (i.e., clonal amplifications of multiple different templates within the same reaction mixture). For such multiplex applications, the use of these extreme denaturing conditions may be counterproductive, as this typically results in the release of one strand of the template from its associated site, leaving the released strand free to migrate within solution and contaminate other amplicons that develop in close proximity. Such cross-contamination typically results in reduced yields of monoclonal amplified populations and increased yields of polyclonal contaminants (which are not typically available for many downstream applications). There is a need for improved nucleic acid amplification methods (and related compositions, systems, and kits) that eliminate the drawbacks associated with conventional amplification methods.

In some embodiments, the present disclosure relates generally to methods, and related compositions, systems, and devices, for nucleic acid amplification, the methods comprising amplifying a nucleic acid template to produce amplicons comprising a substantially monoclonal population of polynucleotides. It is generally considered that monoclonality is desirable in nucleic acid assays because the different properties of different polynucleotides within a polyclonal population can complicate interpretation of assay data. One example relates to nucleic acid sequencing applications, where the presence of a polyclonal population can complicate interpretation of sequencing data; however, many sequencing systems are not sufficiently sensitive to detect nucleotide sequence data from a single polynucleotide template, and therefore require clonal amplification of the template prior to sequencing.

In some embodiments, the amplification methods of the present disclosure can be used to clonally amplify two or more different nucleic acid templates, optionally using and within the same reaction mixture, to generate at least two substantially monoclonal populations of nucleic acids. Optionally, at least one substantially monoclonal population is formed by amplification of a single polynucleotide template.

Optionally, two or more different nucleic acid templates are amplified simultaneously and/or in parallel.

In some embodiments, the present disclosure relates generally to methods (and related compositions, systems, and kits) for nucleic acid synthesis, the methods comprising: providing at least two double-stranded nucleic acid templates in a reaction mixture; and clonally amplifying the at least two double-stranded nucleic acid templates according to any method disclosed herein to form at least two substantially monoclonal populations of nucleic acids.

In some embodiments, clonally amplifying optionally includes forming a reaction mixture. The reaction mixture may comprise a continuous liquid phase. In some embodiments, the continuous liquid phase comprises a single continuous aqueous phase. The liquid phase may comprise two or more polynucleotide templates, which may optionally have the same nucleotide sequence or may have nucleotide sequences that differ from each other. In some embodiments, at least one of the two or more polynucleotide templates may comprise at least one nucleic acid sequence that is substantially non-identical or substantially non-complementary to at least one other polynucleotide template within the reaction mixture.

In some embodiments, two or more different nucleic acid templates are localized, placed, or located at different sites prior to amplification.

In some embodiments, two or more different nucleic acid templates are clonally amplified, optionally within a single reaction mixture, in solution, and following such clonal amplification, the resulting two or more populations of substantially monoclonal nucleic acids are subsequently localized, placed, or located at different sites.

The different sites are optionally members of an array of sites. The array can comprise a two-dimensional array of sites on a surface (e.g., the surface of a flow cell, electronic device, transistor chip, reaction chamber, well, etc.) or a three-dimensional array of sites within a matrix or other medium (e.g., solid, semi-solid, liquid, fluid, etc.).

Optionally, two or more different nucleic acid templates are amplified within a continuous liquid phase, typically a continuous aqueous phase, of the same reaction mixture, thereby producing two or more different and substantially monoclonal polynucleotide populations, wherein each polynucleotide population is produced by amplification of a single polynucleotide template present in the reaction mixture.

Optionally, the continuous liquid phase is contained within a single phase or the same phase of the reaction mixture.

In some embodiments, the present disclosure relates generally to methods (and related compositions, systems, and kits) for nucleic acid synthesis, the methods comprising: providing a double-stranded nucleic acid template; and forming a population of substantially monoclonal nucleic acids by amplifying the double-stranded nucleic acid template. Optionally, amplifying comprises clonally amplifying the double stranded nucleic acid template.

Optionally, the amplifying comprises performing at least one round of amplification under substantially isothermal conditions.

Optionally, the amplifying comprises performing at least two consecutive cycles of nucleic acid synthesis under substantially isothermal conditions.

In some embodiments, the amplifying comprises Recombinase Polymerase Amplification (RPA). For example, amplification may include performing at least one RPA round.

In some embodiments, the amplification comprises template walking. For example, the amplification may comprise performing at least one template walking round.

In some embodiments, amplification optionally comprises performing two different amplification rounds within a site or reaction chamber. For example, amplification may comprise performing at least one RPA round in the locus or reaction chamber and at least one template walking round in the locus or reaction chamber in any order or combination of rounds. In some embodiments, at least two consecutive cycles in any one or more rounds of amplification are performed under substantially isothermal conditions. In some embodiments, at least one of the amplification rounds is performed under substantially isothermal conditions.

Optionally, the nucleic acid template to be amplified is double stranded, or the template is at least partially double stranded prior to amplification using a suitable procedure. (the template to be amplified is used interchangeably herein with a nucleic acid template or a polynucleotide template). In some embodiments, the template is linear. Alternatively, the template may be circular, or comprise a combination of linear and circular regions.

Optionally, the double-stranded nucleic acid template comprises the forward strand. The double-stranded nucleic acid template may further comprise a reverse strand. The forward strand optionally comprises a first primer binding site. The reverse strand optionally comprises a second primer binding site.

In some embodiments, the template already comprises a first and/or second primer binding site. Alternatively, the template optionally does not initially comprise a primer binding site, and the disclosed methods optionally comprise ligating or introducing a primer binding site to the template prior to amplification. For example, the method can optionally include ligating or otherwise introducing a linker comprising a primer binding site to the template. The linker may be attached to or otherwise introduced into the terminus of a linear template or into the body of a linear or circular template. Optionally, the template may be circularized after attachment or introduction of a linker. In some embodiments, a first linker may be attached to or introduced into a first end of the linear template, and a second linker may be attached to or introduced into a second end of the template.

In some embodiments, amplification comprises contacting the partially denatured template with the first primer, with the second primer, or with the first primer and the second primer, in any order or combination.

In some embodiments, the first primer comprises a first primer sequence. The first primer optionally comprises an extendable terminus (e.g., a 3' OH-containing terminus). The first primer can optionally be attached to a compound (e.g., a "drag tag") or to a support (e.g., a bead or site or the surface of a reaction chamber).

In some embodiments, the second primer comprises a second primer sequence. The second primer optionally comprises an extendable terminus (e.g., a 3' OH-containing terminus). The second primer can optionally be attached to a compound (e.g., a "resistance tag") or to a support (e.g., a bead or site or the surface of a reaction chamber).

Optionally, a first primer binds to the first primer binding site to form a first primer-template duplex. The second primer can bind to the second primer binding site to form a second primer-template duplex.

In some embodiments, amplifying comprises extending the first primer to form an extended first primer. For example, amplification can include extending a first primer of a first primer-template double strand to form an extended first primer.

Optionally, the extension is performed by a polymerase. The polymerase may be a strand displacing polymerase.

In some embodiments, amplification comprises contacting the template to be amplified with a recombinase.

In some embodiments, the amplification comprises formation of a partially denatured template. For example, amplification may comprise partially denaturing the double stranded nucleic acid template.

Optionally, partially denaturing comprises subjecting the double stranded nucleic acid template to partial denaturation conditions.

In some embodiments, the partial denaturation conditions comprise a temperature less than the Tm of the nucleic acid template, including, for example, a temperature 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, or 50 ℃ less than the Tm of the nucleic acid template. In some embodiments, the partially denaturing conditions comprise a temperature above (e.g., at least 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, or 50 ℃ higher) the Tm of the first primer, the second primer, or both the first and second primers. In some embodiments, the partial denaturation condition comprises a temperature above (e.g., at least 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, or 50 ℃ higher) the Tm of the first primer binding site, the second primer binding site, or both the first primer binding site and the second primer binding site. In some embodiments, the nucleic acid template may comprise a linker sequence at one or both ends, and the partial denaturation condition may comprise a temperature above the Tm of the linker sequence. In some embodiments, partial denaturation conditions (particularly partial denaturation temperature) are used to selectively amplify nucleic acid templates in a "template walking" process, as further described herein.

In other embodiments, the partial denaturing conditions comprise treating or contacting the nucleic acid template to be amplified with one or more enzymes capable of partially denaturing the nucleic acid template, optionally in a sequence-specific or sequence-directed manner. In some embodiments, at least one enzyme catalyzes strand invasion and/or unwinding, optionally in a sequence-specific manner. Optionally, the one or more enzymes comprise one or more enzymes selected from the group consisting of a recombinase, a topoisomerase, and a helicase. In some embodiments, partially denaturing the template may comprise contacting the template with a recombinase and forming a nucleoprotein complex comprising the recombinase. Optionally, the template is contacted with the recombinase in the presence of the first primer, the second primer, or both the first and second primers. Partial denaturation may include the use of recombinase enzymes to catalyze strand exchange and hybridization of the first primer to the first primer binding site (or hybridization of the second primer to the second primer binding site). In some embodiments, the partial denaturation comprises strand exchange using a recombinase and hybridization of the first primer to the first primer binding site and the second primer to the second primer binding site.

In some embodiments, the partially denatured template comprises a single-stranded portion and a double-stranded portion. In some embodiments, the single stranded portion comprises a first primer binding site. In some embodiments, the single stranded portion comprises a second primer binding site. In some embodiments, the single-stranded portion comprises a first primer binding site and a second primer binding site.

In some embodiments, partially denaturing the template comprises contacting the template with one or more nuclear protein complexes. At least one of the nucleoprotein complexes may comprise a recombinase. At least one of the nucleoprotein complexes can comprise a primer (e.g., a first primer or a second primer, or a primer comprising a sequence complementary to a corresponding primer binding sequence in the template). In some embodiments, partially denaturing the template may comprise contacting the template with a nucleoprotein complex comprising a primer. Partial denaturation can include hybridizing a primer of a nucleoprotein complex to a corresponding primer binding site in the template, thereby forming a primer-template duplex.

In some embodiments, partially denaturing the template may comprise contacting the template with a first nucleoprotein complex comprising a first primer. Partial denaturation can include hybridizing a first primer of a first nucleoprotein complex to a first primer binding site of the forward strand, thereby forming a first primer-template duplex.

In some embodiments, partially denaturing the template may comprise contacting the template with a second nucleoprotein complex comprising a second primer. Partial denaturation can include hybridizing a second primer of a second nucleoprotein complex to a second primer binding site of the reverse strand, thereby forming a second primer-template duplex.

In some embodiments, the disclosed methods (and related compositions, systems, and kits) can further comprise one or more primer extension steps. For example, the method can include extending the primer by incorporating the nucleotide using a polymerase.

In some embodiments, the polymerase is a strand displacing polymerase.

Optionally, extending the primer comprises contacting the primer with a polymerase and one or more types of nucleotides under nucleotide incorporation conditions. In some embodiments, the one or more types of nucleotides do not comprise an exogenous label, in particular an optically detectable label, such as a fluorescent moiety or dye. Optionally, the reaction mixture comprises nucleotides, which are naturally occurring nucleotides. Optionally, the nucleotide does not contain a group that terminates nucleic acid synthesis (e.g., a dideoxy group, a reversible terminator, etc.). Typically, extension of the primer occurs in a template-dependent manner.

Optionally, the disclosed methods (and related compositions, systems, and kits) include extending a first primer by incorporating one or more nucleotides into the first primer-template double stranded first primer using a polymerase, thereby forming an extended first primer.

Optionally, the disclosed methods (and related compositions, systems, and kits) include binding a second primer to a second primer binding site of the first extended primer by any suitable method (e.g., ligation or hybridization).

Optionally, the disclosed methods (and related compositions, systems, and kits) include extending a second primer by incorporating one or more nucleotides into the second primer-template double stranded second primer using a polymerase, thereby forming an extended second primer.

In some embodiments, extending the first primer results in the formation of a first extended primer. The first extended primer may comprise some or all of the reverse strand sequence of the template. Optionally, the first extended primer comprises a second primer binding site.

In some embodiments, extending the second primer results in the formation of a second extended primer. The second extended primer may comprise some or all of the forward strand sequence of the template. Optionally, the second extended primer comprises a first primer binding site.

In some embodiments, the method is performed without subjecting the double-stranded nucleic acid template to extreme denaturing conditions during amplification. For example, the method can be performed without subjecting the nucleic acid template to a temperature greater than or equal to the Tm of the template during amplification. In some embodiments, the method can be performed without contacting the template with a chemical denaturant, such as NaOH, urea, guanidinium salts, and the like, during amplification. In some embodiments, the amplification comprises isothermal amplification.

In some embodiments, the method is performed without subjecting the nucleic acid template to extreme denaturing conditions during at least two, three, four, or more than four consecutive cycles of nucleic acid synthesis. For example, a method can include two, three, four, or more than four consecutive cycles of nucleic acid synthesis without contacting the nucleic acid template with a chemical denaturant. In some embodiments, a method can comprise performing two, three, four, or more than four consecutive cycles of nucleic acid synthesis without subjecting the nucleic acid template to a temperature that is 25, 20, 15, 10, 5, 2, or 1 ℃ above the actual or calculated Tm of the template or population of templates (or the actual or calculated average Tm of the template or population of templates). Two, three, four, or more than four consecutive cycles of nucleic acid synthesis may include intervening partial denaturation and/or primer extension steps.

In some embodiments, the disclosed methods (and related compositions, systems, and kits) can further comprise attaching one or more extended primer strands to a support. Ligation may optionally be performed during amplification or alternatively after amplification is complete. In some embodiments, the support comprises a plurality of second primers, and the method can comprise hybridizing at least one extended first primer strand to a second primer of the support.

In some embodiments, the disclosed methods (and related compositions, systems, and kits) can further comprise attaching one or more extended second primer strands to the support. In some embodiments, the support is attached to a first primer. For example, the support may comprise a plurality of first primers, and the method may comprise hybridizing at least one extended second primer to a first primer of the support, thereby attaching the extended second primer to the support. For example, a first primer can hybridize to a first primer binding site in an extended second primer.

In some embodiments, the support is attached to a second primer. For example, the support may comprise a plurality of second primers, and the method may comprise hybridizing at least one extended first primer to a second primer of the support, thereby attaching the extended first primer to the support. For example, a first primer can hybridize to a second primer binding site in an extended first primer.

In some embodiments, the support comprises at least one first primer and at least one second primer, and the disclosed methods (and related compositions, systems, and kits) comprise attaching the extended first primer and the extended second primer to the support.

Optionally, the support is attached to a target-specific primer. The target-specific primers optionally hybridize (or are capable of hybridizing) to a first subset of templates within the reaction mixture, but are not capable of binding to a second subset of templates within the reaction mixture.

Optionally, the support is attached to a universal primer. The universal primer optionally hybridizes (or is capable of hybridizing) to all or substantially all of the templates within the reaction mixture.

Optionally, the reaction mixture comprises a first support covalently attached to a first target-specific primer and a second support covalently attached to a 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 substantially complementary to a first target nucleic acid sequence and the second target-specific primer is substantially complementary to a second target nucleic acid sequence, and wherein the first and second target nucleic acid sequences are different.

In some embodiments, the disclosed methods comprise forming a first amplicon on a first support by amplifying a first template and a second amplicon on a second support by amplifying a second template, optionally within the same continuous phase of the reaction mixture. The first amplicon is optionally linked or attached to a first support, and the second amplicon is optionally linked or attached to a second support.

The disclosed methods optionally include generating two or more monoclonal or substantially monoclonal amplicons by clonally amplifying two or more polynucleotide templates. Optionally clonally amplifying two or more polynucleotide templates within a continuous liquid phase of an amplification reaction mixture. The continuous liquid phase of the amplification reaction mixture may comprise a continuous aqueous phase. In some embodiments, amplifying comprises generating at least two substantially monoclonal amplified polynucleotide populations, each of the polynucleotide populations formed by amplification of a single polynucleotide template. Optionally, the clonally amplifying comprises at least one RPA round. Optionally, the clonally amplifying comprises at least one template walking round.

In some embodiments, amplifying optionally comprises forming an amplification reaction mixture comprising a continuous liquid phase. In some embodiments, the continuous liquid phase is a single continuous aqueous phase. The liquid phase may comprise two or more polynucleotide templates, which may optionally be different from each other. For example, the two or more polynucleotide templates may comprise at least one nucleic acid sequence that is substantially non-identical or substantially non-complementary to at least one other polynucleotide template within the amplification reaction mixture.

In some embodiments, amplifying optionally comprises forming an amplification reaction mixture comprising a single continuous aqueous phase having two or more polynucleotide templates. Amplification optionally includes forming two or more substantially monoclonal populations of nucleic acids by clonally amplifying two or more polynucleotide templates within a single aqueous phase. Optionally, the clonally amplifying comprises at least one RPA round. Optionally, the clonally amplifying comprises at least one template walking round.

In some embodiments, the present disclosure generally relates to methods (and related compositions, systems, and kits) for optionally parallel amplification of one or more nucleic acid templates using partial denaturation conditions. In some embodiments, such methods are optionally used to amplify two or more templates in an array format. Optionally, the templates are amplified in bulk in solution prior to dispensing into the array. Alternatively, the template is first assigned to a site in the array, followed by amplification of the template in situ at (or within) the site of the array.

Optionally, the template is single-stranded or double-stranded. The template optionally comprises one or more primer binding sites.

In some embodiments, a method may comprise subjecting a double-stranded nucleic acid template comprising a primer binding site on at least one strand to at least one template-based replication cycle using a polymerase.

Optionally, at least one template-based replication cycle comprises a partial denaturation step, an annealing step and an extension step.

In some embodiments, the method comprises amplifying the double-stranded nucleic acid template by subjecting the template to at least two consecutive template-based replication cycles.

In some embodiments, the method comprises partially denaturing the template. Optionally, the method comprises forming a partially denatured template comprising a single stranded region. The partially denatured template may also comprise a double-stranded region. The single-stranded region may comprise a primer binding site.

Optionally, partially denaturing comprises subjecting the template to a temperature at least 20, 15, 10, 5, 2, or 1 ℃ lower than the Tm of the primer binding site.

Optionally, partially denaturing comprises subjecting the template to a temperature that is the same as or higher than the Tm of the primer binding site.

Optionally, partially denaturing comprises contacting the double stranded template with a recombinase and a primer. The recombinase and the primer may form part of a nucleoprotein complex, and partial denaturation comprises contacting the template with the complex.

In some embodiments, the method comprises forming a primer-template duplex by hybridizing a primer to a primer binding site of the single-stranded region. In some embodiments, the template to be primed comprises a double-stranded region. Optionally, the double-stranded region does not comprise a primer binding site.

In some embodiments, the method comprises extending the primer of the primer-template double strand. Optionally, the method comprises forming an extended primer.

In some embodiments, different templates can be clonally amplified on different discrete supports (inflow beads or particles) without partitioning prior to amplification. In other embodiments, the template is partitioned or partitioned into an emulsion prior to amplification. Optionally, the template is dispensed into the microdroplet, forming part of the hydrophilic phase of an emulsion having a discontinuous hydrophilic phase and a continuous hydrophobic phase. In some embodiments, the emulsion droplets of the hydrophilic phase further comprise one or more components necessary for performing PRA. For example, the emulsion droplets may comprise a recombinase. Optionally, the microdroplet comprises a strand displacing polymerase. In some embodiments, the droplet comprises a support-immobilized primer and/or a solution-phase primer. Optionally, the primer may bind to the template or to its amplification product.

In some embodiments, the compositions, systems, methods, devices, and kits for nucleic acid amplification comprising nucleic acid synthesis following partial denaturation of template using emulsion-based amplification disclosed herein provide advantageous aspects over conventional expansion methods, including emulsion-based PCR or emPCR involving traditional thermocycling. For example, a nucleic acid amplification reaction that includes emulsion-based RPA ("emRPA") or emulsion-based template walking can produce longer amplified polynucleotide templates, have fewer amplification steps, reduced time to prepare amplified polynucleotide templates, and/or increased sequencing data quality. Some suitable emulsion compositions for use with the amplification methods disclosed herein can be found, for example, in U.S. patent nos. 7622280, 7601499 and 7323305, which are incorporated herein by reference in their entirety.

In some embodiments, a method comprises providing a double-stranded template comprising a forward strand comprising a first primer binding site, a reverse strand comprising a second primer binding site; partially denaturing the template and forming a partially denatured template comprising a single-stranded region comprising the first primer binding site and at least one double-stranded region; forming a first primer-template duplex by hybridizing a first primer to the first primer binding site of the single-stranded region; extending a first primer of the first primer-template double strand using a polymerase to form an extended first primer comprising a second primer binding site, wherein the extended first primer at least partially hybridizes to the forward strand of the template; partially denaturing the extended first strand from the template to form a single-stranded region comprising a second primer binding site; hybridizing a second primer to the second primer binding site of the single stranded region and forming a second primer-template duplex, and extending the second primer of the second primer-template duplex, thereby forming an extended second primer.

In some embodiments, the present disclosure relates generally to methods (and related compositions, systems, and kits) for methods of synthesizing nucleic acids from a nucleic acid template, comprising: providing a first nucleic acid duplex comprising a forward strand and a reverse strand, wherein the forward strand comprises a forward primer binding site and the reverse strand comprises a reverse primer binding site, and wherein the first duplex has a first melting temperature ("template Tm"), the forward primer binding site has a second melting temperature ("forward primer Tm"), and the reverse primer binding site has a third melting temperature ("reverse primer Tm"); partially denaturing the first duplex, wherein the partially denatured first duplex comprises a single-stranded region comprising a forward primer binding site and at least one duplex region; forming a primed first duplex by hybridizing a forward primer to the forward primer binding site of the partially denatured first duplex; extending the forward primer by contacting the primed first duplex with a strand displacing polymerase and nucleotides under primer extension conditions, thereby forming a second duplex having a fourth melting temperature ("fourth Tm"), the second duplex comprising one strand comprising a forward primer binding site and one strand comprising a reverse primer binding site; partially denaturing the second double strand, wherein the partially denatured second double strand comprises a single-stranded region comprising a reverse primer binding site and at least one double-stranded region; forming a reverse priming second duplex by hybridizing a reverse primer to the reverse primer binding site of the partially denatured second duplex; extending the reverse primer of the reverse priming second double strand by contacting the reverse priming second double strand with a strand displacing polymerase and nucleotides under primer extension conditions.

In some embodiments, methods (and related compositions, systems, and kits) may further include sequencing the amplified template or sequencing the extended primer (e.g., the extended first primer or the extended second primer). Sequencing may comprise any suitable sequencing method known in the art. In some embodiments, sequencing comprises sequencing by synthesis or sequencing by electronic detection (e.g., nanopore sequencing). In some embodiments, sequencing comprises extending the template or amplified template or extending a sequencing primer that hybridizes to the template or amplified template by polymerase-mediated nucleotide incorporation. In some embodiments, sequencing comprises sequencing the template attached to the support or the amplified template by contacting the template or extended primer with a sequencing primer, a polymerase, and at least one type of nucleotide. In some embodiments, sequencing comprises contacting the template or amplified template or extended primer with a sequencing primer, a polymerase, and with only one type of nucleotide that does not comprise an exogenous label or chain termination group.

Optionally, the template (or amplified product) may be placed, localized or located at a site. In some embodiments, the plurality of templates/amplified templates/extended first primers are placed or located at different sites in the array of sites. In some embodiments, the placement, localization, or localization is performed prior to template amplification. In some embodiments, the placing, locating, or localizing is performed after the amplifying. For example, the amplified template or extended first primer may be placed, located or localized at different sites of the array.

The methods disclosed herein result in the production of a plurality of amplicons, at least some of which include a clonally amplified population of nucleic acids. The clonally amplified populations produced by the methods of the present disclosure may be used for a variety of purposes. In some embodiments, the disclosed methods (and related compositions, systems, and kits) optionally further comprise analysis and/or processing of the clonally amplified populations (amplicons). For example, in some embodiments, the number of amplicons that exhibit certain desired properties can be detected and optionally quantified.

In some embodiments, the method can include determining which discrete supports (e.g., beads) comprise amplicons. Similarly, the method may comprise determining which sites of the array contain amplicons. Optionally using DNA-based detection procedures such as UV absorption, staining with DNA-specific dyes,

Assays, qPCR, hybridization to fluorescent probes, etc. to detect the presence of amplicons on the assay support or site. In some embodiments, the method can include determining which bead supports (or sites of the array) have resulted in substantially monoclonal amplicons. For example, the bead supports (or array sites) can be analyzed to determine which supports or sites can produce a detectable and coherent (i.e., analyzable) sequence-dependent signal.

In some embodiments, the amplification is followed by sequencing of the amplified product. The amplified product that is sequenced can include an amplicon comprising a substantially monoclonal population of nucleic acids. In some embodiments, the disclosed methods comprise forming or localizing individual members of multiple amplicons to different sites. The different sites optionally form part of an array of sites. In some embodiments, the sites in the array of sites comprise wells (reaction chambers) on the face of the isFET array.

In some embodiments, the method of downstream analysis comprises sequencing at least some of the plurality of amplicons in parallel. Optionally, multiple templates/amplified templates/extended first primers at different array sites are sequenced in parallel.

In some embodiments, sequencing may comprise binding sequencing primers to nucleic acids of at least two different amplicons or at least two different substantially monoclonal populations.

In some embodiments, sequencing may comprise incorporating the nucleotides into a sequencing primer using a polymerase. Optionally, incorporating comprises forming at least one nucleotide incorporation byproduct.

Optionally, the nucleic acid to be sequenced is located at a site. The site may comprise a reaction chamber or well. The sites may be part of an array of similar or identical sites. The array can comprise a two-dimensional array of sites on a surface (e.g., the surface of a flow cell, electronic device, transistor chip, reaction chamber, well, etc.) or a three-dimensional array of sites within a matrix or other medium (e.g., solid, semi-solid, liquid, fluid, etc.).

In some embodiments, the site is operably coupled to a sensor. The method can include detecting nucleotide incorporation using a sensor. Optionally, the sites and sensors are located in an array of sites coupled to the sensors.

In some embodiments, the site comprises a hydrophilic polymer matrix conformally disposed within a well operatively coupled to the sensor.

Optionally, the hydrophilic polymer matrix comprises a hydrogel polymer matrix.

Optionally, the hydrophilic polymer matrix is an in situ cured polymer matrix.

Optionally, the hydrophilic polymer matrix comprises polyacrylamide, copolymers thereof, derivatives thereof, or combinations thereof.

Optionally, the polyacrylamide is conjugated to an oligonucleotide primer.

Optionally, the pores have a characteristic diameter of 0.1 to 2 microns.

Optionally, the pores have a depth of 0.01 microns to 10 microns.

In some embodiments, the sensor comprises a Field Effect Transistor (FET). The FET may comprise an Ion Sensitive FET (ISFET).

In some embodiments, methods (and related compositions, systems, and kits) can include detecting the presence of one or more nucleotide incorporation by-products at an array site, optionally using a FET.

In some embodiments, the method may include detecting a pH change occurring within at least one reaction chamber, optionally using a FET.

In some embodiments, the disclosed methods comprise introducing a nucleotide into the site; and detecting an output signal from the sensor due to incorporation of the nucleotide into the sequencing primer. The output signal is optionally based on the threshold voltage of the FET. In some embodiments, the FET includes a floating gate conductor coupled to the site.

In some embodiments, the FET includes a floating gate structure comprising a plurality of conductors electrically coupled to each other and separated by a dielectric layer, and the floating gate conductor is the uppermost conductor of the plurality of conductors.

In some implementations, the floating gate conductor includes an upper surface that defines a bottom surface of the site.

In some embodiments, the floating gate conductor comprises a conductive material and the upper surface of the floating gate conductor comprises an oxide of the conductive material.

In some embodiments, the floating gate conductor is coupled to at least one of the reaction chambers through the sensing material.

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

In some embodiments, the amplification reaction mixture may comprise a recombinase. The recombinase may comprise any suitable agent that promotes recombination between polynucleotide molecules. The recombinase may be an enzyme that catalyzes homologous recombination. For example, the amplification reaction mixture may comprise a recombinase comprising or derived from a bacterium, eukaryote, or virus (e.g., bacteriophage).

In some embodiments, the amplification reaction mixture comprises an enzyme that can bind a primer and a polynucleotide template to form a complex or can catalyze strand invasion of a polynucleotide template to form a D-loop structure. In some embodiments, the amplification reaction mixture comprises one or more proteins selected from the group consisting of UvsX, recA, and Rad 51.

In some embodiments, the amplification reaction mixture may comprise a recombinase helper protein, such as UvsY.

In some embodiments, the amplification reaction mixture may comprise a single-stranded binding protein (SSBP).

In some embodiments, the amplification reaction mixture may comprise a polymerase. The polymerase optionally has or lacks exonuclease activity. In some embodiments, the polymerase has 5 'to 3' exonuclease activity, 3 'to 5' exonuclease activity, or both. Optionally, the polymerase lacks any one or more of such exonuclease activities.

In some embodiments, the polymerase has strand displacement activity.

In some embodiments, the amplification reaction mixture may comprise one or more solid or semi-solid supports. At least one of the supports may comprise one or more first primers comprising a first primer sequence. In some embodiments, at least one polynucleotide in the reaction mixture comprises a first primer binding sequence. The first primer binding sequence can be substantially identical or substantially complementary to the first primer sequence. In some embodiments, at least one, some, or all of the supports comprise a plurality of first primers that are substantially identical to each other. In some embodiments, all primers on the support are substantially identical to each other, or all primers comprise substantially identical first primer sequences.

In some embodiments, at least one support comprises two or more different primers attached thereto. For example, at least one support can comprise at least one first primer and at least one second primer.

In some embodiments, the aqueous phase of the reaction mixture comprises a plurality of supports, wherein at least two supports of the plurality of supports are attached to a primer comprising the first primer sequence. In some embodiments, the reaction mixture comprises two or more different polynucleotide templates having a first primer binding sequence.

In some embodiments, the amplification reaction mixture may comprise a diffusion limiting agent. The diffusion limiting agent can be any agent that is effective to prevent or slow diffusion of one or more of the polynucleotide templates and/or one or more of the amplification reaction products through the amplification reaction mixture.

In some embodiments, the amplification reaction mixture may comprise a sieving agent. The sieving agent can be any agent effective to sieve one or more polynucleotides (e.g., amplification reaction products and/or polynucleotide templates) present in an amplification reaction mixture. In some embodiments, the sieving agent limits or slows the migration of polynucleotide amplification products through the reaction mixture.

In some embodiments, the amplification reaction mixture may comprise crowding reagents.

In some embodiments, the amplification reaction mixture comprises a crowding reagent and a sieving reagent.

In some embodiments, the disclosed methods comprise clonally amplifying at least two of the two or more polynucleotide templates by: (a) Forming an amplification reaction mixture comprising a single continuous liquid phase comprising two or more polynucleotide templates, one or more surfaces or supports, and amplification components; and (b) clonally amplifying at least two of the polynucleotide templates on one or more supports. Optionally, clonally amplifying includes forming at least two different substantially monoclonal amplicons. In some embodiments, clonally amplifying comprises subjecting the amplification reaction mixture to amplification conditions. In some embodiments, two or more of the amplicons are each attached to a surface or support. For example, an amplification reaction mixture may comprise a single support or surface such that each polynucleotide template is attached to a given region of the support or surface.

In some embodiments, the methods for nucleic acid amplification can be performed in a single reaction vessel.

In some embodiments, the method for nucleic acid amplification may be performed in a single continuous liquid phase that does not provide for partitioning of multiple nucleic acid amplification reactions occurring in a single reaction vessel. In some embodiments, the method for nucleic acid amplification may be performed in a water-in-oil emulsion (micro-reaction vessel) providing partitioning.

In some embodiments, methods for nucleic acid amplification can be performed to attach a plurality of polynucleotides to a support or surface. For example, a method can include forming a reaction mixture comprising at least one surface, and subjecting the reaction mixture to amplification conditions. In some embodiments, the surface comprises a surface of a bead, a planar surface or an inner wall of a groove or tube.

In some embodiments, a method for nucleic acid amplification comprises: (a) Forming an amplification reaction mixture comprising a single continuous liquid phase comprising a plurality of beads, a plurality of different polynucleotides, and a recombinase; (b) Subjecting the amplification reaction mixture to isothermal amplification conditions, thereby generating a plurality of beads attached to a population of substantially monoclonal nucleic acids attached thereto.

In some embodiments, the present disclosure relates generally to methods (and related compositions, systems, and kits) for performing array-based amplification of nucleic acid templates directly on the surface of an array, resulting in the formation of an array of any of its individual features including amplicons (comprising a population of substantially monoclonal amplification products). These embodiments are in contrast to other embodiments described herein in which nucleic acid templates are optionally amplified in solution on discrete supports (e.g., beads) and subsequently dispensed into an array.

In some embodiments, methods (and related compositions, systems, and kits) for array-based amplification are provided. In some embodiments, different polynucleotide templates are assigned to the array of sites and subsequently amplified in situ. The resulting amplified subarrays are then analyzed using appropriate downstream procedures.

In some embodiments, a method for nucleic acid amplification comprises: a) Assigning at least two different polynucleotides to sites by introducing a single polynucleotide into at least two sites in fluid communication with each other; and (b) forming at least two substantially monoclonal populations of nucleic acids by amplifying the polynucleotides within the at least two sites. The site may optionally include a surface, well, trench, flow cell, reaction chamber, or tank. In some embodiments, amplification is performed without blocking the sites from each other. For example, at least two sites may be in fluid communication with each other during amplification.

In some embodiments, a method for nucleic acid amplification comprises: a) Assigning at least two different polynucleotides to the sites by introducing a single polynucleotide into at least two sites; and (b) forming at least two substantially monoclonal populations of nucleic acids by amplifying the polynucleotides within the at least two sites. The site may optionally include a surface, well, trench, flow cell, reaction chamber, or tank. In some embodiments, amplification is performed without blocking the sites from each other. For example, at least two sites can be in fluid communication with each other during amplification.

In some embodiments, the site comprises a reaction chamber, and the method for nucleic acid amplification comprises: a) Partitioning at least two polynucleotide templates into reaction chambers by introducing a single polynucleotide into at least two reaction chambers in fluid communication with each other; and (b) forming at least two substantially monoclonal populations of nucleic acids by amplifying the polynucleotide templates within the at least two reaction chambers. In some embodiments, amplification is performed without closing the reaction chambers from each other. For example, at least two reaction chambers can be in fluid communication with each other during amplification.

In some embodiments, the site comprises a reaction chamber and the method for nucleic acid amplification comprises: a) Partitioning at least two different polynucleotides into reaction chambers by introducing a single polynucleotide into at least two reaction chambers in fluid communication with each other; and (b) forming at least two substantially monoclonal populations of nucleic acids by amplifying the polynucleotides within the at least two reaction chambers. In some embodiments, amplification is performed without closing the reaction chambers from each other. For example, at least two reaction chambers can be in fluid communication with each other during amplification.

In some embodiments, the amplification steps of any and all methods of the present disclosure can be performed without completely denaturing the polynucleotide during amplification. For example, the disclosed methods can include amplifying at least two different polynucleotides by isothermal amplification. Amplification may comprise amplifying at least two different polynucleotides under substantially isothermal conditions. Optionally, the amplification is performed without contacting the polynucleotide with a chemical denaturant during amplification.

In some embodiments, amplifying comprises contacting the polynucleotide to be amplified with a reaction mixture. Contacting can optionally be done before or after dispensing; it will be understood that the present disclosure includes embodiments in which the polynucleotides are contacted with each component (or combination of components) of the reaction mixture sequentially at different times, as well as embodiments in which any one or some components of the reaction mixture are contacted with at least two different polynucleotides prior to partitioning and the remainder of the reaction mixture are contacted with at least two different polynucleotides after partitioning.

The allocated at least two polynucleotides may optionally be used as templates for nucleic acid synthesis in their respective reaction chambers. In some embodiments, at least two polynucleotides comprise different sequences. In some embodiments, the polynucleotide is double stranded or as single stranded prior to partitioning. In some embodiments, the polynucleotide is linear, circular, or a combination of both. In typical embodiments, the polynucleotide is at least partially double stranded (or partitioned in single stranded form and then rendered at least partially double stranded within the site or reaction chamber after partitioning). The polynucleotide may be made double stranded prior to amplification (particularly in embodiments where amplification includes RPA or template walking).

In some embodiments, the at least two different polynucleotide templates to be amplified each comprise a primer binding site, and amplifying comprises binding a primer to the primer binding site to form a primer-template duplex.

Optionally, amplifying comprises extending the primer-template double strand primer. Extension optionally occurs at or within the site or reaction chamber of the array. Optionally, extending the primer comprises contacting the primer with a polymerase and one or more types of nucleotides under nucleotide incorporation conditions. In some embodiments, the one or more types of nucleotides do not comprise an exogenous label, in particular an optically detectable label, such as a fluorescent moiety or dye. Optionally, the reaction mixture comprises nucleotides, which are naturally occurring nucleotides. Optionally, the nucleotide does not contain a group that terminates nucleic acid synthesis (e.g., a dideoxy group, a reversible terminator, etc.). Typically, extension of the primer occurs in a template-dependent manner.

Optionally, at least two different polynucleotides (i.e., the templates to be amplified) each comprise a first sequence (referred to as a "first primer binding site") that is substantially identical or substantially complementary to at least some portion of the first primer.

In some embodiments, the reaction mixture comprises a first primer comprising a first primer sequence. The first primer optionally comprises an extendable terminus (e.g., a 3' OH-containing terminus). The first primer can optionally be attached to a compound (e.g., a "resistance tag") or to a support (e.g., a bead or site or the surface of a reaction chamber).

In some embodiments, the at least two different polynucleotides comprise a second sequence (referred to as a "second primer binding site") that is substantially identical or substantially complementary to at least some portion of the second primer.

In some embodiments, the second primer comprises a second primer sequence. The second primer optionally comprises an extendable terminus (e.g., a 3' OH-containing terminus). The second primer can optionally be attached to a compound (e.g., "resistance tag") or to a support (e.g., a bead or site or a surface of a reaction chamber).

In some embodiments, the method comprises extending the second primer by incorporating one or more nucleotides into the second primer of the second primer-template double strand using a polymerase, thereby forming an extended second primer.

In some embodiments, the method is performed without subjecting the double-stranded nucleic acid template to extreme denaturing conditions during amplification. For example, the method can be performed without subjecting the nucleic acid template to a temperature greater than or equal to the Tm of the template during amplification. In some embodiments, the method can be performed without contacting the template with a chemical denaturant, such as NaOH, urea, guanidinium, and the like, during amplification. In some embodiments, the amplifying comprises isothermal amplification.

In some embodiments, the method is performed without subjecting the nucleic acid template to extreme denaturing conditions during at least two, three, four, or more than four consecutive cycles of nucleic acid synthesis. For example, a method may comprise two, three, four, or more than four sequential nucleic acid synthesis steps without contacting the nucleic acid template with a chemical denaturant. In some embodiments, a method can comprise performing two, three, four, or more than four consecutive cycles of nucleic acid synthesis without subjecting the nucleic acid template to a temperature that is 25, 20, 15, 10, 5, 2, or 1 ℃ above the actual or calculated Tm of the template or population of templates (or the actual or calculated average Tm of the template or population of templates). Two, three, four, or more than four consecutive cycles of nucleic acid synthesis may include intervening partial denaturation and/or primer extension steps.

In some embodiments, the disclosed methods (and related compositions, systems, and kits) can further comprise attaching one or more extended second primer strands to the support. In some embodiments, the support is attached to a first primer. For example, the support may comprise a plurality of first primers, and the method may comprise hybridizing at least one extended second primer to a first primer of the support, thereby attaching the extended second primer to the support. For example, a first primer can hybridize to a first primer binding site in an extended second primer. The support may comprise, for example, any array of surfaces.

In some embodiments, the disclosed methods comprise forming a first amplicon by amplifying a first template on a first support and a second amplicon by amplifying a second template on a second support, optionally within the same continuous phase of the reaction mixture and at different sites on a surface (e.g., within an array). The first amplicon is optionally linked or attached to a first support, and the second amplicon is optionally linked or attached to a second support.

The disclosed methods optionally include generating two or more monoclonal or substantially monoclonal amplicons at two or more different sites of the site array by clonally amplifying two or more polynucleotide templates, such that at least two sites are formed to each comprise a substantially monoclonal population of nucleic acids. Two or more polynucleotide templates are optionally placed or located at different sites and then clonally amplified within a continuous liquid phase of an amplification reaction mixture that is contacted with the array. The continuous liquid phase of the amplification reaction mixture may comprise a continuous aqueous phase.

In some embodiments, amplifying comprises generating at least two substantially monoclonal amplified polynucleotide populations, each of the polynucleotide populations formed by amplification of a single polynucleotide template.

Optionally, the clonally amplifying comprises at least one RPA round.

Optionally, clonally amplifying includes at least one template walking round.

In some embodiments, amplifying optionally comprises forming an amplification reaction mixture comprising a single continuous aqueous phase having two or more polynucleotide templates. Amplification optionally includes forming two or more substantially monoclonal populations of nucleic acids by clonally amplifying two or more polynucleotide templates within a single aqueous phase. Optionally, clonally amplifying includes at least one round of RPA. Optionally, the clonally amplifying comprises at least one template walking round.

In some embodiments, a plurality of different polynucleotide templates are placed or located at different sites prior to amplification. For example, the amplified template or extended first primer may be placed, located or localized at a different site of the array.

In some embodiments, amplification results in the formation of at least two substantially monoclonal populations of nucleic acids (e.g., amplicons) in at least two different sites on the surface, which can then be analyzed in situ using an appropriate procedure.

In some embodiments, the present disclosure relates generally to methods (and related compositions, systems, and kits) for preparing a surface. Optionally, the surface comprises a plurality of sites comprising a first site and a second site.

In some embodiments, a method comprises forming an array of nucleic acids on a surface, wherein the forming comprises attaching a first nucleic acid to a first site and attaching a second nucleic acid to a second site. The linking can optionally be performed by using any of the methods disclosed herein, including, for example, by linking the nucleic acid to a primer covalently attached to the surface.

In some embodiments, the method comprises contacting at least a first and a second nucleic acid with a single reaction mixture comprising reagents for nucleic acid synthesis. The reaction mixture may optionally comprise any one or more of the components described herein. In some embodiments, the reaction mixture comprises all of the components necessary to carry out RPA. In some embodiments, the reaction mixture comprises all of the components that perform the template walk.

In some embodiments, the method comprises forming a first amplicon at the first site and a second amplicon at the second site by replicating the first or second nucleic acid using reagents for nucleic acid synthesis in the reaction mixture. Replication may include primer extension. Replication may include one or more RPA cycles. Replication may include one or more template walking cycles.

In some embodiments, the replicating includes at least one RPA cycle.

In some embodiments, replication comprises at least one template walking cycle.

In some embodiments, replication comprises at least one RPA cycle and at least one template walk cycle.

In some embodiments, the replicating includes at least one RPA round.

In some embodiments, the replicating includes at least one template walking round.

In some embodiments, the replicating includes at least one RPA round and at least one template walking round.

In some embodiments, the present disclosure relates generally to methods (and related compositions, systems, and kits) for preparing a surface, the methods comprising: (a) Providing a surface having a plurality of sites comprising a first site and a second site; (b) Forming an array of nucleic acids on a surface, wherein the forming comprises attaching a first nucleic acid to a first site and attaching a second nucleic acid to a second site; (c); and (d) forming a first amplicon at the first site and a second amplicon at the second site by replicating the first or second nucleic acid using reagents for nucleic acid synthesis in the reaction mixture.

In some embodiments, the present disclosure relates generally to a method for preparing a surface, the method comprising: providing a surface having a plurality of sites comprising a first site and a second site; forming an array of nucleic acids on a surface, wherein the forming comprises attaching a first nucleic acid to a first site and attaching a second nucleic acid to a second site; contacting at least first and second nucleic acids with a single reaction mixture comprising reagents for nucleic acid synthesis; and forming a first substantially monoclonal amplicon at the first site and a second substantially monoclonal amplicon at the second site by amplifying the first or second nucleic acid using reagents for nucleic acid synthesis in the reaction mixture. Optionally, the first and second sites remain in fluid communication during amplification. Optionally, amplification is performed without completely denaturing the polynucleotide during amplification. For example, the disclosed methods can include amplifying at least two different polynucleotides by isothermal amplification. Amplification may comprise amplifying at least two different polynucleotides under substantially isothermal conditions. Optionally, the amplification is performed without contacting the polynucleotide with a chemical denaturant during amplification.

In some embodiments, at least one site of the plurality of sites comprises a reaction well, a trench, or a chamber.

In some embodiments, at least one site of the plurality of sites is attached to a sensor.

In some embodiments, the sensor is capable of detecting nucleotide incorporation that occurs at or near the at least one site.

In some embodiments, the sensor comprises a Field Effect Transistor (FET).

In some embodiments, at least the first site or the second site or the first and second sites comprise a primer attached to the surface.

In some embodiments, at least one site of the plurality of sites comprises a hydrophilic polymer matrix conformally disposed within a well operatively coupled to the sensor.

Optionally, the hydrophilic polymer matrix comprises a hydrogel polymer matrix.

Optionally, the hydrophilic polymer matrix is an in situ cured polymer matrix.

Optionally, the polyacrylamide is conjugated to an oligonucleotide primer.

Optionally, the pores have a characteristic diameter of 0.1 to 2 microns.

Optionally, the pores have a depth of 0.01 microns to 10 microns.

In some embodiments, the sensor comprises a Field Effect Transistor (FET). The FETs can include Ion Sensitive FETs (ISFETs), chemFETs, bioFETs, and the like.

In some embodiments, the FET is capable of detecting the presence of nucleotide incorporation byproducts at least one site.

In some embodiments, the FET is capable of detecting a chemical moiety selected from hydrogen ions, pyrophosphates, hydroxyl ions, and the like.

In some embodiments, the method may comprise detecting a pH change occurring at the site or within the at least one reaction chamber, optionally using a FET.

In some embodiments, the disclosed methods comprise introducing a nucleotide into at least one site of the plurality of sites; and detecting an output signal from the sensor due to incorporation of the nucleotide into the sequencing primer. The output signal is optionally based on a threshold voltage of the FET. In some embodiments, the FET includes a floating gate conductor coupled to the site.

In some implementations, the floating gate conductor includes an upper surface defining a bottom surface of the site.

In some implementations, the floating gate conductor comprises a conductive material and the upper surface of the floating gate conductor comprises an oxide of the conductive material.

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

In some embodiments, the reaction mixture comprises all of the components necessary to carry out RPA.

In some embodiments, the reaction mixture comprises all of the components necessary to perform the template walk.

In some embodiments, the reaction mixture may comprise one or more solid or semi-solid supports. At least one of the supports can include one or more first primers comprising a first primer sequence. In some embodiments, at least one of the supports comprises two or more different primers attached thereto. For example, at least one support can comprise at least one first primer and at least one second primer.

Alternatively, in some embodiments, the reaction mixture does not comprise any support. In some embodiments, at least two different polynucleotide templates are amplified directly on the surface of the reaction chamber or the sites of the array.

In some embodiments, the reaction mixture may comprise a recombinase. The recombinase may comprise any suitable agent that promotes recombination between polynucleotide molecules. The recombinase may be an enzyme that catalyzes homologous recombination. For example, the reaction mixture may comprise a recombinase comprising or derived from a bacterium, eukaryote, or virus (e.g., bacteriophage).

Optionally, the reaction mixture comprises nucleotides that are not exogenously labeled. For example, the nucleotide may be a naturally occurring nucleotide, or a synthetic analog that does not contain a fluorescent moiety, dye, or other exogenous optically detectable label. Optionally, the reaction mixture comprises nucleotides, which are naturally occurring nucleotides. Optionally, the nucleotide does not contain a group that terminates nucleic acid synthesis (e.g., a dideoxy group, a reversible terminator, etc.).

Optionally, the reaction mixture comprises nucleotides, which are naturally occurring nucleotides. Optionally, the nucleotide does not contain a group that terminates nucleic acid synthesis (e.g., a dideoxy group, a reversible terminator, etc.).

In some embodiments, the reaction mixture comprises an enzyme that can bind the primer and the polynucleotide template to form a complex or can catalyze strand invasion of the polynucleotide template to form a D-ring structure. In some embodiments, the reaction mixture comprises one or more proteins selected from the group consisting of UvsX, recA, and Rad 51.

In some embodiments, the present disclosure relates generally to methods (and related compositions, systems, and kits) for preparing a surface, the method comprising: (a) Providing a surface having a plurality of sites, wherein each site is attached to a nucleic acid primer; (c) Contacting the surface with a plurality of polynucleotide templates and attaching at least one template to the surface; and amplifying the at least one template on the surface, thereby forming at least one population of substantially monoclonal amplified target polynucleotide sequences located at sites on the surface.

In some embodiments, the present disclosure relates generally to methods for nucleic acid amplification, comprising: (1) Providing a surface having a first site and a second site, the first site operably coupled to a first sensor and comprising a first template; a second site operably coupled to a second sensor and comprising a second template; (2) partitioning the reaction mixture into first and second sites; and (3) forming a first amplicon by amplifying the first template at the first site; and forming a second amplicon by amplifying the second template at the second site.

In some embodiments, amplification comprises at least one RPA cycle.

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

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

In some embodiments, the amplification comprises at least one round of RPA.

In some embodiments, the amplification comprises at least one template walking round.

In some embodiments, the amplification comprises at least one RPA round and at least one template walking round.

In some embodiments, any or all of the methods disclosed herein may result in the production of a plurality of amplicons, at least some of which comprise a clonally amplified population of nucleic acids. The clonally amplified populations produced by the methods of the present disclosure may be used for a variety of purposes. In some embodiments, the disclosed methods (and related compositions, systems, and kits) optionally further comprise analysis and/or processing of the clonally amplified populations (amplicons).

In some embodiments, amplicons produced according to the present disclosure may be subjected to downstream analytical methods, such as sequencing.

In some embodiments, the amplified nucleic acids can be further analyzed (e.g., sequenced) at the assigned sites without the need to recover and move the amplified products to a different site or surface for analysis (e.g., sequencing).

In some embodiments, the method of downstream analysis comprises sequencing at least a portion of the plurality of amplicons in parallel. Optionally, multiple templates/amplified templates/extended first primers at different array sites are sequenced in parallel.

For example, in some embodiments, amplification is followed by in situ sequencing of the amplified product. The amplified product that is sequenced can include an amplicon comprising a substantially monoclonal population of nucleic acids. Optionally, the population of monoclonal nucleic acids (amplicons) at different sites of the array is sequenced in parallel.

In some embodiments, sequencing can include incorporating nucleotides into a sequencing primer using a polymerase. Optionally, incorporating includes forming at least one nucleotide incorporation byproduct.

In some embodiments, the sites comprise a hydrophilic polymer matrix conformally disposed within a well operatively coupled to the sensor.

Optionally, the hydrophilic polymer matrix comprises a hydrogel polymer matrix.

Optionally, the hydrophilic polymer matrix is an in situ cured polymer matrix.

Optionally, the polyacrylamide is conjugated to an oligonucleotide primer.

Optionally, the pores have a characteristic diameter of 0.1 to 2 microns.

Optionally, the pores have a depth of 0.01 to 10 microns.

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

In some embodiments, the reaction mixture may comprise a recombinase. The recombinase may comprise any suitable agent that facilitates recombination between polynucleotide molecules. The recombinase may be an enzyme that catalyzes homologous recombination. For example, the reaction mixture may comprise a recombinase comprising or derived from a bacterium, eukaryote, or virus (e.g., bacteriophage).

In some embodiments, the reaction mixture comprises an enzyme that can bind the primer and the polynucleotide template to form a complex or can catalyze strand invasion of the polynucleotide template to form a D-ring structure. In some embodiments, the reaction mixture comprises one or more proteins selected from UvsX, recA, and Rad 51.

In some embodiments, the reaction mixture may comprise a recombinase accessory protein, such as UvsY.

In some embodiments, the reaction mixture may comprise a single-chain binding protein (SSBP).

In some embodiments, the reaction mixture may comprise a polymerase. The polymerase optionally has or lacks exonuclease activity. In some embodiments, the polymerase has 5 'to 3' exonuclease activity, 3 'to 5' exonuclease activity, or both. Optionally, the polymerase lacks any one or more of such exonuclease activities.

In some embodiments, the polymerase has strand displacement activity.

In some embodiments, the reaction mixture may comprise a diffusion limiting agent. The diffusion limiting agent can be any agent effective to prevent or slow diffusion of one or more of the polynucleotide templates and/or one or more of the amplification reaction products through the reaction mixture.

In some embodiments, the reaction mixture may comprise a sieving agent. The sieving agent can be any agent effective to sieve one or more polynucleotides (e.g., amplification reaction products and/or polynucleotide templates) present in a reaction mixture. In some embodiments, the sizing agent limits or slows the migration of polynucleotide amplification products through the reaction mixture.

In some embodiments, the reaction mixture may comprise a crowding reagent.

In some embodiments, the reaction mixture comprises a crowding reagent and a sieving reagent.

In some embodiments, the disclosed methods comprise contacting each of at least two polynucleotides with a recombinase, a support to which a plurality of first oligonucleotide primers are attached (the first oligonucleotide primers being at least partially complementary to at least some portion of the polynucleotides), a polymerase, and a plurality of nucleotides in any order and in any combination.

In some embodiments, the at least two different polynucleotides comprise a forward strand comprising a first primer binding site, and amplification within the at least two sites (or within the at least two reaction chambers) optionally comprises binding a first primer to the first primer binding site at the site or within the reaction chamber to form a first primer-template duplex. Optionally, the binding of the first primer to the at least two different polynucleotide templates is mediated by a recombinase. For example, amplification may include forming a nucleoprotein complex comprising the recombinase and the first primer. Optionally, the first primer is attached to a site or a surface of the reaction chamber. In some embodiments, amplification at a site or within a reaction chamber comprises: a first nucleoprotein complex (or "first nucleoprotein filament") is formed. Amplification optionally further comprises contacting at least one of the polynucleotides in the site or reaction chamber with the first nucleoprotein filament, the polymerase and the plurality of nucleotides in any order or combination.

Optionally, discrete supports (e.g., beads) each comprising a plurality of first primers are each dispensed into a reaction chamber or site prior to amplification, and amplifying comprises amplifying one of at least two different polynucleotides on a support within a site or reaction chamber. In some embodiments, any of the partitioning and/or contacting steps may be repeated prior to amplification, optionally to increase the number and/or yield of sites or reaction chambers that produce monoclonal products.

Optionally, amplifying comprises extending a first primer of the first primer-template double strand within the reaction chamber using a polymerase, thereby forming an extended first primer. Optionally, extending the first primer displaces the reverse strand from the forward strand. The extended first primer optionally comprises a second primer binding site.

Optionally, the amplifying comprises a reverse synthesis step comprising binding a second primer to the second primer binding site of the extended first primer and extending the second primer to form a second primer-template duplex. Optionally, binding of the second primer to the polynucleotide template is mediated by a recombinase. For example, amplification may include formation of a nucleoprotein complex comprising the recombinase and the second primer. Optionally, the second primer is attached to the site or to the surface of the reaction chamber. In some embodiments, amplification within a site or reaction chamber comprises: a second nucleoprotein complex (or "second nucleoprotein filament") is formed. Amplification optionally further comprises contacting at least one of the site or the polynucleotide template in the reaction chamber or at least one of the extended first primer with the second nucleoprotein filament, the polymerase and the plurality of nucleotides in any order or combination.

Optionally, amplifying comprises extending the first primer-template duplex, the second primer-template duplex, or both using a polymerase. The polymerase may have strand displacement activity.

In some embodiments, methods (and related compositions, systems, and kits) can include placing, localizing, or otherwise restricting at least one substantially monoclonal population at a site. The sites may form part of an array of sites.

Optionally, at least one of the sites comprises the inner walls of a reaction chamber, support, particle, microparticle, sphere, bead, filter, flow cell, well, channel reservoir (channel reservoir), gel, or tube.

In some embodiments, at least one site comprises a hydrophilic polymer matrix conformally disposed within a well operatively coupled to the sensor.

Optionally, the hydrophilic polymer matrix comprises a hydrogel polymer matrix.

Optionally, the hydrophilic polymer matrix is an in situ cured polymer matrix.

Optionally, the polyacrylamide is conjugated to an oligonucleotide primer.

Optionally, the pores have a characteristic diameter of 0.1 to 2 microns.

Optionally, the pores have a depth of 0.01 to 10 microns.

In some embodiments, the FET comprises a floating gate structure comprising a plurality of conductors electrically coupled to one another and separated by dielectric layers, and the floating gate conductor is the uppermost conductor of the plurality of conductors.

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

Optionally, the plurality of different polynucleotide templates (or amplified polynucleotides) comprise at least one nucleic acid comprising a selectively cleavable moiety.

Optionally, the selectively cleavable moiety comprises uracil.

Optionally, the method for nucleic acid amplification further comprises cleaving the cleavable moiety with a cleavage agent.

Optionally, cleavage can be performed prior to amplification, e.g., prior to formation of a reaction mixture.

Optionally, cleavage can be performed after amplification, e.g., after the nucleic acid template is amplified.

Optionally, the reaction mixture comprises at least one primer comprising a cleavable moiety.

Optionally, the plurality of different polynucleotides comprises a plurality of amplicons.

Optionally, the plurality of different polynucleotides comprises a plurality of different amplicons.

In some embodiments, amplification can be performed using any of the methods, compositions, systems, and kits disclosed in U.S. provisional patent application No. 61/792247 (incorporated herein by reference in its entirety), filed on 2013, month 3, and day 15.

In some embodiments, amplicons produced according to the present disclosure can be subjected to downstream analytical methods, such as quantification. For example, in some embodiments, the number of amplicons that exhibit certain desired properties may be detected and optionally quantified.

In some embodiments, the amplified nucleic acids may optionally be subjected to additional downstream analysis steps in the disclosed methods.

In some embodiments involving amplification of different polynucleotide templates on discrete and separate supports, the method may comprise determining which discrete supports (e.g., beads) comprise amplicons. Similarly, in embodiments where templates are assigned to an array prior to amplification, the method may comprise determining which sites of the array contain amplicons, and optionally further comprising counting the number of sites containing amplicons. Optionally using DNA-based detection procedures such as UV absorption, staining with DNA-specific dyes,

Assay, qPCR, and fluorescent ProbeHybridization of the needle, etc., to detect the presence of the amplicon on the assay support or site. In some embodiments, the method can include determining which bead supports (or sites of the array) have resulted in substantially monoclonal amplicons. For example, the bead supports (or array sites) can be analyzed to determine which supports or sites can produce a detectable and coherent (i.e., analyzable) sequence-dependent signal.

In some embodiments, the disclosed methods include an additional downstream analysis step that provides the same type of information previously obtained by conventional techniques such as digital PCR or digital RPA (as described, for example, in Shen 2011Analytical Chemistry 83 3533-3540; published U.S. patent applications US2012/0264132 and 2012/0329038 (all of which are incorporated herein by reference in their entirety)). Digital PCR (dPCR) is an improvement over conventional Polymerase Chain Reaction (PCR) and can be used to directly quantitatively and clonally amplify nucleic acids (including DNA, cDNA, methylated DNA or RNA). One difference between dPCR and traditional PCR is the method of measuring the amount of nucleic acid. PCR and dPCR perform one reaction per single sample, dPCR also performs a single reaction within the sample, however the sample is divided into a large number of partitions and the reactions are performed individually in each partition. The separation allows for sensitive measurement of the amount of nucleic acid. dPCR has been demonstrated to be useful for studying changes in gene sequences, such as copy number changes or point mutations.

In contrast to the present method, dPCR generally requires partitioning of the sample prior to amplification; in contrast, several embodiments disclosed herein provide for parallel amplification of different templates within a single continuous phase of a reaction mixture without partitioning. In dPCR, the sample is typically partitioned so that individual nucleic acid molecules within the sample are localized and localized to many discrete regions. The sample is divided by simple dilution so that each portion contains about 1 copy of the DNA template or less. By isolating individual DNA templates, the method effectively enriches for DNA molecules that are present at very low levels in the original sample. Partitioning of the samples facilitates molecular counting using Poisson statistics. As a result, each partition will contain a "0" or "1" molecule, or a negative or positive reaction, respectively. Although the initial copy number of molecules is proportional to the number of amplification cycles in conventional PCR, dPCR generally does not rely on the number of amplification cycles to determine the initial sample size.

Conventional methods of dPCR analysis typically utilize fluorescent exploration and light-based detection methods to identify the amplified products. Such methods require sufficient amplification of the target molecule to produce sufficient signal that can be detected, but can result in additional errors or deviations.

In those embodiments of the present disclosure that include the partitioning of nucleic acid templates into the wells of an isFET array and subsequent amplification of the templates in the wells of the array, an optional downstream analysis step can be performed after amplification that quantifies the number of sites or wells that contain amplified product. In some embodiments, the products of a nucleic acid amplification reaction can be detected to count the number of sites or wells comprising amplified template.

For example, in some embodiments, the present disclosure relates generally to methods of nucleic acid synthesis, comprising: providing a sample comprising a first number of polynucleotides; and dispensing individual polynucleotides of the sample into different sites of the array of sites.

Optionally, the method may further comprise forming a substantially monoclonal population of nucleic acids within their respective sites by amplifying the individual polynucleotides.

Optionally, the sites remain in fluid communication during amplification.

Optionally, the amplification comprises partially denaturing the template.

Optionally, the amplifying comprises subjecting the template to a partial denaturation temperature. In some embodiments, the template comprises a low melting point sequence comprising a primer binding site that is rendered single stranded when the template is subjected to a partial denaturation temperature.

Optionally, the amplification comprises partially denaturing the template.

Optionally, amplifying comprises contacting at least two different templates at two different array sites with a single reaction mixture for nucleic acid amplification.

Optionally, the reaction mixture comprises a recombinase.

Optionally, the reaction mixture comprises at least one primer comprising a "resistance tag".

Optionally, the amplifying comprises performing at least one amplification cycle comprising: partially denaturing the template, hybridizing the primer to the template, and extending the primer in a template-dependent manner. Optionally, the amplifying comprises isothermal amplification. In some embodiments, amplification is performed under substantially isothermal conditions.

In some embodiments, the percentage of sites containing one or more template molecules is greater than 50% and less than 100%.

In some embodiments, the disclosed methods can further comprise detecting a change in ion concentration in at least one site due to at least one amplification cycle.

In some embodiments, the disclosed methods can further comprise quantifying the initial amount of the target nucleic acid.

Some examples of array-based digital PCR using ion-based sensing techniques can be found, for example, in U.S. provisional application No. 61/635584 (incorporated herein by reference in its entirety), filed 4/19/2012.

In some embodiments, the present disclosure relates generally to methods for detecting a target nucleic acid, comprising: dividing the sample into a plurality of sample volumes, wherein more than 50% of the portions contain no more than 1 target nucleic acid molecule per sample volume; subjecting a plurality of sample volumes to conditions for amplification, wherein the conditions comprise partially denaturing conditions; detecting a change in ion concentration in a volume of the sample in which the target nucleic acid is present; counting the number of portions having amplified target nucleic acid; and determining the amount of the target nucleic acid in the sample. The change in ion concentration may be an increase in ion concentration or may be a decrease in ion concentration. In some embodiments, the method may further comprise combining the sample with beads. In some embodiments, a method can include loading a sample onto a substrate, wherein the substrate includes at least one well.

In some embodiments, subjecting the target nucleic acid to partial denaturation conditions comprises contacting the target nucleic acid molecules in their respective sample volumes with a recombinase and a polymerase under RPA conditions.

In some embodiments, subjecting the target nucleic acid to partial denaturation conditions comprises subjecting the target nucleic acid molecule to a partial denaturation temperature.

In some embodiments, the present disclosure relates generally to methods for performing absolute quantification of nucleic acids, comprising: diluting a sample comprising an initial amount of nucleic acid templates and dispensing the nucleic acid templates of the sample into a plurality of sites of an array, wherein the percentage of sites comprising one or more nucleic acid templates is greater than 50% and less than 100%; subjecting a plurality of sites to at least one amplification cycle, wherein the amplification cycle is performed according to any of the amplification methods disclosed herein; detecting a change in ion concentration in at least one of the plurality of sample volumes due to at least one amplification cycle; and quantifying the initial amount of nucleic acid template. The change in ion concentration may be an increase in ion concentration, a decrease in ion concentration, a change in pH, and may involve the detection of positive ions such as hydrogen ions, anions such as pyrophosphate molecules, or cations and anions.

In some embodiments, the disclosure also relates generally to methods (and related compositions, systems, and kits) for attaching individual members of a nucleic acid template population to different supports of a plurality of supports or to different sites of a plurality of sites by using recombinase-mediated strand exchange. These methods, compositions, systems and kits can be used to generate fixed amplicon populations suitable for manipulation in applications where it is desirable to obtain or differentiate different amplicons individually. In some embodiments, a plurality of discrete supports or a plurality of sites in an array each comprise a capture primer. Immobilization of each template to each support (or to each site of the array) can be achieved by contacting the template with the support or site in the presence of a primer ("fusion primer"). In some embodiments, the fusion primer comprises a target-specific portion that is complementary to a portion of the template, and a universal primer binding site that is complementary to at least a portion of the capture primer of the support or site. Optionally, the contacting is performed in the presence of an RPA component. The RPA component may comprise a recombinase. The RPA component may comprise a strand displacing polymerase. In some embodiments, the fusion primer is recombined into the template by recombinase-mediated strand exchange, thereby forming a template-primer adduct comprising a universal primer binding site. In some embodiments, the capture primer is subsequently recombined into the universal primer binding site, forming an immobilized template attached to a support or site.

In some embodiments of bead-based amplification, a library of fusion primers, each comprising a different target-specific portion and a common universal primer binding site, is contacted with a plurality of templates and a plurality of supports in a reaction mixture comprising a polymerase and a strand displacing polymerase. The template library is then attached to a plurality of supports by subjecting the mixture to RPA conditions, thereby generating a plurality of supports each having a different template attached thereto.

In some embodiments of array-based amplification, a library of fusion primers, each comprising a different target-specific moiety and a common universal primer binding site, is contacted with a plurality of templates and a surface comprising a plurality of sites in a reaction mixture comprising a polymerase and a strand displacing polymerase. At least some of the plurality of sites comprise a universal capture primer. The template library is then attached to a plurality of sites on the surface by subjecting the mixture to RPA conditions, thereby generating a plurality of supports each having a different template attached thereto.

In some embodiments, the present disclosure relates generally to compositions comprising reagents for amplifying one or more nucleic acid templates in parallel using partially denaturing conditions (and related methods for making and using the compositions).

In some embodiments, the composition may comprise any of the components described herein for performing RPA.

In some embodiments, the composition may comprise any of the components described herein for performing template walking.

In some embodiments, the present disclosure relates generally to compositions and systems for nucleic acid amplification, comprising: a surface comprising a first site and a second site; and a nucleic acid amplification reaction mixture, wherein the mixture is in contact with the first and second sites.

In some embodiments, the reaction mixture comprises a recombinase.

In some embodiments, the first site is operatively coupled to a first sensor and the second site is operatively connected to a second sensor.

In some embodiments, the first and second sites are operably connected to the same sensor.

Optionally, the first site comprises a first substantially monoclonal population of nucleic acids. The second site optionally comprises a second substantially monoclonal population of nucleic acids.

In some embodiments, the disclosed compositions comprise: a surface comprising a first site and a second site, wherein the first site comprises a first population of substantially monoclonal nucleic acids and the second site comprises a second population of substantially monoclonal nucleic acids; and a nucleic acid amplification reaction mixture, wherein the mixture is in contact with the first and second sites.

In some embodiments, the composition comprises an array of sites comprising a first site comprising (e.g., linked to) a first capture primer and a second site comprising (e.g., linked to) a second capture primer.

In some embodiments, at least one site of the plurality of sites is attached to the sensor.

In some embodiments, the sensor comprises a Field Effect Transistor (FET).

In some embodiments, at least the first site or the second site or the first and second sites comprise a capture primer attached to the surface.

Optionally, the hydrophilic polymer matrix comprises a hydrogel polymer matrix.

Optionally, the hydrophilic polymer matrix is an in situ cured polymer matrix.

Optionally, the hydrophilic polymer matrix comprises polyacrylamide, a copolymer thereof, a derivative thereof, or a combination thereof.

Optionally, the polyacrylamide is conjugated to an oligonucleotide primer.

Optionally, the pores have a characteristic diameter of 0.1 to 2 microns.

Optionally, the pores have a depth of 0.01 to 10 microns.

In some embodiments, the disclosed methods comprise introducing a nucleotide at least one of the plurality of sites; and detecting an output signal from the sensor due to incorporation of the nucleotide into the sequencing primer. The output signal is optionally based on a threshold voltage of the FET. In some embodiments, the FET includes a floating gate conductor coupled to the site.

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

In some embodiments, the reaction mixture comprises all components necessary to carry out RPA.

In some embodiments, the method of amplification can include performing a "template walk" as described in U.S. patent publication No. 2012/0156728 (which is incorporated herein by reference in its entirety) published on day 21/6/2012. For example, in some embodiments, the present disclosure generally relates to methods, compositions, systems, devices, and kits for clonally amplifying one or more nucleic acid templates to form a clonally amplified population of nucleic acid templates. Any of the amplification methods described herein optionally include repeated nucleic acid amplification cycles. The amplification cycle optionally comprises: (ii) hybridization of the primer to the template strand, (b) extension of the primer to form a first extended strand, (c) partial or incomplete denaturation of the extended strand from the template strand. The primer that hybridizes to the template strand (designated as the "forward" primer for convenience) is optionally immobilized on or to the support. The support is, for example, a solid or semi-solid. Optionally, the denatured portion of the template strand from step (c) is free to hybridize with a different forward primer in the next amplification cycle. In embodiments, primer extension in a subsequent amplification cycle comprises displacement of the first extended strand from the template strand. A second "reverse" primer, which hybridizes to the 3' end of the first extended strand, for example, may be included. The reverse primer is optionally non-immobilized.

In embodiments, the template is amplified using primers immobilized on or to one or more solid or semi-solid supports. Optionally the support comprises an immobilized primer complementary to the first portion of the template strand. Optionally, the support does not significantly comprise an immobilized primer homologous to a second non-overlapping portion of the same template strand. Two portions are non-overlapping if they do not contain any subparts that hybridize to each other or to their complements. In another example, the support optionally does not significantly comprise immobilized primers that can hybridize to the complement of the template strand.

Optionally, a plurality of nucleic acid templates are simultaneously amplified in a single continuous liquid phase in the presence of one or more supports, each of which comprises one or more fixation sites. In embodiments, each template is amplified to produce clonal amplicon populations, wherein each clonal population is immobilized in or on a different support or immobilization site than the other amplified populations. Optionally, the amplified population remains substantially monoclonal after amplification.

The template is, for example, amplified to produce a clonal population comprising a template homologous strand (referred to herein as the "template strand" or "reverse strand") and/or a template complementary strand (referred to herein as the "primer strand" or "forward strand"). In embodiments, clonality is maintained in the resulting amplified nucleic acid population by maintaining an association between the template strand and its primer strand, thereby effectively linking or "tying" related clonal progeny together and reducing the likelihood of cross-contamination between different clonal populations. Optionally, one or more amplified nucleic acids in the clonal population are attached to a support. Clonal populations of substantially identical nucleic acids can optionally have spatially localized or discrete macroscopic manifestations. In embodiments, the clonal population can resemble an isolated spot or colony (e.g., when partitioned into a support, optionally on the outer surface of the support).

In some embodiments, the present disclosure generally relates to novel methods of generating a limited clonal population of clonal amplicons, optionally immobilized to or in or on one or more supports. The support may, for example, be a solid or semi-solid (e.g., a gel or hydrogel). The amplified clonal population is optionally attached to the outer surface of the support or may also be within the inner surface of the support (e.g., when the support has a porous or matrix structure).

In some embodiments, amplification is achieved by multiple cycles of primer extension along the template strand of interest (also referred to as the "reverse" strand). For convenience, a primer that hybridizes to the template strand of interest is referred to as a "forward" primer, and is optionally extended in a template-dependent manner to form a "forward" strand that is complementary to the template strand of interest. In some methods, the forward strand itself is hybridized to a second primer, referred to as the "reverse" primer, which is extended to form a new template strand (also referred to as the reverse strand). Optionally, at least a portion of the new template strand is homologous to the original template of interest ("reverse") strand.

As mentioned, one or more primers may be immobilized to or in or on one or more supports. Optionally, one primer is immobilized by attachment to a support. The second primer may be present and optionally not immobilized or attached to the support. Different templates may be amplified simultaneously, e.g. in a single continuous liquid phase, on different support or fixation sites to form a monoclonal population of nucleic acids. The liquid phase may be considered continuous if any portion of the liquid phase is in fluid contact or communication with any other portion of the liquid. In another example, the liquid phase may be considered continuous if no portion is completely subdivided or divided or otherwise completely physically separated from the remainder of the liquid. Optionally, the liquid phase is flowable. Optionally, the continuous liquid phase is not within the gel or matrix. In other embodiments, the continuous liquid phase is within a gel or matrix. For example, the continuous liquid phase occupies pores, interstices or other voids of the solid or semi-solid support.

When the liquid phase is in a gel or matrix, one or more primers are optionally immobilized on a support. Optionally the support is a gel or the matrix itself. Alternatively, the support is not a gel or matrix itself. In an example, one primer is immobilized on a solid support contained within a gel and is not immobilized to a gel molecule. The support is for example in the form of a planar surface or one or more particles. Optionally, the ground plane surface or plurality of microparticles comprise a forward primer having substantially the same sequence. In embodiments, the support does not comprise a significant amount of a second, different primer. Optionally, the second non-immobilized primer is in solution within the gel. The second non-immobilized primer binds to, for example, the template strand (i.e., the reverse strand), while the immobilized primer binds to the forward strand.

Embodiments of template walking include methods of primer extension comprising: (a) a primer-hybridizing step, (b) an extending step, and (c) a walking step. Optionally, the primer-hybridizing step comprises hybridizing a first primer molecule ("first forward primer") to a complementary forward primer binding sequence ("forward PBS") on the nucleic acid strand ("reverse strand"). Optionally, the extending step comprises generating an extended first forward strand that is the full-length complement of the reverse strand and hybridizes thereto. The extended first forward strand is generated, for example, by extending a first forward primer molecule in a template-dependent manner using the reverse strand as a template. Optionally, the walking step comprises hybridizing a second primer ("second forward primer") to the forward PBS, wherein the reverse strand is also hybridized to the first forward strand. For example, the walking step comprises denaturing at least a portion of the forward PBS ("free portion") from the forward strand, while another portion of the reverse strand remains hybridized to the forward strand.

In embodiments, the primer extension method is an amplification method comprising template walking, wherein any one or more steps of primer-hybridization, extension, and/or walking are repeated at least once. For example, the method can include amplifying the forward strand through one or more amplification cycles. The amplification cycle optionally includes extension and walking. An exemplary amplification cycle comprises or consists essentially of extension followed by walking. Optionally, the second forward primer of the first amplification cycle is used as the first forward primer of a subsequent amplification cycle. For example, the second forward primer of a walking step in a first amplification cycle is used as the first forward primer of an extension step of a subsequent amplification cycle.

Optionally, the method of primer extension or amplification further comprises extending or amplifying the reverse strand by: (a) Hybridizing a first reverse primer molecule to a complementary reverse primer binding sequence ("reverse PBS") on the extended forward strand; (b) Generating an extended first reverse strand by extending a first reverse primer molecule in a template-dependent manner using the forward strand as a template, the strand being the full-length complement of the forward strand and hybridized thereto; and (c) hybridizing a second primer ("second reverse primer") to the reverse PBS, wherein the forward strand is also hybridized to the first reverse strand. Optionally performing one or more repetitions of steps (b) - (c), wherein the second reverse primer of step (c) is the first reverse primer of repeated step (b); and wherein a substantial portion of the forward strand hybridizes to the reverse strand during or at all times between the one or more repetitions. In embodiments, the majority is optionally at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

Optionally, the reverse strand and/or the forward strand are not exposed to complete denaturing conditions during amplification, which can result in complete separation of a significant portion (e.g., more than 10%, 20%, 30%, 40%, or 50%) of the plurality of strands from their extended and/or full-length complements.

In embodiments, a substantial portion of the forward and/or reverse strands optionally hybridize to the extended and/or full-length complement at all times during or between one or more amplification cycles (e.g., 1, 5, 10, 20, or all amplification cycles performed). In embodiments, the majority of the chain is optionally at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the chain. In embodiments, this is accomplished by maintaining the amplification reaction at a T above that of the non-extended primer _m And less than the T of the primer-complementary strand _m Is achieved by the temperature of (1). For example, the amplification conditions are maintained at a temperature above the T of the non-extended forward primer _m But below the T of the extended or full length reverse strand _m . Likewise, for example, the amplification conditions are maintained at a temperature above the T of the non-extended reverse primer _m But below the T of the extended or full length forward strand _m 。

Optionally, one or more of the forward primers and/or one or more of the reverse primers are breathable (e.g., have a low T) _m . In an example, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the nucleotide bases of the respirable primers are adenine, thymine, or uracil or are complementary to adenine, thymine, or uracil.

In some embodiments, the present disclosure generally relates to methods, compositions, systems, devices, and kits for clonally amplifying nucleic acid templates on a support in an amplification reaction solution. Optionally, the nucleic acid template is contacted with the support in a solution comprising a continuous liquid phase. The support can include a primer population that includes at least a first primer and a second primer. The population of primers can be immobilized on the support, for example, by covalent attachment to the support. In some embodiments, the nucleic acid template comprises a primer binding sequence proximal to the target sequence. The primer binding sequence may be complementary to the sequence of the first primer and optionally the sequence of the second primer. The target sequence may not be complementary to a primer in the primer population. In some embodiments, the primer binding sequence of the nucleic acid template hybridizes to the first primer. The first primer can be extended along the template using a polymerase, thereby forming an extended first primer. At least a portion of the primer binding sequence of the template may be separated (e.g., denatured or melted) from the extended first primer. Optionally, separation is performed while maintaining hybridization between the portion of the template and the extended first primer. The isolated portion of the primer binding sequence can then hybridize to a second primer. Optionally, such hybridization is performed while maintaining hybridization between other portions of the template and the extended first primer. The second primer can be extended along the template using a polymerase, thereby forming a support comprising the extended first primer and the extended second primer. The extended portion of the extended first primer and/or the extended second primer may comprise a sequence complementary to the target sequence.

In some embodiments, the present disclosure generally relates to methods for clonally amplifying nucleic acid templates on a support in an amplification reaction solution, comprising: contacting a nucleic acid template with a support in a liquid solution, wherein the support comprises an immobilized primer population comprising at least a first primer and a second primer, and wherein the nucleic acid template comprises a primer binding sequence proximal to a target sequence, wherein the primer binding sequence is complementary to the sequence of the first primer and the sequence of the second primer, and the target sequence is not complementary to the primers in the primer population; hybridizing a primer binding sequence of a nucleic acid template to a first primer; extending the first primer along the template using a polymerase, thereby forming an extended first primer; denaturing at least a portion of the primer binding sequence of the template with the extended first primer while maintaining hybridization between another portion of the template and the extended first primer; hybridizing portions of the denatured primer binding sequence to the second primer while maintaining hybridization between other portions of the template and the extended first primer; and extending the second primer along the template using a polymerase, thereby forming a support comprising an extended first primer and an extended second primer, wherein the extension of the extended first primer and the extended second primer each comprise a sequence complementary to the target sequence. The population of primers can comprise substantially identical primers that differ in sequence by no more than 1, 2, 3, 4, or 5 nucleotides. In some embodiments, the population of primers comprises different primers, at least some of which comprise a sequence that is complementary to the primer binding sequence of the template. In some embodiments, the primers of the primer population are not complementary to the 5' half of the sequence of the template. In some embodiments, the primers of the primer population are not complementary to the 3' half of the sequence of any extended primer of the support. In some embodiments, the primers of the primer population are not complementary to any sequence of the template other than the primer binding sequence.

In some embodiments, the present disclosure generally relates to methods for clonally amplifying a population of nucleic acid templates on a population of supports in an amplification reaction solution, comprising: clonally amplifying a first template on a first support according to any of the methods disclosed herein, and clonally amplifying a second nucleic acid template on a second support according to the same method, wherein all supports are contained within a single continuous liquid phase during amplification.

Provided, among other things, is a method of generating a confined clonal population of immobilized clonal amplicons of a single-stranded template sequence, comprising: (a) Ligating a single-stranded template sequence ("template 1") to a fixed site ("IS 1"), wherein IS1 comprises multiple copies of an immobilized primer ("IS 1 primer") that can substantially hybridize to template 1, and template 1 binds to IS1 by hybridization to the IS1 primer, and (b) amplifying template 1 in solution using the IS1 primer and a non-immobilized primer ("SP 1 primer"), wherein an amplified strand complementary to single-stranded template 1, when single-stranded, cannot substantially hybridize to a primer on IS1, wherein amplifying a limited clonal population of immobilized clonal amplicons generates around the initial hybridization point of template 1 to IS 1.

Also provided IS a method of generating an isolated and fixed clonal population of a first template sequence ("template 1") and a second template sequence ("template 2"), the method comprising amplifying the first and second template sequences to generate a clonal amplicon population of template 1 that IS substantially linked to a first fixing site ("IS 1") and not a second fixing site ("IS 2"), or a clonal amplicon population of template 2 that IS substantially linked to IS2 and not IS1, wherein: (a) both templates and all amplicons are contained within the same continuous liquid phase, wherein the continuous liquid phase IS in contact with first and second immobilization sites ("IS 1" and "IS2", respectively), and wherein IS1 and IS2 are spatially separated, (b) template 1 when in single-stranded form comprises a first subsequence ("T1-FOR") at one end and a second subsequence ("T1-REV") at its opposite end, (c) template 2 when in single-stranded form comprises a first subsequence ("T2-FOR") at one end and a second subsequence ("T2-REV") at its opposite end, (d) IS1 comprises multiple copies of an immobilized nucleic acid primer ("IS 1 primer") that can substantially hybridize to T1-FOR and T2-FOR when T1 and T2 are single-stranded, (e) IS2 comprises multiple copies of an immobilized primer ("IS 2") that can substantially hybridize to the immobilized primer in T1 and T2-immobilized phases to T1-fr, but IS not substantially hybridize to the reverse strand primer ("IS 1-2"), and IS a complementary primer, and IS not substantially hybridize to the reverse strand, but IS a reverse strand, which IS not capable of hybridizing to a reverse strand, and IS not capable of hybridizing to a reverse strand; and (g) the reverse complement of T2-REV, when single-stranded, does not substantially hybridize to a primer on IS2, but may substantially hybridize to a non-immobilized primer ("SP 2")

Optionally, in any of the methods described herein, any nucleic acid that has been dissociated from one fixation site is capable of substantially hybridizing to the two fixation sites and any movement (e.g., movement by diffusion, convection) of the dissociated nucleic acid to the other fixation site in the continuous liquid phase is substantially unimpeded.

Optionally, in any of the processes described herein, the continuous liquid phase IS contacted with IS1 and IS2 simultaneously.

Optionally, in any of the methods described herein, the first portion of the template to which the immobilized primer binds does not overlap with a second portion of the template, the complement of which is bound by the non-immobilized primer.

Optionally, in any of the methods described herein, at least one template to be amplified is generated from the input nucleic acid after contacting the nucleic acid with the at least one immobilization site.

Optionally, any of the methods described herein comprise the steps of: (a) Contacting a support comprising an immobilized primer with a single-stranded nucleic acid template, wherein: hybridizing the first immobilized primer to a Primer Binding Sequence (PBS) on the template; (b) Extending the hybridized first primer with template-dependent extension to form an extended strand that is complementary to and at least partially hybridized to the template; (c) Denaturing the template from the extended complementary strand portion so that at least part of the PBS is in single stranded form ("free portion"); (d) Hybridizing the free portion to an unextended, immobilized second primer; (e) Extending the second primer with template-dependent extension to form an extended strand complementary to the template; (f) Optionally, the annealed extended immobilized nucleic acid strands are separated from each other.

Optionally, in any of the methods described herein, (a) during amplification, a nucleic acid duplex is formed so as to comprise the starting template and/or the amplified strand; the duplexes do not undergo conditions during amplification that would result in complete denaturation of a substantial number of the duplexes.

Optionally, in any of the methods described herein, the single stranded template is generated by obtaining a plurality of input double or single stranded nucleic acid sequences to be amplified (which sequences may be known or unknown) and adding or generating a first universal adaptor sequence and a second universal adaptor sequence on the end of at least one input nucleic acid; wherein the first universal adaptor sequence hybridizes to an IS1 primer and/or an IS2 primer and the reverse complement of the second universal adaptor sequence hybridizes to at least one non-immobilized primer. The adaptor may be double stranded or single stranded.

Optionally, in any of the methods described herein, first and second nucleic acid linker sequences are provided at the first and second ends of the single stranded template sequence.

Optionally, in any of the methods described herein, a tag is also added to one or more nucleic acid sequences (e.g., a template or primer or amplicon), the tag enabling identification of the nucleic acid comprising the tag.

Optionally, in any of the methods described herein, all primers on at least one of the fixation sites or supports have the same sequence. Optionally, the immobilization site or support comprises a plurality of primers having at least two different sequences. In some embodiments, the fixation site or support comprises at least one target-specific primer.

Optionally, in any of the methods described herein, the continuous medium is flowable. Optionally, the mixing of non-immobilized nucleic acid molecules is substantially unimpeded in the continuous liquid phase during at least part of the amplification process, e.g., during any one or more of the steps or cycles described herein.

Optionally, in any of the methods described herein, the mixing is substantially unimpeded over a period of time during the amplification. For example, mixing is substantially unimpeded throughout the duration of amplification.

In embodiments, amplification is achieved using RPA, i.e., recombinase-polymerase amplification (see, e.g., WO2003072805, which is incorporated herein by reference). RPA is optionally performed without substantial change in temperature or reagent conditions. In embodiments herein, partial denaturation and/or amplification may be achieved using a recombinase and/or a single-chain binding protein, including any one or more of the steps or methods described herein. Suitable recombinases include RecA and its prokaryotic or eukaryotic analogs or functional fragments or variants thereof, optionally in combination with single-chain binding proteins (SSBs). In embodiments, the recombinase agent is optionally coated with single-stranded DNA (ssDNA), e.g., amplification primers, to form a nucleoprotein filament strand that invades a double-stranded region of homology on the template. This optionally results in short hybrids and replacement strand bubbles (called D-loops). In embodiments, the free 3' -end of the silk strand in the D-loop is extended by a DNA polymerase to synthesize a new complementary strand. The complementary strand displaces the originally paired strand of the template upon extension. In embodiments, one or more amplification primer pairs are contacted with one or more recombinase agents prior to contact with the template, which is optionally double-stranded.

In any of the methods described herein, amplification of the template (target sequence) comprises contacting the recombinase agent with one or more of the at least one amplification primer pair, thereby forming one or more "forward" and/or "reverse" RPA primers. Optionally removing any recombinase agent not associated with the primer or primers. Optionally, one or more forward RPA primers are then contacted with a template strand, optionally having a region complementary to at least one RPA primer. The template strand may be hybridized to the contact of the RPA primer and the complementary template, which optionally results in hybridization between the primer and template. Optionally, one or more polymerases (e.g., in the presence of dntps) are used to extend the 3' end of the primer along the template to generate a double-stranded nucleic acid and displace the template strand. The amplification reaction may comprise repeated cycles of such contacting and extending until the desired degree of amplification can be obtained. Optionally, displaced strands of the nucleic acid are amplified by parallel RPA reactions. Optionally, the displaced strand of nucleic acid is amplified by sequentially contacting it with one or more complementary primers; and (b) extending the complementary primer by any of the strategies described herein.

In embodiments, the one or more primers comprise a "forward" primer and a "reverse" primer. Contacting both primers with a template optionally results in a first double-stranded structure on a first portion of said first strand and a double-stranded structure on a second portion of said second strand. Optionally, the 3' end of the forward and/or reverse primers are extended with one or more polymerases to produce first and second double-stranded nucleic acids and first and second displaced strands of nucleic acid. Optionally, the second displaced strands are at least partially complementary to each other and can hybridize to form a daughter double-stranded nucleic acid, which can be used as a double-stranded template nucleic acid in a subsequent amplification cycle.

Optionally said first and said second displaced strands are at least partially complementary to said first or said second primer and can hybridize to said first or said second primer.

In an alternative embodiment of any of the methods or steps or compositions or arrays described herein, the support optionally comprises immobilized primers having more than one sequence. After the template nucleic acid strand is hybridized to the first complementary immobilized primer, then the first primer can be extended and the template and primer can be partially or completely separated from each other. The extended primer may then be annealed to a second immobilized primer having a different sequence than the first primer, and the second primer may be extended. The two extended primers can then be separated (e.g., denatured, either completely or partially, from each other) and can then be used as templates for extending additional immobilized primers. The process can be repeated to provide amplified, immobilized nucleic acid molecules. In embodiments, the amplification results in immobilized primer extension products having two different sequences that are complementary to each other, wherein all of the primer extension products are immobilized to the support at the 5' end.

In some embodiments, the disclosed methods comprise amplifying, wherein amplifying comprises strand flipping. In the "flip" embodiments described below, two or more primers are extended to form two or more corresponding extended strands. Optionally, the two or more primers being extended comprise or consist essentially of substantially the same sequence and the extended portions of the corresponding extended strands are at least partially non-identical and/or complementary to each other.

One exemplary embodiment of the flipping is as follows. The starting template is amplified, for example, by template walking, to produce a plurality of primer-extended strands (for convenience, see which will be referred to as "forward" strands). Optionally, the forward strand is complementary to the starting template. Optionally, the forward strand is immobilized on a support. Optionally, the forward strands comprise substantially the same sequence, e.g., the forward strands are substantially identical to each other. In embodiments, the forward strand is formed by extending one or more primers (the "forward" primers) immobilized on a support. The forward primer and/or forward strand is optionally attached to the support at or near its 5' end. Optionally, one or more of the primer-extended forward strands comprises a 3 'sequence, referred to as a self-hybridizing sequence, which is not present in the non-extended primer and can hybridize to a 5' sequence under selected conditions (this process will be referred to as "self-hybridization"). The 5' sequence is optionally part of an unextended forward primer. In an example, the forward extension product forms a "stem-loop" structure upon such hybridization. Optionally, the non-extended forward primer comprises a "cleavable" nucleotide at or near its 3' end that is susceptible to cleavage. In embodiments, a cleavable nucleotide is linked to at least one other nucleotide by a "scissile" internucleoside linkage that can be cleaved under conditions that do not substantially cleave phosphodiester linkages.

Following extension, the forward-primer extension product (i.e., the forward strand) is optionally allowed to self-hybridize. In other embodiments, after allowing self-hybridization, the forward strand is cleaved at a scissile junction of cleavable nucleotides (e.g., nucleotides that form a scissile junction with an adjacent nucleotide). Cleavage results in two fragments of the primer-extension product (i.e., the extended forward strand). In embodiments, the first fragment comprises at least part of the original, non-extended forward primer. Optionally, the first fragment does not comprise any extended sequences. Optionally, the first fragment is immobilized (e.g., because the non-extended forward primer is already immobilized). In embodiments, the second fragment comprises an extended sequence. Optionally, the second fragment comprises any 3' portion of the unextended primer outside of the cleavable nucleotide or no portion of the unextended primer. Optionally, the second fragment hybridizes to the first portion through its self-hybridizing sequence.

In an example, a cleavable nucleotide is a nucleotide that is removed by one or more enzymes. The enzyme may for example be a glycosylase. The glycosylase optionally has an N-glycosylase activity, which releases a cleavable nucleotide from the double stranded DNA. Optionally, removal of the cleavable nucleotide results in an abasic, purine-free, or pyrimidine-free site. The abasic site may optionally be further modified, for example by another enzymatic activity. Optionally, the abasic site is modified by a lyase to generate a base gap. The lyase, for example, cleaves 3 'and/or 5' of abasic sites. Cleavage by the lyase optionally occurs at the 5 'and 3' ends, resulting in removal of abasic sites and leaving base gaps. Exemplary cleavable nucleotides such as 5-hydroxy-uracil, 7, 8-dihydro-8-hydroxyguanine (8-hydroxyguanine), 8-hydroxyadenine, copy-guanine, methyl-copy-guanine, copy-adenine, aflatoxin B1-copy-guanine, 5-hydroxy-cytosine, can be recognized and removed by a variety of glycosylases to form an apurinic site. One suitable enzyme is formamidopyrimidine [ copy ] -DNA glycosylase, also known as 8-hydroxyguanine DNA glycosylase or FPG. FPG is useful as an N-glycosylase and AP-lyase. N-glycosylase activity optionally releases the damaged purine from the double stranded DNA, thereby producing an apurinic (AP site), wherein the phosphodiester backbone is optionally intact. The AP-lyase activity cleaves 3 'and 5' to the AP site thereby removing the AP site and leaving a single base gap. In an example, the cleavable nucleotide is 8-hydroxyadenine, which is converted to a single base notch by FPG having glycosylase and lyase activities.

In another embodiment, the cleavable nucleotide is uridine. Optionally, the uridine is cleaved by a "USER" reagent comprising Uracil DNA Glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII, wherein UDG catalyzes the excision of uracil bases forming abasic (pyrimidine-free) sites while leaving the phosphodiester backbone intact, and wherein the lyase activity of endonuclease VIII disrupts the phosphodiester backbone 3' and 5' to the abasic sites so as to release abasic deoxyribose, followed by conversion of the phosphate group on the 3' terminus of the cleavage product to an-OH group, optionally using a kinase.

Optionally contacting at least one cleaved fragment with a polymerase. Optionally the first immobilised fragment may be extended by a polymerase. If so desired, the second hybridized fragment may serve as a template for extension of the first fragment. In embodiments, "inverted" double-stranded extension products are formed. The inverted product may optionally undergo template walking in any manner described herein. When both inverted and non-inverted undergo template walking, two different populations of extension products are formed, where the two extension products have the same portion (corresponding to the non-extended primer) and portions complementary to each other (corresponding to the extended portions of the extension products).

In embodiments, a sequence of interest, e.g., a self-hybridizing sequence or a new primer binding site, may optionally be added at the 3' end of the extended forward strand by contacting the extended forward strand with a single-stranded "splicing" linker sequence in the presence of an extension reagent (e.g., a polymerase and dntps). The splice sequence optionally comprises a 3' portion substantially complementary to the 3' terminal portion of the extended forward strand and a 5' portion substantially complementary to the sequence of interest to be added. After hybridizing the splice linker to the 3' end of the extended forward strand, the forward strand is subjected to template-dependent polymerase extension using the splice linker as a template. Such extension results in the addition of the sequence of interest to the 3' end of the extended forward strand.

Thus, any of the methods of primer extension and/or amplification described herein may comprise any one or more of the following steps: (a) Extending the immobilized forward primer by template walking to produce a plurality of extended forward strands, the forward strands optionally being identical; (b) Optionally hybridizing a splice linker to the 3' end of the extended forward strand and subjecting the forward strand to template-dependent polymerase extension using the splice linker as a template, thereby adding additional 3' sequence to the further extended forward strand, wherein part of the added 3' sequence is complementary to part of the non-extended forward primer and hybridizes thereto to form a stem-loop structure; (c) Cleaving the forward strand at a scissile junction of cleavable nucleotides located at or near the junction of the unextended forward primer sequence and the extended forward strand sequence; and optionally removing the cleavable nucleotides, thereby generating two cleaved fragments, wherein the first fragment comprises a portion of the unextended forward primer that hybridizes to the 3' primer-complementary sequence on the second fragment; (d) Optionally subjecting the first fragment to polymerase extension using the second fragment as a template to produce an inverted forward strand; (e) Optionally hybridizing a second splice linker to the 3' end of the inverted forward strand and subjecting the forward strand to template-dependent extension using the splice linker as a template, thereby adding additional 3' sequence to the inverted forward strand, wherein part of the added 3' sequence is a new primer binding sequence not present in the inverted strand; (f) The inverted strand comprising the new primer binding sequence is extended or amplified selectively by contacting with the new primer and extending or amplifying in any method (e.g., as described herein). The new primer will not bind to the non-inverted strand or to the inverted strand that was not further extended in step (e).

Fig. 8 shows a schematic depiction of an exemplary chain flipping and walking strategy. (A) template walking, (B) strand inversion to produce an inverted strand, (C) addition of a new primer binding sequence Pg' on the final inverted strand.

Optionally, a single support is used for any of the amplification methods herein, wherein the single support has a plurality of primers that can hybridize to a template. In such embodiments, the concentration of the collection of templates is adjusted prior to contacting the collection of templates with the solid support such that the individual template molecules in the collection are at least 10 ² 、10 ³ 、10 ⁴ 、10 ⁵ 、4x 10 ⁵ 、5x 10 ⁵ 、6x 10 ⁵ 、8x 10 ⁵ 、10 ⁶ 、5x 10 ⁶ Or 10 ⁷ Molecule/mm ² Is linked or associated (e.g., by hybridization to primers immobilized on a solid support).

Optionally, individual template molecules are amplified in situ on the support, resulting in a population of clones spatially localized around the point of hybridisation of the starting template. Optionally, amplification produces no more than about 10 from a single amplified template ² 、10 ³ 、10 ⁴ 、10 ⁵ 、10 ⁶ 、10 ⁷ 、10 ⁸ 、10 ⁹ ,10 ¹⁰ 、10 ¹¹ 、10 ¹² 、10 ¹⁵ Or 10 ²⁰ The amplicon of (1). Optionally, cloning the colony of amplicons to be at least 10 ² 、10 ³ 、10 ⁴ 、10 ⁵ 、4x 10 ⁵ 、5x 10 ⁵ 、6x 10 ⁵ 、8x 10 ⁵ 、10 ⁶ 、5x 10 ⁶ Or 10 ⁷ Molecule/mm ² Is located on the solid support.

In some embodiments, a collection of nucleic acids can be contacted with one or more supports under conditions in which multiple nucleic acids are bound to the same support. Such contacting may be particularly useful in methods involving parallel clonal amplification of nucleic acids in different regions of the same support. The ratio of the number of nucleic acids to the surface area of the support can be adjusted to promote monoclonal formation by, for example, ensuring that the nucleic acids are appropriately spaced in the support to promote the formation of a single clonal population of amplified nucleic acids without substantial cross-contamination between different clonal populations. For example, when a single support is used, the collection of nucleic acids to be amplified is adjusted to such a dilution that the resulting amplified clonal populations generated from the individual nucleic acids are generally non-contiguous or separated, e.g., non-overlapping. For example, 50%, 70%, 80%, or 90% or more of the individual nucleic acids within the amplified clonal population are not interspersed with substantially non-identical nucleic acids. Optionally, the different amplification populations do not contact or completely overlap the other amplification populations, or may be distinguished from each other using a selected detection method.

In some embodiments, the nucleic acid is attached to the surface of the support. In some embodiments, the nucleic acid may be attached within a support. For example, for a support comprising a hydrogel or other porous matrix, nucleic acids can be attached throughout the volume of the support, including on the surface and within the support.

In some embodiments, a support (or at least one support in a population of supports) can be attached to at least one primer, optionally to a population of primers. For example, the support (or at least one support) may comprise a population of primers. The primers of the primer population may be substantially identical to each other or may comprise substantially identical sequences. One, some, or all of the primers may comprise a sequence that is complementary to a sequence within one or more nucleic acid templates. In some embodiments, the population of primers can comprise at least two non-complementary primers.

The primer may be attached to the support via its 5 'end and has a free 3' end. The support may be the surface of a slide or the surface of beads. Primers having a low melting temperature, e.g. oligo (dT) ₂₀ And low T hybridizable to the collector adaptor _m A region. The distance between the primers needs to be shorter than the linker length to allow the template to walk, or alternatively, a long primer with a long linker at the 5' end will increase the chance of walking.

In some embodiments, the support is ligated and/or contacted with the primer and template (or reverse strand) under conditions in which the primer and template hybridize to each other to form a nucleic acid duplex. The duplex can include a double-stranded portion comprising the complementary sequences of the template and the primer, wherein at least one nucleotide residue of the complementary sequences is base-paired with each other. In some embodiments, the double strand may further comprise a single stranded portion. The double strand may also comprise a single stranded portion. The single stranded portion may comprise any sequence within the template (or primer) that is not complementary to any other sequence in the primer (or template).

Non-limiting exemplary methods for amplification of nucleic acids cloned on a support are described below. Nucleic acids (which will be referred to as the reverse strand for convenience) are clonally amplified on a support to which multiple copies of complementary forward primers are attached. An exemplary nucleic acid is one of a plurality of DNA collector molecules, e.g., a plurality of nucleic acid members having one or more sequences in common ("linkers") at their 5 'and/or 3' ends and variable sequences therebetween, e.g., gDNA or cDNA. In embodiments, the 3' common portion, e.g., the junction, is respirable (e.g., low T) _m ) The region, and the 5' common sequence (e.g., linker) optionally have a less respirable (e.g., higher T) _m ) Regions, or vice versa. In another embodiment, both the 5 'and 3' common sequences are respirable. Breathable (e.g. low T) _m ) A region is for example an A-, T-and/or U-rich region, for example an AT (or U) -rich sequence, for example polyT, polyA, polyU and A, T and U bases or any combination of bases complementary to such bases. Exemplary methods are described herein.

One non-limiting exemplary method of nucleic acid amplification of clones by "template walking" on a support is shown in FIG. 1. A non-limiting description of an exemplary method of template walking is as follows.

Double-stranded DNA library molecules are denatured and single-stranded DNA is attached to a support by hybridization to primers on the support. The ratio of the number of DNA molecules to the area of the support or the number of beads is set to promote monoclonal formation.

The primer is attached to the primer via its 5 'end and has a free 3'. The support may be the surface of a slide or the surface of beads. Primers having a low melting temperature, e.g. oligo (dT) ₂₀ Or oligo (dA) ₃₀ And low T hybridizable to library adaptors _m And (4) a region. The distance between the primers may be shorter than the linker length to allow template walking, or alternatively, a long primer with a long linker at the 5' end will increase the chance of walking.

Nucleic acids are clonally amplified on a support to which multiple copies of primers are attached. An exemplary nucleic acid is one of a plurality of DNA library molecules, e.g., having one or more sequences in common (e.g., "linkers") at their 5 'and/or 3' ends and variable sequences therebetween, e.g., gDNA or cDNA. In embodiments, the 3' linker has a low T _m Region, and the 5' linker optionally has a higher T _m Regions, or vice versa. Low T _m A region is, for example, a pyrimidine-rich region, such as an AT (or U) -rich sequence, e.g., polyT, polyA, polyU and A, T and U bases or any combination of bases complementary to such bases. Exemplary methods are described herein.

One or more primers, whether in solubilized form or attached to a support, are incubated using a DNA polymerization or extension reaction mixture, optionally containing any one or more reagents such as enzymes, dntps, and buffers. Extending the primer (e.g., forward primer). Optionally, extension is template-dependent extension of the primer along the template, including the continuous incorporation of nucleotides each complementary to a continuous nucleotide on the template, such that the extended or unextended forward primer is complementary to the reverse strand (also referred to as antiparallel or complementary). Optionally, extension is achieved by an enzyme, such as a polymerase, having polymerase activity or other extension activity. The enzyme may optionally have other activities including 3'-5' exonuclease activity (proofreading activity) and/or 5'-3' exonuclease activity. Alternatively, in some embodiments, the enzyme may lack one or more of these activities. In embodiments, the polymerase has strand displacement activity. Examples of useful strand displacing polymerases include bacteriophage Φ 29DNA polymerase and Bst DNA polymerase. Optionally, the enzyme is active at elevated temperatures, e.g., at or above 45 ℃, at or above 50 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, or 85 ℃.

An exemplary polymerase is Bst DNA polymerase (exonuclease negative), which is a 67kDa Bacillus stearothermophilus DNA polymerase protein (large fragment) (exemplified by accession No. 2bdp _a) having 5'-3' polymerase activity and strand displacement activity single lack of 3'-5' exonuclease activity. Other polymerases include Taq DNA polymerase I from Thermus aquaticus (exemplified by accession number 1 Taq), eco DNA polymerase I from Escherichia coli (accession number P00582), aea DNA polymerase I from hyperthermophiles (Aquifex aeolicus) (accession number 067779), or functional fragments or variants thereof, e.g., functional fragments or variants having at least 80%, 85%, 90%, 95%, or 99% sequence identity at the nucleotide level.

Typically, the extension step produces a nucleic acid comprising a double-stranded duplex portion in which two complementary strands hybridize to each other. In one embodiment, walking comprises subjecting the nucleic acid to partial denaturing conditions that denature a portion of the nucleic acid strand but not enough to completely denature the nucleic acid over its entire length. In embodiments, the nucleic acid is not subjected to fully denaturing conditions during part or the entire duration of the walking procedure.

In embodiments, the sequences of the negative and/or positive strands are designed such that the primer binding sequences, or portions thereof, are respirable, i.e., susceptible to denaturation under selected conditions (e.g., amplification conditions). The respirable portion is optionally more susceptible than a majority of similar length nucleic acids having random sequences, or more susceptible than at least another portion of the strand comprising the respirable sequence. Optionally, the respirable sequences exhibit a significant amount of denaturation under the selected amplification conditions (e.g., at least 10%, 20%, 30%, 50%, 70%, 80%, 90%, or 95% of the molecules are completely denatured across the respirable sequence). For example, the respirable sequences are designed to be completely denatured in 50% of the strand molecules at 30, 35, 40, 42, 45, 50, 55, 60, 65, or 70 ℃ under selected conditions (e.g., amplification conditions).

Exemplary breathable PBS's can be pyrimidine-rich (e.g., having high contents of a and/or T and/or U) when partial denaturation is achieved by heating or elevated temperatures. PBS contains, for example, poly-A, poly-T or poly-U sequences or polypyrimidine tracts. One or more amplification or other primers (e.g., immobilized primers) are optionally designed to be correspondingly complementary to these primer binding sequences. Exemplary PBSs for a nucleic acid strand comprise a poly-T sequence, e.g., a segment of at least 10, 15, 20, 25, or 30 thymidine nucleotides, while corresponding primers have the complement of PBS, e.g., a segment of at least 10, 15, 20, 25, or 30 adenosine nucleotides. Exemplary low melting primers optionally have a high proportion (e.g., at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of nucleobases: which typically (e.g., under the selected amplification conditions) form no more than two hydrogen bonds with a complementary base when the primer hybridizes to a complementary template. Examples of such nucleobases include A (adenine), T (thymine) and U (uracil). Exemplary low melting primers optionally have a high proportion of any one or more of a (adenine), T (thymine) and/or U (uracil) or derivatives thereof. In embodiments, the derivative comprises a nucleobase complementary to a (adenine), T (thymine) and/or U (uracil). The portion of the primer that hybridizes to PBS optionally has at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% A (adenine), T (thymine), or U (uracil) nucleotides or Any combination thereof. In another example, the portion of the primer that hybridizes to PBS comprises a polyA sequence (e.g., at least 5, 10, 15, 20, 25, or 30 nucleotides in length). Other exemplary primers comprise (NA) _x ) _n The sequence is repeated. Optionally, n (lower case) is from 2 to 30, such as from 3 to 10, such as from 4 to 8."N" (capital) is any nucleotide-and optionally, N is C or G. "A" is a short notation for adenine and "x" indicates the number of adenine residues in the repeat sequence, e.g., 2, 3, 4, 5, 6, 10 or more. Exemplary primers Comprise (CAA) _n 、CA) _n 、(CAAA) _n Or even (GAA) _n A plurality of repeated sequences of (a).

Optionally only one strand (e.g. the forward or reverse strand) has a respirable PBS. In another embodiment, both the forward and reverse chains have a breathable PBS. The respirable PBS is optionally complementary to a primer that is immobilized to a support or not immobilized (e.g., in dissolved form). Optionally, the chain comprising the respirable PBS is immobilized to a support or is non-immobilized (e.g., in dissolved form). Optionally both primers are immobilized, or both strands are immobilized. Optionally neither primer nor strand is immobilized.

The amplification cycle optionally includes respiration (breaking), annealing, and extension. Optionally subjecting the nucleic acid to be amplified to conditions suitable or optimal for at least one of these steps. In embodiments, the nucleic acid is subjected to conditions suitable for more than one of these steps (e.g., annealing and extension, or respiration and extension). In some examples, all three of these steps may occur simultaneously under the same conditions.

In an exemplary method, the nucleic acid can be subjected to conditions that allow or promote respiration. In embodiments, "respiration" is considered to occur when the two strands of a double-stranded double helix are substantially hybridized to each other but denatured in the local portion of interest. A first complementary strand (e.g., a forward or reverse strand) to which one or more respirable sequences of a nucleic acid (e.g., a forward and/or reverse PBS having a low Tm portion) hybridize is locally denatured ("respired") from the first strand, and thus available for hybridization to another second strand. An exemplary first strand is a primer extension product from a first primer. An exemplary second strand is, for example, a second unextended primer (e.g., a PBS complementary oligonucleotide comprising, for example, a dT or dA sequence). Optionally, the first and second strands are immobilized on a support, and can be placed in close proximity (e.g., sufficiently close proximity to allow walking). The conditions for respiration are optionally partially denaturing conditions under which the PBS is substantially denatured but another portion of the nucleic acid remains hybridized or double stranded. Optionally, a DNA helicase may be included in the reaction mixture to facilitate partial denaturation.

Optionally, the nucleic acid is then subjected to conditions that promote annealing, such as a reduction in temperature, to enable hybridization between the respirable PBS and the second strand. In embodiments, the same conditions are used to promote respiration and elongation. In another embodiment, the annealing conditions are different from the respiration conditions-e.g., the annealing conditions are non-denaturing conditions or conditions that promote denaturation less than the respiration conditions. In an example, the annealing conditions include a lower temperature (e.g., 37 ℃) than the breathing conditions in which a higher temperature (e.g., 60-65 ℃) is used. Optionally, complete denaturation conditions are avoided during one or more amplification cycles (e.g., most amplification cycles or substantially all amplification cycles).

Optionally, one or more PBS-breathing and primer extension steps are repeated multiple times to amplify the starting nucleic acid. When one or more nucleic acid reagents (e.g., primers) are immobilized to the support, the primer-extension products substantially remain attached to the support, e.g., due to attachment of extended primers that were not extended prior to amplification to the support or by hybridization to such primers.

Optionally, one or more samples of the nucleic acid population to be amplified are prepared. The population of nucleic acids may be in single-stranded or double-stranded form; optionally, the one or more nucleic acids each comprise a nucleic acid strand having a known 3 'end sequence and a known 5' end sequence that is substantially identical or complementary to the one or more primers used for amplification. The 3 'portion of the nucleic acid strand may, for example, be complementary to the immobilized primer, while the 5' portion may be the same as the solubilized primer. The 5 'and/or 3' portions may be common ("universal") or invariant between individual nucleic acids within a population. Optionally, the nucleic acids within a population each comprise different (e.g., unknown) sequences between common portions, e.g., genomic DNA, cDNA, mRNA, mate-pair fragments, exomes, and the like. Collections can, for example, have sufficient members to ensure coverage of greater than 50%, 70%, or 90% of the corresponding genetic source.

In some embodiments, the present disclosure relates generally to compositions for nucleic acid amplification comprising reaction mixtures for nucleic acid amplification and related systems, devices, kits, and methods.

In some embodiments, the present disclosure relates generally to compositions, and associated systems, devices, kits, and methods, for nucleic acid amplification comprising a reaction mixture comprising a continuous liquid phase comprising (i) a polymerase and (ii) a plurality of supports, at least one of which is attached to a substantially monoclonal population of nucleic acids.

In some embodiments, the present disclosure relates generally to compositions, and associated systems, devices, kits, and methods, for nucleic acid amplification comprising a reaction mixture comprising a continuous liquid phase comprising (i) a polymerase and (ii) a plurality of supports including a first support and a second support.

In some embodiments, the present disclosure relates generally to compositions (and related systems, devices, kits, and methods) for nucleic acid amplification comprising: a reaction mixture comprising a continuous liquid phase comprising (i) a plurality of supports including a first support and a second support, (ii) a plurality of different polynucleotides comprising a first polynucleotide and a second polynucleotide, and (iii) reagents for isothermal nucleic acid amplification. In some embodiments, the reagents for nucleic acid amplification comprise a polymerase and one or more types of nucleotides (e.g., a plurality of nucleotides). Optionally, the reagents for isothermal nucleic acid amplification comprise a recombinase.

Optionally, the first and second polynucleotides have different sequences.

Optionally, at least one end of at least one of the plurality of different polynucleotides is linked to at least one oligonucleotide linker.

Optionally, at least one end of at least some of the plurality of different polynucleotides comprises a common sequence.

Optionally, at least two of the different polynucleotides in the reaction mixture comprise a common sequence.

Optionally, the first and second polynucleotides are different.

In some embodiments, the solution phase comprises one or more supports of a plurality comprising primers.

Optionally, the reaction mixture comprises a continuous liquid phase.

Optionally, the reaction mixture can be used to perform isothermal or thermocycling nucleic acid amplification.

Optionally, the continuous liquid phase comprises any one or any combination of (i) one or more polymerases and/or (ii) at least one support.

Optionally, the continuous liquid phase comprises a plurality of supports.

Optionally, the continuous liquid phase comprises a first support.

Optionally, the continuous liquid phase comprises a second support.

Optionally, at least one support of the plurality can be attached to a substantially monoclonal population of nucleic acids.

Optionally, a first support can be attached to the first substantially monoclonal population of nucleic acids.

Optionally, a second support can be attached to the second substantially monoclonal population of nucleic acids.

Optionally, the first and second substantially monoclonal populations of nucleic acids comprise different sequences or substantially the same sequence.

Optionally, the first and second substantially monoclonal populations of nucleic acids hybridize to each other or do not hybridize under stringent hybridization conditions.

Optionally, the first and second substantially monoclonal nucleic acid populations are not identical.

Optionally, the first and second substantially monoclonal nucleic acid populations are non-complementary.

Optionally, the reaction mixture comprises nucleotides that are not exogenously labeled. For example, the nucleotide may be a naturally occurring nucleotide, or a synthetic analog that does not contain a fluorescent moiety, dye, or other exogenous optically detectable label.

Optionally, the reaction mixture is contained in a single reaction vessel.

Optionally, the reaction mixture comprises an isothermal or thermocycling reaction mixture.

Optionally, the plurality of supports comprises beads, particles, microparticles, spheres, gels, filters, or the inner wall of a tube.

Optionally, at least one support of the plurality can be attached to a plurality of nucleic acids.

Optionally, at least one support of the plurality can be attached to one or more primers. The primers may be the same (or comprise a common sequence) or different.

Optionally, at least one support can be attached to a plurality of first primers.

Optionally, at least one support can be attached to a plurality of first primers and a plurality of second primers.

Optionally, the plurality of first primers comprise substantially identical sequences.

Optionally, the plurality of first primers comprises at least one first primer comprising a sequence that is identical or complementary to at least a portion of a polynucleotide of the plurality of different polynucleotides.

Optionally, the plurality of second primers comprises at least one second primer comprising a sequence that is identical or complementary to at least a portion of a polynucleotide of the plurality of different polynucleotides.

In some embodiments, at least one polynucleotide of the plurality of different polynucleotides comprises a first sequence that is substantially identical to or substantially complementary to a sequence within the first primer. In some embodiments, at least one polynucleotide further comprises a second sequence that is substantially identical or substantially complementary to a sequence within the second primer. In some embodiments, substantially all of the polynucleotides in the plurality of different polynucleotides comprise the first sequence and the second sequence.

Optionally, at least one support of the plurality is attached to 2-10 different pluralities of primers.

Optionally, the 2-10 different pluralities of primers comprise different sequences.

Optionally, the 2-10 different pluralities of primers comprise at least one sequence that hybridizes to at least a portion of different polynucleotides.

Optionally, the 2-10 different pluralities of primers comprise at least one sequence that hybridizes to at least a portion of a common sequence in different polynucleotides.

Optionally, at least one of the supports is attached to at least one unique identifying barcode sequence.

Optionally, the first and second substantially monoclonal populations of nucleic acids have substantially the same or different sequences.

Optionally, the reaction mixture comprises at least one recombinase.

Optionally, the recombinase may catalyze homologous recombination, strand invasion, and/or D-loop formation.

Optionally, the recombinase is part of a nucleoprotein filament comprising the recombinase linked to a primer attached to a support in a reaction mixture. Primers linked by a recombinase can be attached to a support or in solution.

Optionally, the reaction mixture comprises a nuclear protein complex or a plurality of nuclear protein complexes.

Optionally, the reaction mixture comprises a first nuclear protein complex.

Optionally, the reaction mixture comprises a second nucleoprotein complex.

Optionally, at least one nucleoprotein complex of the plurality comprises at least one recombinase attached to the primer.

Optionally, the reaction mixture comprises a first nucleoprotein complex comprising at least one recombinase enzyme linked to a first primer.

Optionally, the reaction mixture comprises a second nucleoprotein complex comprising at least one recombinase linked to a second primer.

Optionally, the recombinase comprises a phage recombinase from T4, T2, T6, rb69, aeh1, KVP40, acinetobacter (Acinetobacter) phage 133, aeromonas (Aeromonas) phage 65, blue-green algae phage P-SSM2, blue-green algae phage PSSM4, blue-green algae phage S-PM2, rb14, rb32, aeromonas phage 25, vibrio (Vibrio) phage nt-1, phi-1, rb16, rb43, phage 31, phage 44RR2.8t, rb49, phage Rb3, or phage LZ 2.

Optionally, the recombinase comprises uvsX recombinase from a T4 bacteriophage or recA recombinase from e.

Optionally, the reaction mixture further comprises a polymerase.

Optionally, the polymerase lacks 5 'to 3' exonuclease activity.

Optionally, the polymerase comprises strand displacement activity.

Optionally, the polymerase comprises a thermostable or thermostable polymerase.

Optionally, the polymerase comprises a DNA polymerase or an RNA polymerase.

Optionally, the reaction mixture further comprises at least one type of nucleotide.

Optionally, at least one support of the plurality comprises at least one primer.

Optionally, the first support is attached to a first substantially monoclonal population of nucleic acids and the second support is attached to a second substantially monoclonal population of nucleic acids.

Optionally, the first and second substantially monoclonal nucleic acid populations have different nucleic acid sequences.

Optionally, the first and second substantially monoclonal populations of nucleic acids do not hybridize to each other under stringent hybridization conditions.

Optionally, the population of substantially monoclonal nucleic acids of the first and second nucleic acids are non-identical and non-complementary.

Optionally, the reaction mixture comprises (i) at least two polynucleotide templates to be amplified and/or (ii) at least one nucleoprotein filament complex.

Optionally, the reaction mixture comprises at least one polynucleotide, primer, template, or amplification product linked to a resistance compound. As used herein, the term "drag compound" and variants thereof describe any chemical composition that: the compositions can attach to nucleic acids and impede their diffusion through the reaction mixture, but still allow for nucleic acid synthesis using such polynucleotides, primers, templates, or amplification products in a nucleic acid synthesis reaction. The attachment of such resistance compounds to nucleic acids within a synthesis reaction generally reduces the mobility of such nucleic acids in the reaction mixture and can be used to prevent cross-contamination of amplification products or templates between different synthesis reactions that occur using the same reaction mixture. In some embodiments, the attachment of a resistance component to one or more nucleic acid components can increase the number or proportion of monoclonal products.

In some embodiments, the present teachings provide methods for nucleic acid amplification comprising at least one mobility-altered nucleic acid (e.g., a primer). In some embodiments, the mobility-altered nucleic acid exhibits increased or decreased mobility through an aqueous medium. In some embodiments, the modified nucleic acid comprises a nucleic acid (e.g., a primer) linked at any position along the length of the nucleic acid to one or more compounds that alter the flow of the nucleic acid through an aqueous medium (e.g., a resistance compound). In some embodiments, a resistance compound that alters the mobility of nucleic acids can be attached to any primer in a nucleic acid amplification reaction, including a first, second, third, fourth, or any other primer. For example, one or more resistance compounds can be attached to a nucleic acid at any one or any combination of the 5 'end, 3' end, and/or internal position. In some embodiments, the modified nucleic acid may be covalently or non-covalently linked to a resistance compound that alters the mobility of the nucleic acid through aqueous media. For example, a resistance compound, when linked to a nucleic acid, can provide aqueous hydrodynamic resistance by altering the physical size, length, radius, shape, or charge of the modified nucleic acid compared to a nucleic acid lacking the linked compound. In some embodiments, a resistance compound attached to a nucleic acid can alter the interaction between the nucleic acid and an aqueous medium (as compared to the interaction between the aqueous medium and a nucleic acid lacking the attached compound). In some embodiments, the resistance compound may be synthetic, recombinant, or naturally occurring. In some embodiments, the drag compound may be dotted, uncharged, polar or hydrophobic. In some embodiments, the drag compound may be linear, branched, or have a dendritic polymeric structure. In some embodiments, the drag compound may comprise a single moiety or polymer of a nucleoside, sugar, lipid, or amino acid.

Optionally, the drag compound comprises a sugar moiety, a polysaccharide, a protein, a glycoprotein, or a polypeptide. Optionally, the resistance compound comprises BSA, lysozyme, beta-actin, myosin, whey protein, ovalbumin, beta-galactosidase, lactate dehydrogenase, or an immunoglobulin (e.g., igG).

Optionally, the drag compound that alters the flow of the nucleic acid through the aqueous medium comprises one or more polyethylene oxide (PEO) or polypropylene oxide (PPO) moieties, including polymers of polyethylene oxide (PEO) or polypropylene oxide (PPO). Non-limiting examples of such polymers include triblock copolymersSubstances (e.g. PEO-PPO-PEO), pluronics ^TM -type polymers and hydrophobically modified PEO polymers. Optionally the resistance compound comprises one or more amino acid moieties, polypeptides and clusterin. Optionally, the drag compound comprises a sugar moiety, a polysaccharide, a hydrophobically modified polysaccharide, a cellulose derivative, sodium carboxymethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, or hydroxypropyl methyl cellulose. Optionally, the resistant compound comprises hydrophobically modified alkali-soluble associative (HASE) polymer, hydrophobically modified polyacrylamide, heat-sensitive polymer, or N-isopropylacrylamide (NTPAAm), and optionally, the resistant compound comprises poly (ethylene glycol) methyl ether acrylate (PEGMEA), tetraethylene glycol diacrylate (TEGDA), poly (ethylene glycol) dimethacrylate (EGDMA), or N, N' -methylene-bis-acrylamide (NMBA).

Optionally, the resistance compound comprises a protein or polypeptide, including BSA, lysozyme, β -actin, myosin, whey protein, ovalbumin, β -galactosidase, or lactate dehydrogenase. In some embodiments, the resistance compound can be attached to the nucleic acid through an amine or thiol linkage.

In some embodiments, the mobility-altering nucleic acid comprises a nucleic acid linked to a binding partner (e.g., an affinity moiety for interacting with a receptor moiety). In some embodiments, receptor moieties can be used as a resistance compound. In some embodiments, an affinity moiety can be attached to a nucleic acid, and the affinity moiety (which acts as a resistance compound) interacts with a receptor moiety. For example, the nucleic acid can be linked to a biotin moiety that can bind to an avidin-like moiety. Avidin-like moieties can be used as a resistive compound. Avidin-like moieties include avidin as well as any derivative, analog, and other non-natural form of avidin that can bind to a biotin moiety. Other examples of binding partners include epitopes (e.g., protein a) and their respective antibodies (e.g., anti-FLAG antibodies), as well as fluorescein and anti-fluorescein antibodies. One skilled in the art will readily identify other binding partner combinations for attaching a resistance compound to a nucleic acid.

Optionally, the resistance compound may be attached to the primer by attaching the resistance compound and the primer to each of the two members of the binding partner pair.

Optionally, at least one primer in the reaction mixture comprises biotin.

Optionally, the resistance compound comprises avidin or streptavidin.

Optionally, the resistant compound comprises a sugar moiety, a polysaccharide, a protein, a glycoprotein, or a polypeptide.

Optionally, the resistance compound comprises BSA, lysozyme, beta-actin, myosin, whey protein, ovalbumin, beta-galactosidase, lactate dehydrogenase, or an immunoglobulin (e.g., igG).

Optionally, the reaction mixture further comprises a helper protein.

Optionally, the helper protein comprises a helicase, a single-chain binding protein, or a recombinase loading factor.

Optionally, the helicase comprises uvsW from a T4 bacteriophage.

Optionally, the single-stranded binding protein comprises an Sso SSB from Sulfolobus solfataricus, an MjA SSB from Methanococcus jannaschii, or an Escherichia coli SSB protein.

Optionally, the single-chain binding protein comprises gp32 protein from a T4 bacteriophage or a modified gp32 protein from a T4 bacteriophage.

Optionally, the recombinase loading protein comprises uvsY from T4 bacteriophage.

Optionally, the reaction mixture further comprises ATP.

Optionally, the reaction mixture further comprises an ATP regeneration system.

Optionally, the ATP regeneration system comprises creatine phosphate.

Optionally, the ATP regeneration system comprises creatine kinase.

Optionally, the reaction mixture further comprises an adduct for increasing the efficiency or yield of the nucleic acid amplification reaction.

Optionally, the adduct comprises betaine, DMSO, proline, trehalose, MMNO (4-methylmorpholine N-oxide), or a PEG-like compound.

Optionally, at least one polynucleotide template in the reaction mixture comprises a first sequence that is complementary or identical to at least some portion of the first primer, and a second sequence that is complementary or identical to at least some portion of the second primer. Optionally, the reaction mixture comprises a plurality of double-stranded polynucleotides comprising a first sequence that is complementary or identical to at least a portion of the first primer, and a second sequence that is complementary or identical to at least a portion of the second primer. Optionally, the first sequence is located at or near an end of at least one double-stranded polynucleotide in the plurality and the second sequence is located at or near another end of at least one double-stranded polynucleotide in the plurality.

Optionally, the reaction mixture further comprises a diffusion limiting agent.

Optionally, the diffusion limiting agent reduces the rate of diffusion of the polynucleotide away from the support.

Optionally, the diffusion limiting agent reduces the level of polyclonal nucleic acid population attached to the support.

Optionally, the diffusion limiting agent comprises a polymeric compound.

Optionally, the diffusion limiting agent comprises a sugar polymer.

Optionally, the diffusion limiting agent comprises a cellulose-based compound.

Optionally, the diffusion limiting agent comprises a glucose or galactose polymer.

Optionally, the carbohydrate polymer comprises cellulose, dextran, starch, glycogen, agar or agarose.

Optionally, the diffusion limiting agent comprises a block copolymer compound.

Optionally, the diffusion limiting agent comprises a poly (propylethylene oxide) central chain flanked by two poly (ethylene oxide) hydrophilic chains.

Optionally, the diffusion limiting agent forms micelles.

Optionally, the diffusion limiting agent forms micellar liquid crystals.

Optionally, the diffusion limiting agent comprises Pluronics ^TM Compound (I)。

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

Optionally, the compositions for nucleic acid amplification and related systems, devices, kits, and methods further comprise a surface, substrate, or medium comprising a plurality of sites, wherein at least one site is operably coupled to one or more sensors.

Optionally, the plurality of sites comprises the inner walls of a reaction chamber, support, particle, microparticle, sphere, bead, filter, flow cell, well, channel, sink container, gel, or tube.

Optionally, the plurality of sites may be arranged in a random array or a programmed array.

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

Optionally, at least one of the plurality of sites comprises a three-dimensional chemistry matrix.

Optionally, at least one of the plurality of sites can be covalently attached to the three-dimensional chemistry matrix.

Optionally, at least one of the plurality of sites comprises an acrylamide layer. Optionally, at least one of the plurality of sites comprises a nucleic acid covalently linked to the acrylamide layer.

Optionally, the hydrophilic polymer matrix comprises a hydrogel polymer matrix.

Optionally, the hydrophilic polymer matrix is an in situ cured polymer matrix.

Optionally, the polyacrylamide is conjugated to an oligonucleotide primer.

Optionally, the pores have a characteristic diameter of 0.1 to 2 microns.

Optionally, the pores have a depth of 0.01 to 10 microns.

In some embodiments, the sensor comprises a Field Effect Transistor (FET). The FET may comprise an Ion Sensitive FET (ISFET), a chemically sensitive field effect transistor (chemFET), or a biologically active field effect transistor (bioFET).

Optionally, one or more sensors are configured to detect byproducts of nucleotide incorporation.

Optionally, one or more sensors may be configured to detect the presence of a chemical moiety at one or more of the plurality of sites.

Optionally, the one or more sensors comprise a Field Effect Transistor (FET), an Ion Sensitive Field Effect Transistor (ISFET), a chemosensitive field effect transistor (chemFET), or a biologically active field effect transistor (bioFET).

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

Optionally, the by-products from the nucleotide incorporation reaction include pyrophosphate, hydrogen ions, or protons.

Also provided are compositions comprising any, any subgroup, or all of the following: at least one reverse nucleic acid strand, a plurality of forward primers immobilized on at least one support, a plurality of reverse primers in solution, and a polymerase. The forward and/or reverse primers are optionally low melting or are adenine, thymine or uracil rich as described herein. Exemplary compositions include a solid support comprising a plurality of spatially separated clonal populations that each comprise a low melting primer binding sequence at the 3 'end and a low melting primer sequence at the 5' end. Optionally, the composition further comprises a recombinase. Alternatively, the composition optionally does not comprise another enzyme that is not a polymerase, such as a recombinase or a reverse transcriptase or a helicase or a nickase. Another exemplary composition comprises any one or more of the following components: (1) a reverse nucleic acid strand, (2) a plurality of low melting forward primers immobilized on a support, (3) a plurality of low melting reverse primers in solution, and (4) a polymerase. Optionally, the forward primer is hybridizable (e.g., complementary) to a 3' portion or end of the reverse strand. Optionally, the reverse primer is substantially identical to the 5' portion or end of the reverse strand. The composition may comprise any one or more of the agents described herein, and/or may be subjected to any one or more of the procedures or conditions described herein.

In some embodiments, the present disclosure relates generally to compositions comprising amplified nucleic acids produced by any of the methods of the present disclosure. In some embodiments, a limited clonal population of clonal amplicons is formed around discrete sites on a support. Exemplary discontinuous sites are the point of attachment of an initial nucleic acid strand to a support, and from which other nucleic acids within a clonal population are generated directly or indirectly by primer extension using the initial nucleic acid or a copy thereof as a template.

Optionally, the composition comprises a collection of nucleic acids that can be produced by any one or more of the methods described herein. For example, a collection can comprise immobilized nucleic acids occupying one or more discrete regions on a surface. In some embodiments, each region comprises a plurality of identical nucleic acid strands and optionally a plurality of identical complementary strands hybridized thereto, wherein the complementary strands are not attached or linked or associated with a solid support except as a result of hybridization to an immobilized nucleic acid. Optionally, the individual nucleic acid strands within such a region are positioned such that the other nucleic acid strand is located within a distance of the length of the strand on the surface. Optionally per mm of the surface on which the nucleic acid is immobilized ² In which at least one separate zone is presentA domain. E.g. number of separate areas/mm ² A surface having immobilized thereon nucleic acids greater than 10 ² Greater than 10 ³ Greater than 10 ⁴ Greater than 10 ⁵ Greater than 10 ⁶ Greater than 10 ⁷ Or greater than 10 ⁸ 。

The collection of amplified clonal populations can form an array, which can be one-dimensional (e.g., an array of generally monoclonal microbeads) or two-dimensional (e.g., amplified clonal populations on a planar support) or three-dimensional. The individual clonal populations of the array are optionally, but not necessarily, positioned or arranged so as to be addressed or addressable. Optionally, the different clonal populations are spaced apart from each other by a suitable distance, typically sufficient to allow the different clonal populations to be distinguished from each other. In embodiments, a confined population of clones is spread out over a planar substrate in an ordered or disordered (e.g., random) manner.

The features of the exemplary array are populations of individually distinguishable nucleic acid clones, wherein the features are optionally distributed on one or more supports. In an exemplary microbead embodiment, the array comprises a plurality of microbeads, wherein individual microbeads typically comprise a monoclonal population of nucleic acids, and different microbeads typically comprise different clonal populations (e.g., which differ in sequence). Optionally, the microbeads are distributed or filled in a monolayer on a planar substrate. In other embodiments, the array comprises a single (e.g., planar) support comprising a plurality of spatially discrete populations of nucleic acid clones, wherein the different clonal populations optionally differ in sequence.

Optionally, one or more nucleic acids within an individual clonal population can be directly attached to a planar substrate. In another example, the nucleic acids of the individual clonal populations are attached to microbeads, e.g., as discussed herein. Clonal microbeads are optionally packed closely together in a random or ordered fashion on a planar substrate. Optionally, more than 20%, 30%, 50%, 70%, 80%, 90%, 95%, or 99% of the microbeads are in contact with at least one, two, four, or six other microbeads. Optionally, less than 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or 99% of the microbeads are in contact with one, two, four, or six other microbeads.

In some embodiments, the present disclosure relates generally to methods for nucleic acid amplification and related compositions, systems, kits, and devices, including multiplex nucleic acid amplification using any of the amplification methods, compositions, or systems disclosed herein.

In some embodiments, the methods comprise performing multiplex amplification using a polymerase (e.g., polymerase-mediated multiplex nucleic acid amplification reactions).

In some embodiments, the method may further comprise re-amplifying amplicons from the multiplex nucleic acid amplification using a nucleic acid amplification reaction.

Optionally, multiplex nucleic acid amplification can be performed in a single reaction mixture.

Optionally, a sample comprising a plurality of different nucleic acid target sequences can be subjected to multiplex nucleic acid amplification.

Optionally, a plurality of different nucleic acid target sequences can be amplified in a single reaction mixture.

Optionally, at least a few dozen or at least hundreds or at least thousands (or more) of nucleic acid target sequences can be amplified in a single reaction mixture.

Optionally, at least fifty or at least one hundred nucleic acid target sequences can be amplified in a single reaction mixture.

Optionally, the multiplex amplification may comprise contacting at least a portion of the sample with any one or any combination of a recombinase, a polymerase, and/or at least one primer.

Optionally, the multiplex amplification can be performed under isothermal or thermocycling conditions.

In some embodiments, the present disclosure relates generally to methods and related compositions, systems, kits, and devices for nucleic acid amplification, including multiplex nucleic acid amplification comprising amplifying at least fifty different nucleic acid target sequences (or more) from a sample comprising a plurality of different nucleic acid target sequences within a single reaction mixture, the amplifying comprising contacting at least a portion of the sample with a recombinase and a plurality of primers under isothermal amplification conditions.

In some embodiments, the present disclosure relates generally to methods and related compositions, systems, kits, and devices for nucleic acid amplification, including multiplex nucleic acid amplification comprising amplifying different nucleic acid target sequences from a sample comprising a plurality of different nucleic acid target sequences within a single reaction mixture, the amplification comprising generating at least fifty of the plurality of different amplified target sequences (or more) by contacting at least a portion of the sample with a recombinase and a plurality of primers under isothermal amplification conditions.

In some embodiments, the present disclosure relates generally to methods for nucleic acid amplification and related compositions, systems, kits, and devices, including generating a substantially monoclonal population of nucleic acids by re-amplifying amplicons from a multiplex of nucleic acid amplifications using a nucleic acid amplification reaction (e.g., a recombinase).

Optionally, the method for multiplex nucleic acid amplification may further comprise a recombinase-mediated nucleic acid amplification method comprising re-amplifying at least some of the at least fifty different amplified target sequences by: (a) Forming a reaction mixture comprising a single continuous liquid phase comprising (i) a plurality of supports, (ii) at least one of the fifty different amplified target sequences and (iii) a recombinase; and (b) subjecting the reaction mixture to amplification conditions, thereby generating a plurality of supports attached to a population of nucleic acids that are substantially monoclonal attached thereto.

Optionally, in the method for multiplex nucleic acid amplification, different nucleic acid target sequences from a sample can be amplified under substantially non-depleted conditions.

Optionally, in the method for multiplex nucleic acid amplification, different nucleic acid target sequences from a sample can be amplified under substantially depleted conditions.

Optionally, in the method for multiplex nucleic acid amplification, the single reaction mixture comprises an isothermal or thermocycling reaction mixture.

In some embodiments, two or more templates or targets may be amplified within separate chambers, wells, cavities, or sites of an array that are in fluid communication with each other or occupied by the same single continuous liquid phase of an amplification reaction mixture. Such embodiments include embodiments for array-based nucleic acid amplification.

For example, in some embodiments, the present disclosure relates to methods for nucleic acid amplification comprising: the method includes the steps of dispensing a target polynucleotide to a reaction chamber or site in an array of reaction chambers or sites, and amplifying individual target polynucleotides within the reaction chambers or sites. Optionally, two or more target polynucleotides are partitioned into two or more reaction chambers or sites of the array, and the two or more partitioned target polynucleotides are amplified in parallel within their respective reaction chambers or sites. Optionally, at least two of the reaction chambers or sites each receive a single target polynucleotide during dispensing (one or more of the reaction chambers or sites can optionally receive zero or more than one target polynucleotide during dispensing). At least two target polynucleotides may be clonally amplified within their respective reaction chambers. At least one reaction chamber comprising a target polynucleotide may be in fluid communication with at least one other reaction chamber comprising a target polynucleotide during amplification.

In some embodiments, the present disclosure relates generally to methods (and related compositions, systems, devices, and kits) for nucleic acid amplification, comprising: (a) Partitioning at least two different polynucleotides into an array of reaction chambers by introducing a single polynucleotide into at least two reaction chambers in fluid communication with each other; and (b) forming at least two substantially monoclonal populations of nucleic acid by amplifying the polynucleotides within the at least two reaction chambers. Typically, at least two reaction chambers are in fluid communication with each other during amplification.

In some embodiments, the present disclosure relates generally to methods (and related compositions, systems, devices, and kits) for nucleic acid amplification, comprising: (a) In an array of reaction chambers comprising first and second reaction chambers, dispensing a first template polynucleotide into the first reaction chamber and a second template polynucleotide into the second reaction chamber, and (b) forming at least two substantially monoclonal populations of nucleic acids by clonally amplifying the first and second template polynucleotides within their respective reaction chambers, wherein a single polynucleotide is dispensed from a nucleic acid sample having a plurality of different polynucleotides. Optionally, the first and second reaction chambers comprise different portions of a single continuous liquid phase during amplification. For example, the first and second reaction chambers of the array may be in fluid communication during amplification.

In some embodiments, the present disclosure relates generally to methods for nucleic acid amplification comprising (a) assigning a different single polynucleotide into each of a plurality of reaction chambers, and (b) forming a population of monoclonal nucleic acids in each of the reaction chambers by amplifying the different single polynucleotides within the plurality of reaction chambers, wherein the single different polynucleotide is assigned from a nucleic acid sample having a plurality of different polynucleotides.

In some embodiments, the present disclosure relates generally to methods for nucleic acid amplification comprising (a) partitioning at least two different polynucleotides into an array of reaction chambers by introducing a single polynucleotide into at least two reaction chambers in fluid communication with each other; and (b) forming at least two substantially monoclonal populations of nucleic acid by amplifying the polynucleotides within the at least two reaction chambers.

In some embodiments, the method can further include introducing one or more supports (e.g., beads or particles, etc.) into at least one reaction chamber or site of the array. One or more supports can be introduced into at least one reaction chamber or site before, during, or after the polynucleotides are dispensed into the array. In some embodiments, at least one reaction chamber or site of the array receives a single support. In some embodiments, a majority of the reaction chambers or sites receive a single support. In some embodiments, the support can be mixed with the polynucleotides prior to dispensing and dispensed with the polynucleotides into an array. At least one support may optionally be attached to a nucleic acid molecule comprising a primer sequence that is substantially complementary or substantially identical to a portion of a polynucleotide present in a reaction chamber or site during amplification. In some embodiments, at least one support comprises a nucleic acid molecule comprising a primer sequence that is substantially complementary or substantially identical to a portion of a target polynucleotide or template in a reaction chamber or well. In some embodiments, at least one support comprises a nucleic acid molecule comprising a primer sequence that is substantially complementary or substantially identical to at least a portion of another primer present in a reaction chamber or site during amplification.

In some embodiments, amplification may comprise introducing a reaction mixture into at least one reaction chamber or site of an array. Optionally, the reaction mixture is introduced into the reaction chamber or site before, during, or after the partitioning of the polynucleotides to the array or the introduction of the support to the array. The reaction mixture, support, and polynucleotides may be introduced or distributed into the array in any order or in any combination. In some embodiments, at least one reaction chamber or site of the array receives a single support, a single polynucleotide, and a reaction mixture sufficient to support amplification of the polynucleotide within the reaction chamber or site.

In some embodiments, the method may comprise hybridizing at least part of the polynucleotide to the support by contacting the support with the polynucleotide under hybridization conditions. Hybridization can occur before, during, or after introduction of the support and/or polynucleotides to the reaction chambers or sites of the array. In some embodiments, at least one support attached to a first primer sequence is introduced into at least one reaction chamber or site of the array, followed by introduction of a polynucleotide into the reaction chamber or site, where the polynucleotide hybridizes to the support. Alternatively, the support may be hybridised to an amplification product produced by amplification of a polynucleotide within a reaction chamber or site.

The reaction mixture may include any of the reaction mixtures and components described herein. In some embodiments, the reaction mixture may include any one or more of the following components: isothermal amplification reagents (e.g., one or more recombinant enzymes, helicases, i.e., associated cofactors, polymerases, etc.), screening agents, nucleotides, and the like.

In some embodiments, the present disclosure relates generally to methods (and related compositions, kits, systems, and devices) for nucleic acid amplification, comprising: (a) Introducing a first polynucleotide template and a first support, in any order or combination, into a first reaction chamber or site of an array of reaction chambers or sites, and a second polynucleotide template and a second support into a second reaction chamber or site of the array; and (b) clonally amplifying a first polynucleotide template on a first support within a first reaction chamber or site and clonally amplifying a second polynucleotide template on a second support within a second reaction chamber or site, while the first reaction chamber or site is in fluid communication with the second reaction chamber or site during amplification. Clonally amplifying may include generating a first support attached to a first amplicon from a first polynucleotide template, and a second support attached to a second amplicon from a second polynucleotide template. Optionally, the first and second sites (or reaction chambers) comprise the same continuous liquid phase of the same reaction mixture during amplification. For example, the reaction mixture may comprise a single continuous liquid phase comprising the first and second polynucleotide templates and the first and second supports. The reaction mixture may be introduced into the reaction chambers or sites of the array before, during, or after the introduction of the polynucleotide templates and/or supports. In some embodiments, the disclosed methods further comprise introducing the reaction mixture into the first and second reaction chambers or sites after introducing the first and second polynucleotide templates and the first and second supports. In some embodiments, the reaction mixture comprises a recombinase or helicase or a recombinase and a helicase. The recombinase may be derived from myovirus (e.g., uvsX), bacterial, yeast, or human, or analogs thereof from other species. In some embodiments, the reaction mixture comprises a polymerase. In some embodiments, the reaction mixture comprises a sieving agent, such as polyacrylamide, agarose, or a cellulose polymer (e.g., HEC, CMC, or MC, or derivatives thereof). In some embodiments, the reaction mixture comprises a diffusion limiting agent.

In some embodiments, amplification comprises hybridizing a first primer binding sequence of a polynucleotide template to a first primer sequence of a first primer of a support to attach the polynucleotide template to a support or surface (e.g., a particle or bead) having a first primer comprising a first primer sequence.

In embodiments where amplification is performed within a reaction chamber or site of the array, as well as embodiments where amplification is performed within a single reaction vessel, the surface or support optionally includes at least a first primer comprising a first primer sequence. In some embodiments, the one or more polynucleotide templates comprise a first primer binding sequence. The first primer binding sequence can be identical or substantially identical to the first primer sequence. Alternatively, the first primer binding sequence may be complementary or substantially complementary to the first primer sequence. In some embodiments, the first primer sequence and the first primer binding sequence do not exhibit significant identity or complementarity, but are substantially identical or substantially complementary to another nucleotide sequence present in the reaction mixture. In such embodiments, amplification may include the formation of an amplification reaction intermediate comprising a nucleotide sequence having substantial identity or complementarity to the first primer sequence, the first primer binding sequence, or both.

In some embodiments, at least two different polynucleotide templates are present in the reaction mixture and amplification results in the formation of at least two different substantially monoclonal populations each derived from amplification of a single said polynucleotide template. In some embodiments, two or more of the at least two substantially monoclonal populations are attached to the same support or surface. Each of the two or more substantially monoclonal populations can be attached to the same support or to different unique locations on the surface. Alternatively, each of the two or more substantially monoclonal populations can each be attached to a different support or surface. The separation of different monoclonal populations to different supports or surfaces can be advantageous in applications where it is desirable to separate the populations prior to analysis. In some embodiments, the support or surface is part of a bead or particle, which may be spherical or spheroidal in shape. In some embodiments, the support or surface forms part of a two-dimensional or three-dimensional array.

In some embodiments, the disclosed methods for nucleic acid amplification can be performed while a sieving agent or a diffusion reducing agent is included in the reaction mixture. These reagents may increase the total number and/or proportion (e.g., percentage) of monoclonal populations formed during amplification. In some embodiments, the methods comprise using a reaction mixture that provides an increased yield of monoclonal or substantially monoclonal amplicons relative to conventional reaction mixtures.

In some embodiments, the method comprises amplification by partially denaturing the template. For example, amplification includes template walking. For example, the template to be amplified may include an adaptor sequence comprising a primer binding site that has a relatively low Tm compared to the template as a whole. In some embodiments, amplification is performed at a temperature substantially above the Tm of the linker sequence and substantially below the Tm of the template, as described in more detail herein. In some embodiments, amplification is performed at a temperature of at least 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, or 50 ℃ below the Tm of the nucleic acid template. In some embodiments, amplification is performed at a temperature above (e.g., at least 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, or 50 ℃ higher) the Tm of the first primer, the second primer, or both the first and second primers.

In some embodiments, the reaction mixture may optionally include any one or more of the following components: (a) One or more supports optionally comprising at least a first primer sequence; (2) a recombinase; (3) a polymerase; (4) a diffusion limiting agent; (5) screening agent; (6) crowding reagents; (7) an ATP regeneration system; (8) single-chain binding protein (SSBP); and (8) recombinase cofactors, e.g., recombinase loading proteins. In some embodiments, the yield of a monoclonal or substantially monoclonal population is increased by amplifying (e.g., by attaching an amplification primer to a surface) a polynucleotide template on the surface in the presence of a diffusion limiting agent and/or a sieving agent. The diffusion limiting or sizing agent can provide increased yield of the monoclonal population by reducing diffusion or migration of amplified product polynucleotides away from the surface during amplification.

In some embodiments, the reaction mixture comprises one or more isothermal amplification reagents. Such reagents may include, for example, a recombinase or helicase.

In some embodiments, methods for nucleic acid amplification can be performed using an enzyme that catalyzes homologous recombination, e.g., an enzyme that can bind to a first primer to form a complex or can catalyze strand invasion or can form a D-loop structure. In some embodiments, the enzyme that catalyzes homologous recombination comprises a recombinase.

In some embodiments, the amplification conditions comprise isothermal conditions or thermocycling conditions.

In some embodiments, a method for nucleic acid amplification comprises: (a) Forming a reaction mixture comprising a single continuous liquid phase comprising (i) an enzyme that catalyzes homologous recombination, (ii) one or more surfaces, and (iii) a plurality of different polynucleotides; and (b) subjecting the reaction mixture to conditions suitable for nucleic acid amplification.

In some embodiments, a method for nucleic acid amplification comprises: (a) Forming a reaction mixture comprising a single continuous liquid phase comprising (i) an enzyme that catalyzes homologous recombination, (ii) one or more beads each linked to a plurality of first primers, and (iii) a plurality of different polynucleotides; (b) Two or more substantially monoclonal amplified nucleic acid populations are formed by subjecting the reaction mixture to amplification conditions. The amplification conditions may include isothermal or thermocycling conditions. In some embodiments, the first primer can hybridize to at least a portion of a polynucleotide.

In some embodiments, the present disclosure relates generally to methods for nucleic acid amplification comprising (a) forming a reaction mixture comprising a single continuous liquid phase comprising one or more supports (or surfaces), a plurality of polynucleotides, and a recombinase; (b) Clonally amplifying at least two of the plurality of different polynucleotides on at least one support (or surface) by subjecting the reaction mixture to amplification conditions. In some embodiments, the amplification conditions may comprise isothermal or thermocycling amplification conditions. The reaction mixture may optionally include a recombinase. In some embodiments, the reaction mixture comprises a polymerase. In some embodiments, the reaction mixture includes primers, which may be in solution. Optionally, at least one of the support or surface may comprise a primer.

In some embodiments, the present disclosure relates generally to methods for nucleic acid amplification comprising (a) forming a reaction mixture comprising a single continuous liquid phase comprising (i) a recombinase enzyme, (ii) a plurality of beads linked to one or more first primers comprising a first primer sequence, and (iii) a plurality of different polynucleotide templates; (b) Hybridizing at least one of said first primers to at least one of a plurality of different polynucleotide templates; (c) Subjecting the reaction mixture to nucleic acid amplification conditions and generating at least one substantially monoclonal population of polynucleotides by amplifying at least one polynucleotide template to form at least a first amplified population. In some embodiments, at least 30%, 90% of the polynucleotides in at least one substantially monoclonal population are substantially identical (or substantially complementary) to at least one polynucleotide template originally present in the reaction mixture. In some embodiments, at least a portion of the first amplified population is linked to one bead of the plurality of beads.

In some embodiments, the forming a reaction mixture in step (a) comprises: a nucleoprotein complex is formed by contacting a recombinase with at least one of a plurality of first primers linked to a plurality of beads.

In some embodiments, subjecting the reaction mixture to nucleic acid amplification conditions in step (b) comprises performing a nucleotide polymerization reaction. For example, a nucleotide polymerization reaction can include optionally incorporating a nucleotide into a first primer sequence when the first primer sequence hybridizes to one polynucleotide template in a reaction mixture.

In some embodiments, subjecting the reaction mixture to nucleic acid amplification conditions comprises contacting the first primer with the polynucleotide template, the recombinase, the polymerase, and the nucleotides in any order or in any combination.

In some embodiments, the nucleic acid amplification conditions comprise repeating such a cycle: forming a nucleoprotein complex comprising the recombinase, at least a portion of the first primer, and at least a portion of the first polynucleotide template, and contacting the nucleoprotein complex with a polymerase that catalyzes the incorporation of one or more nucleotides to the first primer.

In some embodiments, a cycling nucleic acid amplification reaction can be performed to produce a plurality of beads each linked to a substantially monoclonal population of polynucleotides.

In some embodiments, the methods for nucleic acid amplification can be performed under isothermal conditions or thermal cycling conditions.

In some embodiments, the plurality of different polynucleotides may be single-stranded or double-stranded polynucleotides. In some embodiments, thermal or chemical denaturation of the double-stranded polynucleotide is not necessary, as the recombinase can produce localized strand denaturation by catalyzing strand invasion.

In some embodiments, the method for nucleic acid amplification may be performed in a single reaction vessel. In some embodiments, the nucleic acid amplification reaction may be performed in a single reaction vessel comprising a single continuous liquid phase. For example, a single continuous liquid phase can comprise an amplification mixture comprising a plurality of beads each linked to a plurality of first primers, a plurality of different polynucleotides, and a plurality of recombinases. In some embodiments, the amplification mixture may further comprise a polymerase and a plurality of nucleotides. In some embodiments, the amplification mixture may further comprise ATP, nucleotides, and cofactors. Non-limiting examples of individual reaction vessels include tubes, wells, or similar structures.

In some embodiments, the polynucleotide and the reagent may be placed into the reaction vessel in any order, including sequentially or substantially simultaneously or a combination of both. In some embodiments, the reagents include a bead to which a plurality of first primers are linked, a recombinase, a polymerase, nucleotides, ATP, a divalent cation, and a cofactor.

In some embodiments, the method for nucleic acid amplification may be performed in a single continuous liquid phase. A single continuous liquid phase may include any liquid phase in which any given portion or region of a single continuous liquid phase is in fluid communication with any other portion or region of the same single continuous liquid phase. Generally, components dissolved or suspended in a single continuous liquid phase may freely diffuse or migrate to any other point in the liquid phase. In some embodiments, however, the single continuous liquid phase may include a diffusion limiting agent that slows the rate of diffusion in the single continuous liquid phase. An exemplary embodiment of a single continuous liquid phase is a single aqueous droplet in a water-in-oil emulsion; in such an emulsion, each droplet will form a separate phase; two droplets may merge to form a single phase.

In some embodiments, the single continuous liquid phase consists essentially of a single aqueous phase. In some embodiments, the single continuous liquid phase lacks a non-aqueous phase; for example, the continuous liquid phase does not include oils or organic solvents. In some embodiments, multiple nucleic acid amplification reactions occur in the aqueous phase of a single reaction vessel. In some embodiments, a single continuous liquid phase does not divide multiple nucleic acid amplification reactions to occur in a single reaction vessel.

In some embodiments, the method for nucleic acid amplification may be performed in providing a divided water-in-oil emulsion.

When nucleic acid amplification is performed using multiple polynucleotide templates, clonal amplification using conventional amplification methods typically relies on techniques such as partitioning the reaction mixture into separate portions or components that are not in fluid communication with each other in order to preserve clonality and prevent cross-contamination of different amplification populations as well as to maintain sufficient yields of monoclonal amplification product. Using such conventional amplification methods, clonally amplifying polynucleotide templates within the same reaction mixture without division or partitioning of the reaction mixture into separate compartments or vessels is generally not feasible, as any polynucleotides (including templates and/or amplification products) within the reaction mixture will tend to migrate randomly through the mixture during such amplification due to diffusion and/or brownian motion. Such diffusion or migration generally increases the occurrence of polyclonal amplification, and will result in little, if any, monoclonal population.

One suitable technique to reduce the generation of polyclonal populations in conventional amplification methods uses physical barriers to separate individual amplification reactions into discrete compartments. For example, emulsion PCR uses a water-in-oil microreaction vessel in which the oil phase comprises a plurality of separate, i.e., discrete, aqueous reaction compartments. Each compartment serves as an independent amplification reaction vessel, so that the entire emulsion is able to support a number of separate amplification reactions in separate (discontinuous) liquid phases of a single reaction vessel (e.g. an Eppendorf tube or well). Similarly, an amplification "master mixture" can be prepared and distributed into separate reaction chambers (e.g., an array of wells) to create a set of discrete and separate phases, each of which defines a separate amplification reaction. Such separate phases may be further blocked from each other prior to amplification. Such a closure can be used to prevent cross-contamination between parallel and separate reactions. Exemplary forms of closure may include the use of caps or phase barriers (e.g., a layer of mineral oil over the aqueous reaction) to partition the PCR reaction into individual and discrete compartments between which transfer of reaction components does not occur.

Other techniques to prevent cross-contamination and reduce polyclonality rely on the immobilization of one or more reaction components (e.g., one or more templates and/or primers) during amplification to prevent cross-contamination of amplification reaction products and the resulting reduction in monoclonality. One such example includes bridge PCR, in which all primers (e.g., forward and reverse primers) required for amplification are attached to the surface of a substrate support. In addition to such immobilization, additional immobilization components may be included in the reaction mixture. For example, the polynucleotide template and/or amplification primers may be suspended in a gel or other matrix during amplification to prevent migration of the amplification reaction products from the synthesis site. Such gels and matrices often need to be removed afterwards, which requires the use of appropriate "melting" or other recovery steps, thereby losing yield.

In some embodiments, the present disclosure provides methods for performing substantially monoclonal amplification of multiple polynucleotide templates in parallel in a single continuous liquid phase of a reaction mixture without requiring partitioning or immobilization of multiple reaction components (e.g., two primers) during amplification. Alternatively, the mixture of polynucleotide templates in solution may be contacted directly with the amplification reaction components and an appropriate surface or support having the first primer attached thereto, and the other components required for amplification, including the polymerase, one or more types of nucleotides, and optionally the second primer, may be provided in the same continuous liquid phase. In some embodiments, the reaction mixture further comprises a recombinase. Optionally, the reaction mixture further comprises at least one reagent selected from the group consisting of a diffusion limiting agent, a sieving agent, and a crowding reagent. Examples of amplification mixtures suitable for effecting monoclonal amplification of a template contained in a single continuous liquid phase are further described herein. Optionally, different templates may be amplified at different locations on a single surface or support, or different templates may be amplified on different surfaces or different supports within the same reaction mixture.

In some embodiments, the reaction mixture may include one or more sieving agents. The sizing agent optionally includes any compound that can provide a physical barrier to migration of the polynucleotide template or its corresponding amplification product. (migration may include any movement of template or amplification product within the reaction mixture; diffusion includes forms of migration involving movement along a concentration gradient). In some embodiments, the sieving agent comprises any compound that can provide a matrix with a plurality of pores that are small enough to reduce movement of the nucleic acid synthesis reaction mixture or any one or more specific components of the nucleic acid reaction mixture.

In some embodiments, the sieving agent provides a molecular sieve. For example, a sizing agent can reduce the movement of a polynucleotide (or a polynucleotide associated with a surface or bead) through a reaction mixture comprising the sizing agent. The sieving agent may optionally have small pores.

The inclusion of a sieving agent can be advantageous when two or more template polynucleotides are clonally amplified within a single continuous liquid phase of a reaction mixture. For example, the sizing agent can prevent or slow diffusion of the template or amplified polynucleotide produced by replication of at least some portion of the template within the reaction mixture, thereby preventing the formation of polyclonal contaminants without the need to partition the reaction mixture during amplification by physical methods or encapsulation methods (e.g., emulsions). Such a method of clonally amplifying templates in a single continuous liquid phase of a single reaction mixture without partitioning greatly reduces the cost, time and effort associated with generating libraries suitable for use in high throughput methods such as digital PCR, next generation sequencing, and the like.

In some embodiments, the average pore size of the sieving agent is such that movement of the target component (e.g., polynucleotide) within the reaction mixture is selectively impeded or prevented. In one example, a sizing agent includes any compound that can provide a matrix with a plurality of pores that are small enough to slow or impede the movement of a polynucleotide through a reaction mixture containing the sizing agent. Thus, the sieving agent can reduce brownian motion of the polynucleotide.

In some embodiments, the sieving agent selectively acts to block migration of molecules having an average molecular size or weight above a particular threshold or range, while not blocking migration of other molecules having an average molecular size or weight below the threshold or range.

In some embodiments, the sieving agent acts selectively to impede migration of molecules having an average molecular size or weight below a particular threshold or range, while not impeding migration of other molecules having an average molecular size or weight above the threshold or range.

In some embodiments, the sieving agent may be selected to selectively impede, slow, reduce, or prevent the movement of a polynucleotide through a reaction mixture, but large enough to allow movement of smaller components (e.g., cations, nucleotides, ATP, and cofactors) through the reaction mixture. In some embodiments, the sieving agent has an average pore size or range of average pore sizes that can be adjusted by increasing or decreasing the concentration of the sieving agent. For example, the molecular weight, intrinsic viscosity, and concentration of a sieving agent (or combination of sieving agents) can be selected to prepare a nucleic acid reaction mixture in a particular solvent (e.g., water) to produce such a matrix: it has a desired ability to prevent migration of a target polynucleotide having a particular size or length, or a desired average pore size or viscosity. In some embodiments, the sieving agent can reduce the overall flow rate by increasing the viscosity of the nucleic acid reaction mixture. In some embodiments, the sieving agent may be water soluble. In some embodiments, a substrate having a plurality of small pores can be prepared by mixing a sieving agent with a solvent (e.g., an aqueous solvent, such as water). In some embodiments, the sieving agent does not interfere with recombinase nucleoprotein complex formation or nucleotide polymerization.

In some embodiments, the present disclosure relates generally to methods for performing a nucleic acid amplification reaction comprising generating two or more substantially monoclonal populations by amplifying target polynucleotides on a surface or support in the presence of one or more screening agents, optionally in the presence of a recombinase, polymerase, or any other suitable agent capable of catalyzing or promoting nucleic acid amplification.

In some embodiments, the inclusion of a sieving agent in the reaction mixture can reduce the movement of polynucleotides away from a given support or surface (e.g., reduce sloughing off) and can increase the likelihood that polynucleotides will hybridize to the support or surface and provide a site for initiation of nucleotide polymerization, thereby increasing the proportion of substantially monoclonal amplicons produced during the amplification reaction.

In some embodiments, amplifying comprises amplifying a plurality of different polynucleotide templates on a plurality of different bead supports in the presence of a screening agent, and recovering a percentage of substantially monoclonal bead supports, each such substantially monoclonal bead support comprising a bead support attached to a population of substantially monoclonal polynucleotides. In some embodiments, the percentage of substantially monoclonal bead supports recovered is substantially greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 89%, 90%, or 95% of the total amplified bead supports (i.e., total bead supports including polyclonal or monoclonal populations) recovered from the reaction mixture. In some embodiments, the percentage of substantially monoclonal bead supports recovered is substantially higher than the percentage of substantially monoclonal bead supports recovered after amplification is performed in the absence of a screening agent but under otherwise substantially similar or identical amplification conditions.

In some embodiments, the sieving agent comprises a polymeric compound. In some embodiments, the sieving agent comprises a crosslinked or non-crosslinked polymer compound. By way of non-limiting example, the sieving agent may include polysaccharides, polypeptides, organic polymers, and the like.

In some embodiments, the sieving agent comprises a linear or branched polymer. In some embodiments, the sieving agent comprises a dotted or neutral polymer.

In some embodiments, the sizing agent may comprise a mixture of one or more polymers each having an average molecular weight and a viscosity.

In some embodiments, the sieving agent comprises a polymer having an average molecular weight of about 10,000 to 2,000,000, or about 12,000 to 95,000, or about 13,000 to 95,000.

In some embodiments, the sizing agent may exhibit an average viscosity range of about 5 centipoise to about 15,000 centipoise measured at about 25 ℃ when dissolved in water at 2 weight percent, or about 10 centipoise to about 10,000 centipoise measured as a 2% aqueous solution at about 25 ℃, or about 15 centipoise to about 5,000 centipoise measured as a 2% aqueous solution at about 25 ℃.

In some embodiments, the sieving agent comprises about 25 to about 1,5000kM _v Or about 75 to 1,000kM _v Or about 85-800kM _v Viscosity average molecular weight (M) of _v ). In some embodiments, the reaction mixture comprises about 0.1 to about 20% weight/volume or about 1-10% w/v or about 2-5% w/v of the sieving agent.

In some embodiments, the sieving agent includes an acrylamide polymer, such as polyacrylamide.

In some embodiments, the sieving agent comprises a polymer of one or more amino acids. For example, the sieving agent may include polylysine, polyglutamic acid, actin, myosin, keratin, tropomyosin (tropmyosin), and the like. In some embodiments, the screening agent may comprise a derivative of any of these polypeptides.

In some embodiments, the sieving agent comprises a polysaccharide polymer. In some embodiments, the sieving agent comprises a polymer of glucose or galactose. In some embodiments, the sizing agent comprises one or more polymers selected from the group consisting of cellulose, dextran, starch, glycogen, agar, chitin, pectin, or agarose. In some embodiments, the sieving agent comprises a glucopyranose polymer.

In some embodiments, the sieving agent comprises a polymer having one or more groups that are polar or charged under the amplification reaction conditions. For example, the polymer may comprise one or more cationic groups, one or more anionic groups, or both. In some embodiments, the sieving agent is a polysaccharide comprising one or more charged groups. In some embodiments, the sieving agent is a polysaccharide comprising one or more carboxyl groups that are or tend to be negatively charged under the amplification reaction conditions. For example, the sizing agent may include a carboxymethyl cellulose (CMC) polymer. In some embodiments, the sieving agent may include spermine and/or spermidine. In some embodiments, the sieving agent comprises polylysine and/or polyarginine. For example, the sieving agent may include poly-L-lysine, poly-D-glutamic acid, and the like. In some embodiments, the sieving agent comprises one or more histones or histone-nucleic acid complexes or derivatives thereof. Histones are highly basic proteins that are capable of binding nucleic acids and include proteins H1, H2A, H2B, H3, and H4. In some embodiments, histones are modified, e.g., by methylation, acetylation, phosphorylation, ubiquitination, SUMO, citrullination, ribosylation (including ADP-ribosylation), and the like.

In some embodiments, the sieving agent comprises a polymer, including a chemically substituted polymer. The polymer may include reactive groups (e.g., the reactive groups may be reacted with an appropriate substituent to produce a substituted polymer). In some embodiments, the polymer comprises a fluorescent-, carboxy-, amino-, or alkoxy-substituted polymer. In some embodiments, the polymer is modified by methylation, acetylation, phosphorylation, ubiquitination, carboxylation, and the like. The substituent may comprise a charged group, for example an anionic or cationic group, the substitution of which into the polymer chain may result in the production of a charged polymer. The degree of substitution can vary from about 0.2 to about 1.0 derivative/monomer unit, typically from about 0.4 to about 1.0, more typically from about 0.6 to about 0.95.

In some embodiments, the sieving agent comprises a cellulose derivative, such as sodium carboxymethylcellulose, sodium carboxymethyl 2-hydroxyethyl cellulose, methyl cellulose, hydroxyethyl cellulose, 2-hydroxypropyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxybutyl methyl cellulose, (hydroxypropyl) methyl cellulose, or hydroxyethyl ethyl cellulose, or a mixture comprising any one or more of such polymers.

In some embodiments, the nucleic acid reaction mixture comprises a mixture of different sieving agents, such as a mixture of different cellulose derivatives, starch, polyacrylamide, and the like.

In some embodiments, the sizing agent comprises one or more complexing polymers comprising subparts of any two different polymers (including any of the polymers described herein). For example, the complexing polymer can include a polysaccharide polymer linked to a polynucleotide polymer, such as a polysaccharide linked to DNA or RNA. In some embodiments, the sizing agent can include a polymer comprising a cellulose portion and a nucleic acid portion, such as DNA-cellulose. In other embodiments, the complexing polymer may comprise polyacrylamide linked to the polynucleotide and/or polypeptide. The inclusion of such complexing polymers in the reaction mixture can serve to further hinder the movement of the target polynucleotide through the reaction mixture.

In some embodiments, the sieving agent may comprise a polymer that has been first contacted or reacted with a suitable crosslinking agent. For example, the sieving agent may comprise acrylamide that has been reacted with bisacrylamide and/or cystdi (acryloyl) amine.

In some embodiments, the reaction mixture includes at least one diffusion reducing agent. In some embodiments, the diffusion reducing agent comprises any compound that reduces migration of a polynucleotide from a region of higher concentration to a region with lower concentration. In some embodiments, the diffusion reducing agent comprises any compound that reduces migration of any component (regardless of size) of the nucleic acid amplification reaction. In some embodiments, the components of the nucleic acid amplification reaction include beads/primers, polynucleotides, recombinases, polymerases, nucleotides, ATP, and/or cofactors.

It should be noted that the concepts of the sieving agent and the diffusion reducing agent are not necessarily mutually exclusive; the sieving agent can often be effective in reducing diffusion of the target compound through the reaction mixture, while the diffusion reducing agent can often have a sieving effect on the reaction components. In some embodiments, the same compound or reaction mixture additive may be used as a sieving agent and/or a diffusion reducing agent. Any of the sizing agents disclosed herein can, in some embodiments, be used as a diffusion reducing agent in a hook and vice versa.

In some embodiments, the diffusion reducing agent and/or sieving agent comprises polyacrylamide, agar, agarose, or a cellulosic polymer such as hydroxyethyl cellulose (HEC), methyl Cellulose (MC), or carboxymethyl cellulose (CMC).

In some embodiments, the sieving agent and/or diffusion reducing agent is included in the reaction mixture at a concentration of at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 74%, 90%, or 95% w/v (weight of reagents/unit volume of reaction mixture).

In some embodiments, the reaction mixture comprises at least one crowding reagent. For example, crowding reagents can increase the concentration of one or more components in a nucleic acid amplification reaction by creating a crowded reaction environment. In some embodiments, the reaction mixture includes a sieving agent and a crowding reagent.

In some embodiments, the method for nucleic acid amplification comprises one or more surfaces. In some embodiments, a plurality of first primers can be attached to the surface, the first primers in the plurality having a common first primer sequence.

In some embodiments, the surface may be an external or uppermost layer or boundary of the object. In some embodiments, the surface may be inside the boundary of the object.

In some embodiments, the reaction mixture comprises a plurality of different surfaces, e.g., the reaction mixture can comprise a plurality of beads (e.g., particles, nanoparticles, microparticles, etc.) and at least two different polynucleotide templates can be clonally amplified on the different surfaces, thereby forming at least two different surfaces, each of which is attached to an amplicon. In some embodiments, the reaction mixture comprises a single surface (e.g., the surface of a slide or an array of reaction chambers) and at least two different polynucleotide templates are amplified at two different regions or locations on the surface, thereby forming a single surface linked to two or more amplicons.

In some embodiments, the surface may be porous, semi-porous, or non-porous. In some embodiments, the surface may be a planar surface as well as a concave surface, a convex surface, or any combination thereof. In some embodiments, the surface can be the inner wall of a bead, particle, microparticle, sphere, filter, flow cell, well, trench, well container, gel, or capillary. In some embodiments, the surface comprises a capillary, a groove, a well, a trench, a groove, an inner wall of a container. In some embodiments, the surface may include texture (e.g., etched, cavitated, pores, three-dimensional scaffolds, or protrusions).

In some embodiments, the particles can have a spherical, hemispherical, cylindrical, barrel, annular, rod, disk, conical, triangular, cubic, polygonal shape. Tubular, linear or irregular shapes.

In some embodiments, the surface may be prepared from any material including glass, borosilicate glass, silica, quartz, fused silica, mica, polyacrylamide, plastic polystyrene, polycarbonate, polymethacrylate (PMA), polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), silicon, germanium, graphite, ceramic, silicon, semiconductor, high index dielectric, crystal, gel, polymer, or thin film (e.g., thin film of gold, silver, aluminum, or diamond).

In some embodiments, the film may be magnetic or paramagnetic beads (e.g., magnetic or paramagnetic nanoparticles or microparticles). In some embodiments, the paramagnetic microparticles can be streptavidin-linked paramagnetic beads (e.g., dynabeads from Invitrogen, carlsbad, CA) ^TM M-270). The particles may have an iron core or comprise a hydrogel or agarose (e.g. Sepharose) ^TM )。

In some embodiments, a plurality of first primers can be attached to the surface. The surface may be coated with acrylamide, carboxyl or amine compounds to attach nucleic acids (e.g., first primer). In some embodiments, amino-modified nucleic acids (e.g., primers) can be attached to a surface coated with a carboxylic acid. In some embodiments, the amino-modified nucleic acid can be reacted with EDC (or EDAC) to attach to a carboxylic acid-coated surface (with or without NHS). The first primer may be immobilized to an acrylamide compound coating on the surface. The particles may be coated with an avidin-like compound (e.g. streptavidin) to bind biotinylated nucleic acids.

In some embodiments, the surface comprises the surface of a bead. In some embodiments, the beads comprise a polymeric material. For example, the beads include gels, hydrogels, or acrylamide polymers. The beads may be porous. The particles may have cavities or pores, or may comprise a three-dimensional scaffold. In some embodiments, the particle may be an Ion Sphere ^TM And (3) granules.

In some embodiments, the disclosed methods (and related compositions, systems, and kits) include immobilizing one or more nucleic acid templates on one or more supports. Nucleic acids can be immobilized on a solid support by any method including, but not limited to, physisorption, by ionic or covalent bond formation, or a combination thereof. The solid support may comprise a polymeric, glass or metallic material. Examples of solid supports include membranes, planar surfaces, microwell plates, beads, filters, test strips, slides, coverslips, and test tubes. Meaning any solid phase material upon which an oligomer is synthesized, attached, linked, or otherwise immobilized. The support may optionally comprise a "resin", "phase", "surface", and "support". The support may be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyvinyl fluoride, polyethyleneoxy (polyethyleneoxy), and polyacrylamide, as well as copolymers and grafts thereof. The support may also be inorganic, such as glass, silica, controlled Pore Glass (CPG) or reverse phase silica. The structure of the support may be in the form of beads, spheres, particles, granules, gels or surfaces. The surface may be planar, substantially planar, or non-planar. The support may be porous or non-porous and may have swelling or non-swelling characteristics. The support may be shaped to comprise one or more apertures, recesses or other reservoirs, containers, features or locations. Multiple supports can be configured at different locations in the array. The support is optionally addressable (e.g., for robotic delivery of reagents), or by detection means including scanning by laser irradiation and confocal or deflected light collection. The amplification support (e.g., beads) can be placed in or on another support (e.g., within the wells of a second support).

In embodiments, the solid support is a "microparticle," "bead," "microbead," or the like (optionally but not necessarily spherical in shape) having a minimum cross-sectional length (e.g., diameter) of 50 microns or less, preferably 10 microns or less, 3 microns or less, about 1 micron or less, about 0.5 microns or less, such as about 0.1, 0.2, 0.3, or 0.4 microns, or less (e.g., less than 1 nanometer, about 1-10 nanometers, about 10-100 nanometers, or about 100-500 nanometers). Microparticles (e.g., dynabeads from Dynal, oslo, norway) can be made from a variety of inorganic or organic materials including, but not limited to, glass (e.g., controlled pore glass), silica, zirconium, crosslinked polystyrene, polyacrylate, polymethylmethacrylate, titanium dioxide, latex, polystyrene, and the like. Magnetization can aid in the collection and concentration of microparticle-bound reagents (e.g., polynucleotides or ligases) after amplification, and can also aid in additional steps (e.g., washing, reagent removal, etc.). In certain embodiments of the invention, populations of microparticles having different shape sizes and/or colors may be used. The microparticles may optionally be encoded, for example with quantum dots, so that such microparticles can be individually or uniquely identified.

In some embodiments, the bead surface may be functionalized to attach a plurality of first primers. In some embodiments, the beads may be of any size that can fit into a reaction chamber. For example, one bead may be assembled into a reaction chamber. In some embodiments, more than one bead can be assembled into the reaction chamber. In some embodiments, the beads may have a minimum cross-sectional length (e.g., diameter) of about 50 microns or less, or about 10 microns or less, or about 3 microns or less, about 1 micron or less, about 0.5 microns or less, such as about 0.1, 0.2, 0.3, or 0.4 microns, or less (e.g., less than 1 nanometer, about 1-10 nanometers, about 10-100 nanometers, or about 100-500 nanometers).

In some embodiments, the beads may have attached thereto a plurality of one or more different primer sequences. In some embodiments, the bead may have attached thereto a plurality of one primer sequence, or may have attached thereto a plurality of two or more different primer sequences. In some embodiments, the beads may have attached thereto at least 1,000 primers, or from about 1,000 to 10,000 primers, or from about 10,000 to 50,000 primers, or from about 50,000 to 75,000 primers, or a plurality of primers from about 75,000 to 100,000 primers or more.

In some embodiments, the reaction mixture comprises a recombinase. The recombinase may include any agent capable of inducing a recombination event or increasing the frequency of occurrence of a recombination event. Recombination events include any event in which two different polynucleotide strands recombine with each other. Recombination may include homologous recombination. The recombinase may be an enzyme or a genetically engineered derivative thereof. The recombinase may optionally be associated with (e.g., bound to) the single-stranded oligonucleotide (e.g., the first primer). In some embodiments, the enzyme that catalyzes homologous recombination can form a nucleoprotein complex by binding a single-stranded oligonucleotide. In some embodiments, a cognate recombinase as part of a nuclear protein complex can bind to a cognate portion of a double-stranded polynucleotide. In some embodiments, a homologous portion of the polynucleotide can hybridize to at least a portion of the first primer. In a certain embodiment, the homologous portion of the polynucleotide may be partially or fully complementary to at least a portion of the first primer.

In some embodiments, homologous recombinases can catalyze strand invasion by forming nucleoprotein complexes and binding to homologous portions of a double-stranded polynucleotide to form recombinant intermediates (D-loop formation) with triple-stranded structures (U.S. Pat. nos. 5,223,414,sena 5,273,881 and 5,670,316 to Zarling, and U.S. Pat. nos. 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, the homologous recombinase includes a wild-type, mutant, recombinant, fusion, or fragment thereof. In some embodiments, homologous recombinases include enzymes from any organism including myoviridae (myoviridae) (e.g., uvsX, RB69, etc. from bacteriophage T4), escherichia coli (e.g., recA), or human (e.g., RAD 51).

In some embodiments, the method for nucleic acid amplification comprises one or more accessory proteins. For example, helper proteins can improve the activity of a recombinase (U.S. Pat. No. 8,071,308 to Piepenburg, et al). In some embodiments, the helper protein may bind to a single strand of the nucleic acid or the recombinase may be loaded on the nucleic acid. In some embodiments, the helper protein comprises a wild type, mutant, recombinant, fusion, or fragment thereof. In some embodiments, the helper proteins may be derived from any combination of species that are the same or different from the recombinase used to perform the nucleic acid amplification reaction. The helper protein may be derived from any bacteriophage, including myococcal phages. Examples of myococcal virus phages include T4, T2, T6, rb69, aeh1, KVP40, acinetobacter phage 133, aeromonas phage 65, blue-green algae phage P-SSM2, blue-green algae phage PSSM4, blue-green algae phage S-PM2, rb14, rb32, aeromonas phage 25, vibrio phage nt-1, phi-1, rb16, rb43, phage 31, phage 44RR2.8t, rb49, phage Rb3, and phage LZ2. The helper protein may be derived from any bacterial species including E.coli, sulfolobus (e.g.sulfolobus solfataricus) or Methanococcus (e.g.Methanococcus jannaschii).

In some embodiments, a method for nucleic acid amplification may comprise a single-stranded binding protein. Single-chain binding proteins include myococcal gp32 (e.g., T4 or RB 69), sso SSB from sulfolobus solfataricus, mjA SSB from Methanococcus jannaschii, or E.coli SSB proteins.

In some embodiments, a method for nucleic acid amplification can include a protein that can increase recombinase loading on a nucleic acid. For example, recombinase loading proteins include UsvY proteins (U.S. patent 8,071,308 to pineburg).

In some embodiments, methods for nucleic acid amplification can include at least one protein that binds nucleic acids, including proteins that unwind double-stranded nucleic acids (e.g., helicases).

In some embodiments, the method for nucleic acid amplification may comprise at least one cofactor for recombinase or polymerase activity. In some embodiments, the cofactor comprises one or more divalent cations. Examples of divalent cations include magnesium, manganese, and calcium.

In some embodiments, the nucleic acid amplification reaction may be preincubated under conditions that inhibit premature reaction initiation. For example, one or more components of a nucleic acid amplification reaction can be withheld from the reaction vessel to prevent premature reaction initiation. To start the reaction, a divalent cation (e.g., magnesium or manganese) may be added. In another example, the nucleic acid amplification reaction can be pre-incubated at a temperature that inhibits enzyme activity. The reaction may be preincubated at about 0-15 deg.C or about 15-25 deg.C to inhibit premature reaction initiation. The reaction can then be incubated at a higher temperature to initiate enzyme activity.

In some embodiments, a method for nucleic acid amplification may include at least one cofactor for recombinase assembly on nucleic acids or for homologous nucleic acid pairing. In some embodiments, the cofactor comprises any form of ATP including ATP and ATP γ S.

In some embodiments, a method for nucleic acid amplification can include at least one co-factor that produces ATP. For example, cofactors include enzyme systems that convert ADP to ATP. In some embodiments, the co-factor comprises phosphocreatine and creatine kinase.

In some embodiments, any of the nucleic acid amplification methods disclosed herein can be performed under isothermal or substantially isothermal amplification conditions or can include steps performed under such conditions. In some embodiments, the isothermal amplification conditions comprise a nucleic acid amplification reaction that undergoes such a temperature change: the temperature change is limited to a limited range during at least some portion of the amplification (or the entire amplification process), including, for example, a temperature change of equal to or less than about 20 ℃, or about 10 ℃, or about 5 ℃, or about 1-5 ℃, or about 0.1-1 ℃, or less than about 0.1 ℃.

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

In some embodiments, the isothermal nucleic acid amplification reaction can be performed at about 15-25 ℃, or about 25-35 ℃, or about 35-40 ℃, or about 40-45 ℃, or about 45-50 ℃, or about 50-55 ℃, or about 55-60 ℃.

In some embodiments, the method for nucleic acid amplification comprises a plurality of different polynucleotides. In some embodiments, the plurality of different polynucleotides comprises single-stranded or double-stranded polynucleotides or a mixture of both. In some embodiments, the plurality of different polynucleotides comprises polynucleotides having the same or different sequences. In some embodiments, the plurality of different polynucleotides comprises polynucleotides having the same or different lengths. In some embodiments, the plurality of different polynucleotides comprises 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 10 ³ –10 ⁶ Or about 10 ⁶ -10 ¹⁰ A plurality of different polynucleotides. In some embodiments, the plurality of different polynucleotides comprises a polymer of deoxynucleotides, ribonucleotides, and/or analogs thereof. In some embodiments, the plurality of different polynucleotides comprises naturally occurring, synthetic, recombinant, cloned, amplified, unamplified, or archived (e.g., preserved) forms. In some embodiments, the plurality of different polynucleotides comprises DNA, cDNA, RNA, or chimeric RNA/DNA and nucleic acid analogs A compound (I) is provided.

In some embodiments, the plurality of different polynucleotide templates may comprise a double-stranded polynucleotide library construct having one or both ends with a nucleic acid adaptor sequence attached thereto. For example, a polynucleotide library construct may comprise a first and second end, wherein the first end is linked to a first nucleic acid linker. The polynucleotide library construct may further comprise a second end linked to a second nucleic acid linker. The first and second linkers may have the same or different sequences. In some embodiments, at least a portion of the first or second nucleic acid linker (i.e., as part of the polynucleotide library construct) can hybridize to the first primer. In some embodiments, a homologous recombinase as part of a nucleoprotein complex can be associated with a polynucleotide library construct having a first or second nucleic acid linker sequence.

In some embodiments, polynucleotide library constructs may be suitable for use in any type of sequencing platform including chemical degradation, chain termination, sequencing by synthesis, pyrophosphate, massively parallel, ion sensitive and single molecule platforms.

In some embodiments, the method for nucleic acid amplification comprises diluting the amount of polynucleotides reacted with beads (e.g., beads with a plurality of first primers attached) to reduce the percentage of beads reacted with more than one polynucleotide. In some embodiments, nucleic acid amplification reactions can be performed using a polynucleotide-to-bead ratio selected to optimize the percentage of beads having a population of monoclonal polynucleotides attached thereto. For example, a nucleic acid amplification reaction can be performed at any polynucleotide to bead ratio in the range of about 1. In some embodiments, the polynucleotide to bead ratio comprises about 1. In some embodiments, a nucleic acid amplification reaction can produce beads that do not have a polynucleotide attached to them, other beads that have one type of polynucleotide attached to them, and other beads that have more than one type of polynucleotide attached to them.

In some embodiments, the reaction mixture comprises one or more primers. For example, the reaction mixture may comprise at least a first oligonucleotide primer. In some embodiments, the first primer can include a forward amplification primer that hybridizes to at least a portion of one strand of the polynucleotide. In some embodiments, the first primer comprises an extendable 3' end for nucleotide polymerization.

In some embodiments, the method for nucleic acid amplification comprises an additional primer that hybridizes to the template. For example, the second primer can be a reverse amplification primer that hybridizes to at least a portion of one strand of the polynucleotide. In some embodiments, the second primer comprises an extendable 3' end. In some embodiments, the second primer is not attached to the surface.

In some embodiments, the third primer can be a forward amplification primer that hybridizes to at least a portion of one strand of the polynucleotide. In some embodiments, the third primer comprises an extendable 3' end. In some embodiments, the third primer is not attached to the surface. In some embodiments, the third primer comprises a binding partner or affinity moiety (e.g., biotin) for enriching the amplified nucleic acid.

In some embodiments, the primers (e.g., the first, second, and third primers) comprise single-stranded oligonucleotides.

In some embodiments, at least a portion of the primer can hybridize to a portion of at least one strand of the polynucleotide in the reaction mixture. For example, at least a portion of the primer can hybridize to a nucleic acid adaptor that is attached to one or both ends of the polynucleotide. In some embodiments, at least a portion of a primer may be partially or fully complementary to a portion of a polynucleotide or a nucleic acid linker. In some embodiments, primers may be suitable for use in any type of sequencing platform including chemical degradation, chain termination, sequencing-by-synthesis, pyrophosphate, massively parallel, ion sensitive, and single molecule platforms.

In some embodiments, a primer (e.g., a first, second, or third primer) can have a 5 'or 3' protruding tail (tailed primer) that does not hybridize to a portion of at least one strand of a polynucleotide in a reaction mixture. In some embodiments, the tailed primer can be of any length, including lengths of 1-50 or more nucleotides.

In some embodiments, the primer comprises a polymer of deoxynucleotides, ribonucleotides, and/or analogs thereof. In some embodiments, the primers include naturally occurring, synthetic, recombinant, cloned, amplified, or unamplified forms. In some embodiments, the primer comprises DNA, cDNA, RNA, or chimeric RNA/DNA and nucleic acid analogs.

In some embodiments, the primer may be any length, including about 5-10 nucleotides, or about 10-25 nucleotides, or about 25-40 nucleotides, or about 40-55 nucleotides, or longer.

In some embodiments, the methods for nucleic acid amplification may comprise one or more different polymerases. In some embodiments, the polymerase includes any enzyme or fragment or subunit thereof that can catalyze the polymerization of nucleotides and/or nucleotide analogs. In some embodiments, the polymerase requires an extendable 3' terminus. For example, polymerase requires terminal 3' OH of a nucleic acid primer to initiate nucleotide polymerization.

Polymerases include any enzyme that catalyzes the polymerization of nucleotides (including analogs thereof) into a nucleic acid strand. Typically, but not necessarily, such nucleotide polymerization may occur in a template-dependent manner. In some embodiments, the polymerase may be a high fidelity polymerase. Such polymerases can include, but are not limited to, naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fused, or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives, or fragments thereof that retain the ability to catalyze such polymerization. Optionally, the polymerase may be a mutant polymerase comprising one or more mutations including the substitution of one or more amino acids with other amino acids, the insertion or deletion of one or more amino acids from the polymerase, or the ligation of two or more portions of the polymerase. As used herein, the term "polymerase" and variants thereof also refers to fusion proteins comprising at least two moieties linked to each other, wherein a first moiety comprises a peptide that can catalyze the polymerization of nucleotides into a nucleic acid strand and is linked to a second moiety comprising a second polypeptide, e.g., a reporter enzyme or processivity-promoting enzyme. Typically, polymerases contain one or more active sites on which nucleotide binding and/or catalysis of nucleotide binding can occur. In some embodiments, the polymerase comprises or lacks other enzyme activities such as 3 'to 5' exonuclease activity or 5 'to 3' exonuclease activity. In some embodiments, the polymerase may be isolated from a cell or produced using recombinant DNA techniques or chemical synthesis methods. In some embodiments, the polymerase may be expressed in prokaryotic, eukaryotic, viral, or phage organisms. In some embodiments, the polymerase may be a post-translationally modified protein or fragment thereof.

In some embodiments, the polymerase can include any one or more polymerases or biologically active fragments of polymerases described in U.S. patent publication No. 2011/0262903 to Davidson et al, published at 27.10.2011 and/or international PCT publication No. WO 2013/023176 to Vander Horn et al, published at 14.2.2013.

In some embodiments, the polymerase can be a DNA polymerase and includes, but is not limited to, bacterial DNA polymerases, eukaryotic DNA polymerases, archaeal DNA polymerases. Viral DNA polymerase and bacteriophage DNA polymerase.

In some embodiments, the polymerase can be a replicase, a DNA-dependent polymerase, a primase, an RNA-dependent polymerase (including RNA-dependent DNA polymerases such as reverse transcriptase), a thermostable polymerase, or a thermostable polymerase. In some embodiments, the polymerase can be any family a or B type polymerase. Many types of family A (e.g., E.coli Pol I), B (e.g., E.coli Pol II), C (e.g., E.coli Pol III), D (e.g., E.coli Pol II), X (e.g., human Pol. Beta.), and Y (e.g., E.coli Umuc/DinB and eukaryote RAD 30/xeroderma pigmentosum variant) polymerases are described in Rothwell and Watsman 2005Advances in Protein Chemistry 71-440. In some embodiments, the polymerase may be a T3, T5, T7, or SP6RNA polymerase.

In some embodiments, a nucleic acid amplification reaction may be performed using one type or mixture of polymerases, recombinases, and/or ligases. In some embodiments, a low fidelity or high fidelity polymerase may be used to perform the nucleic acid amplification reaction.

In some embodiments, the nucleic acid amplification reaction may be catalyzed by a thermostable or thermolabile polymerase.

In some embodiments, the polymerase may lack 5'-3' exonuclease activity. In some embodiments, the polymerase may have strand displacement activity.

In some embodiments, the ancient biological DNA polymerase may be, but is not limited to, a thermostable or thermophilic DNA polymerase such as: bacillus subtilis (Bsu) DNA polymerase I large fragment; thermus aquaticus (Taq) DNA polymerase; thermus filamentous (Thermus filiformis) (Tfi) DNA polymerase; phi29DNA polymerase; bacillus stearothermophilus (Bst) DNA polymerase; hyperthermophilic archaea (Thermococcus sp.) 9 ℃ N-7DNA polymerase; bacillus smithii (Bsm) DNA polymerase large fragment; thermococcus litoralis (Tli) DNA polymerase or Vent ^TM (exo-) DNA polymerase (from New England Biolabs); or "Deep Vent" (exo-) DNA polymerase (New England Biolabs).

In some embodiments, a method for nucleic acid amplification can include one or more types of nucleotides. Nucleotides include any compound that can selectively bind to or be polymerized by a polymerase. Typically, but not necessarily, selective binding of nucleotides to a polymerase is followed by polymerization of the nucleotides by the polymerase into a nucleic acid strand; occasionally, however, a nucleotide may dissociate from a polymerase without being incorporated into a nucleic acid strand, an event referred to herein as a "non-productive" event. Such nucleotides include not only naturally occurring nucleotides but also any analogs (regardless of structure) that can selectively bind to or be polymerized by a polymerase. Although naturally occurring nucleotides typically comprise base, sugar and phosphate moieties, the nucleotides are not limited to theseNucleotides of the present disclosure may include compounds lacking any, some, or all of such moieties. In some embodiments, the nucleotide may optionally comprise a chain of phosphorus atoms comprising 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorus atoms. In some embodiments, the phosphorus chain may be attached to any carbon of the sugar ring, for example, the 5' carbon. The phosphorus chain can be linked to the sugar with an inserted O or S. In one embodiment, one or more of the phosphorus atoms in the chain may be part of a phosphate group with P and O. In another embodiment, the insertion of O, NH, S, methylene, substituted methylene, ethylene, substituted ethylene, CNH may be utilized ₂ 、C(O)、C(CH ₂ )、CH ₂ CH ₂ Or C (OH) CH ₂ R (where R may be 4-pyridine or 1-imidazole) links together the phosphorus atoms in the chain. In one embodiment, the phosphorus atoms in the chain may have a structure comprising O, BH ₃ Or a pendant group of S. In the phosphorus chain, phosphorus atoms having pendant groups other than O may be substituted phosphate groups. In the phosphorus chain, the phosphorus atom having an intervening atom other than O may be a substituted phosphate group. Some examples of nucleotide analogs are described in U.S. Pat. No. 7,405,281 to Xu.

Some examples of nucleotides that can be used in the disclosed methods and compositions include, but are not limited to, ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotide polyphosphates, modified ribonucleotide polyphosphates, modified deoxyribonucleotide polyphosphates, peptide nucleotides, modified peptide nucleotides, metal nucleosides, phosphonate nucleosides, and modified phosphate-sugar backbone nucleotides, analogs, derivatives, or variants of the foregoing, and the like. In some embodiments, a nucleotide may comprise a non-oxygen moiety such as a sulfur-or borane-moiety in place of the oxygen moiety of the alpha phosphate and the sugar of the bridged nucleotide, or the alpha and beta phosphates of the nucleotide, or the beta and gamma phosphates of the nucleotide, or between any other two phosphates of the nucleotide, or any combination thereof.

In some embodiments, the nucleotide is unlabeled. In some embodiments, the nucleotide comprises a label and is referred to herein as a "labeled nucleotide". In some embodiments, the label may be in the form of a fluorescent dye attached to any portion of the nucleotide, including the base, the sugar, or any intervening phosphate group or terminal phosphate group, i.e., the phosphate group furthest from the sugar.

In some embodiments, the nucleotide (or analog thereof) may be attached to a label. In some embodiments, the label comprises an optically detectable moiety. In some embodiments, the label includes a moiety not normally present in a naturally occurring nucleotide. For example, the label may include a fluorescent, luminescent, or radioactive moiety.

In some embodiments, the method for nucleic acid amplification may further comprise an enrichment step. In some embodiments, a method for nucleic acid amplification can produce at least one bead having attached thereto a plurality of polynucleotides (e.g., amplified nucleic acids) having sequences complementary to a template polynucleotide. At least one of the polynucleotides attached to the beads can hybridize to a polynucleotide having a biotinylated moiety (e.g., a reverse amplification product having a third primer). In some embodiments, the enriching step comprises enriching by combining a polynucleotide having a biotinylated moiety with purified beads (e.g., paramagnetic beads) linked to a streptavidin moiety (e.g., myOne from Dynabeads) ^TM Bead) to form a purified complex. In some embodiments, the purification complex may be separated/removed from the reaction mixture by utilizing the attractive force of a magnet.

In some embodiments, the amplicons comprising a population of substantially monoclonal nucleic acids are each placed, distributed, or localized at different sites in a site array.

In some embodiments, the disclosed methods include dispensing, placing, or otherwise positioning a single template molecule (e.g., a single target polynucleotide of a sample) into a reaction chamber or site (e.g., in an array). A single polynucleotide can be dispensed from a sample into a reaction chamber by flowing a fluid having a polynucleotide sample through the reaction chamber. The individual polynucleotides dispensed into the reaction chambers may be single-stranded or double-stranded. In some embodiments, the nucleic acid is amplified in the reaction chamber or site after dispensing.

In some embodiments, different individual target polynucleotides can be dispensed from a sample into each of the different reaction chambers arranged in an array. Different individual polynucleotides can be distributed from a sample to each of the different reaction chambers by flowing a fluid with a polynucleotide sample through the reaction chambers. The different individual polynucleotides apportioned into each of the different reaction chambers may be single-stranded or double-stranded.

In some embodiments, a method comprises dispensing a single polynucleotide into a reaction chamber, and amplifying the single polynucleotide within the reaction chamber, thereby generating a population of monoclonal nucleic acids in the reaction chamber.

In some embodiments, the methods for dispensing a single target polynucleotide into a reaction chamber and amplifying a single target polynucleotide comprise a nucleic acid sample. In some embodiments, a single polynucleotide or different single polynucleotides may be partitioned from a nucleic acid sample having multiple polynucleotides. For example, a nucleic acid sample can 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 10 ³ -10 ⁶ Or more different polynucleotides. Different polynucleotides may have the same or different sequences. Different polynucleotides may be of the same or different lengths. The sample may comprise single-stranded or double-stranded polynucleotides or a mixture of both.

In some embodiments, the method for dispensing a single target polynucleotide into a reaction chamber and amplifying a single target polynucleotide comprises dispensing a single polynucleotide. In some embodiments, a single polynucleotide may include both single-stranded and double-stranded nucleic acid molecules. In some embodiments, the nucleic acid may comprise a polymer of deoxynucleotides, ribonucleotides, and/or analogs thereof. In some embodiments, a nucleic acid may comprise a naturally occurring, synthetic, recombinant, cloned, amplified, unamplified, or archived (e.g., preserved) form. In some embodiments, the nucleic acid may comprise DNA, cDNA, RNA, or chimeric RNA/DNA and nucleic acid analogs. In some embodiments, a single polynucleotide may comprise a nucleic acid library construct comprising a nucleic acid linked at one or both ends to an oligonucleotide linker. In some embodiments, the nucleic acid library constructs may be suitable for use in any type of sequencing platform including chemical degradation, chain termination, sequencing by synthesis, pyrophosphate, massively parallel, ion sensitive and single molecule platforms.

In some embodiments, the array of sites can include one or more reaction chambers (can be a solid surface on the hole). The reaction chamber may have an aperture of defined width and depth. The dimensions of the reaction chamber may be sufficient to allow deposition of reagents or for carrying out reactions. The reaction chamber may have any shape including cylindrical, polygonal, or a combination of different shapes. Any wall of the reaction chamber may have a smooth or irregular surface. The reaction chamber may comprise a bottom having a planar, concave or convex surface. The bottom and side walls of the reaction chamber may comprise the same or different materials and/or may be coated with chemical groups that can react with biomolecules, such as nucleic acids, proteins, or enzymes.

In some embodiments, the reaction chamber can be arranged in a grid or array of a plurality of reaction chamber. The array may comprise two or more reaction chambers. The plurality of reaction chambers may be arranged randomly or in an ordered array. An ordered array may comprise reaction chambers arranged in rows or in a two-dimensional grid having rows and columns.

The array may include any number of reaction chambers for depositing reagents and performing a plurality of individual reactions. For example, the array may comprise at least 256 reaction chambers, or at least 256,000, or at least 1-3 million, or at least 3-5 million, or at least 5-7 million, or at least 7-9 million, at least 9-11 million, at least 11-13 million reaction chambers, or even higher densities comprising 13-700 million reaction chambers or more. The reaction chambers arranged in the grid can have a center distance (e.g., pitch) between adjacent reaction chambers of less than about 10 microns, or less than about 5 microns, or less than about 1 micron, or less than about 0.5 microns.

The array can include reaction chambers having any width and depth dimensions. For example, the reaction chamber may have dimensions to accommodate a single microparticle (e.g., a microbead) or a plurality of microparticles. The reaction chamber can hold a water volume of 0.001-100 picoliters.

In some embodiments, at least one reaction chamber may be coupled to one or more sensors or may be welded over one or more sensors. A reaction chamber coupled to the sensor may provide a confinement of the reagents deposited therein so that products from the reaction may be detected by the sensor. The sensor can detect a change in a product from any type of reaction, including any nucleic acid reaction, such as a primer extension, or nucleotide incorporation reaction. The sensor can detect changes in ions (e.g., hydrogen ions), protons, phosphate groups such as pyrophosphate groups. In some embodiments, at least one reaction chamber may be coupled to one or more Ion Sensitive Field Effect Transistors (ISFETs). Examples of arrays of reaction chambers coupled to ISFET sensors can be found in U.S. patent No. 7,948,015 and U.S. serial No. 12/002,781.

In some embodiments, the amplification methods (and related compositions, systems, and devices) described herein can be performed in an array of reaction chambers, wherein the reaction chambers of the array form part of a single fluidic system. In some embodiments, a plurality of reaction chamber array can include a fluidic interface, the fluidic interface to make fluid (such as liquid or gas) in a controlled laminar flow through the reaction chamber. In some embodiments, the array of reaction chambers may include a fluid head space above the reaction chambers for laminar flow. In some embodiments, the array of reaction chambers may be a flow cell or a portion of a flow chamber, wherein the reaction chambers are in fluid communication with each other. For example, a fluid can flow over the array to at least partially or completely fill one or more reaction chambers of the array. In some embodiments, the fluid may completely fill a plurality of reaction chambers, and excess fluid may flood the top of the reaction chambers to form a fluid layer over the reaction chambers. The fluid layer above the reaction chamber can provide fluid communication for a plurality of reaction chambers in the array. In some embodiments, the array of a plurality of reaction chamber between fluid communication can be used in a plurality of reaction chamber separation parallel reaction. For example, fluidic communication can be used to deliver polynucleotides and/or reagents to multiple reaction chambers for parallel nucleic acid amplification reactions.

In some embodiments, a sample having a plurality of different polynucleotides can be applied to a flow chamber to distribute a single polynucleotide to each reaction chamber in an array. In some embodiments, additional reagents can be applied to the flow chamber to distribute to each reaction chamber in the array. For example, the additional reaction reagents may include microparticles, one or more enzymes, enzyme cofactors, primers, and/or nucleoside triphosphates. In some embodiments, polynucleotides and reagents may be delivered to the array of reaction chambers in any order, including sequentially or substantially simultaneously or a combination of both. For example, in some embodiments, the polynucleotide may be dispensed first to the array of reaction chambers followed by the reagent, or the reverse order may be used, or the polynucleotide and reagent may be dispensed substantially simultaneously.

In some embodiments, any method (including flow chambers) can be used to deliver polynucleotides and/or reagents to a percentage of the reaction chambers in an array. For example, the percentage of reaction chambers loaded in the array includes a percentage of about 1-25%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or higher. In some embodiments, the percentage of reaction chambers loaded with polynucleotides and/or reagents may be increased by performing two or more rounds of loading steps. For example, (a) in a first round, polynucleotides and/or reagents may be dispensed to multiple reaction chambers in an array, and (b) in a second round, polynucleotides and/or reagents may be dispensed to the same array. Additional load rounds (e.g., a third, fourth, or more rounds) may be performed. In some embodiments, any type of reaction may be performed between any loading rounds and/or may be performed after multiple loading rounds are completed. For example, a nucleic acid amplification reaction may be performed between any loading rounds or may be performed after completion of multiple loading rounds. In some embodiments, after each loading round, compounds may be layered on the array to prevent migration of polynucleotides and beads out of the reaction chamber. For example, after each loading round, a solution comprising at least one sieving agent may be layered on the array. In some embodiments, the sizing agent comprises a cellulose derivative. Alternatively, an oil layer may be layered over the reaction mixture in the wells or chambers of the array.

In some embodiments, the disclosed methods (and related compositions, systems, and kits) further comprise ligating the nucleic acid template to a site of the array prior to amplifying the template. Optionally, the site comprises a primer, and the ligating comprises hybridizing the primer to a primer binding site of the template. For example, a site within an array may comprise at least one immobilized primer comprising a sequence complementary to at least a portion of a primer binding site of a template that is assigned or placed at the site. The primers facilitate ligation of the template to the array. In some embodiments, a majority of the sites in the array comprise at least one primer. The primers at different sites may be identical to each other. Alternatively, the primers at different sites may be different from each other. In an exemplary embodiment, each of the at least two sites includes a different target-specific primer.

Any suitable method may be used to attach the primers to the sites of the array. Microparticles attaching primers to the surface of a nanoarray of reaction chambers (e.g., of the ISFET array type used for ion-based sequencing) it may be useful to first synthesize or prepare a three-dimensional matrix within at least some of the reaction chambers of the array. In embodiments, the polymer matrix precursor may be applied to an array of wells associated with one or more sensors. The polymer matrix precursor can be polymerized to form an array of polymer matrices. These polymer matrices can be conjugated to oligonucleotides and can be used in a variety of analytical techniques including genetic sequencing techniques.

In some embodiments, the hydrophilic polymer matrix is dispensed in a well associated with a sensor (e.g., a sensor of a sensor array). In an example, the hydrophilic matrix is as a hydrogel matrix. The hydrophilic matrix may correspond to the sensors of the sensor array in a one-to-one configuration. In other examples, the sensors of the sensor array may include Field Effect Transistor (FET) sensors, such as Ion Sensitive Field Effect Transistors (ISFETs). In particular, the matrix material is cured in situ and adapted to the structure of the individual pores of the sensing device. The interstitial regions between the pores may be substantially free of polymer matrix. In an example, the matrix material can be bonded, e.g., covalently bonded, to the surface of the pores. In an example, the pores have a depth or thickness in a range of 100 nanometers to 10 micrometers. In another example, the pores may have a characteristic diameter in a range of 0.1 microns to 2 microns.

In an exemplary method, the polymer matrix can be formed by applying an aqueous solution comprising a polymer precursor to the wells of the array of wells. The volume of aqueous material defined by the array of wells can be isolated using an immiscible fluid disposed over the array of wells. The isolated volume of solution can be initiated to facilitate polymerization of the matrix precursor, resulting in an array of matrices dispensed within the wells. In an example, an aqueous solution comprising a matrix precursor is dispensed to the pores of a sensing device by flowing an aqueous precursor through the pores. In another example, the aqueous solution is included as a dispersed phase in the emulsion. The dispersed phase may settle or be excited in the pores of the pore array. The polymerization of the matrix precursor may be initiated using an initiation factor placed in the aqueous phase or in an immiscible fluid. In another example, the polymerization may be initiated thermally.

In another exemplary method, a matrix array can be formed within a well of a sensing device by anchoring a starter molecule to a surface within the well of the well array. A solution comprising a matrix precursor may be provided over the wells of the array of wells. The initiation factor may initiate polymerization of the matrix precursor, resulting in the formation of a polymer matrix within the wells of the well array. In other examples, aspects of the above methods are combined to further enhance formation of the matrix array.

In particular embodiments, the sequencing system includes a flow cell in which the sensor array is disposed, includes communication circuitry in electronic communication with the sensor array, and includes a container and a fluid control in fluid communication with the flow cell. In an example, fig. 13 shows an expanded view and a cross-sectional view of the flow cell 100 and shows a portion of the flow chamber 106. Reagent stream 108 flows over the surface of aperture array 102, where reagent stream 108 flows over the open ends of the apertures of aperture array 102. The array of wells 102 and the sensor array 105 together may form an integrated unit constituting the lower wall (or bottom surface) of the flow cell 100. Reference electrode 104 may be fluidly coupled to flow cell 106. In addition, flow cell cover 130 encloses flow chamber 106 to contain reagent stream 108 within a confined area.

Fig. 14 shows an expanded view of the aperture 201 and sensor 214 (as shown at 110 of fig. 13). Pore volume, shape, aspect ratio (e.g., base width to pore depth ratio), and other dimensional characteristics may be selected based on the nature of the reaction taking place and the labeling techniques (if any) or reagents, byproducts used. The sensor 214 may be a chemical field effect transistor (chemFET), more particularly an Ion Sensitive FET (ISFET), having a floating gate 218 with a sensor plate 220 separated from the interior of the hole, optionally by a layer of material 216. Additionally, a conductive layer (not shown) may be placed on the sensor board 220. In an example, the material layer 216 comprises an ion sensitive material layer. The material layer 216 may be a ceramic layer, such as an oxide of zirconium, hafnium, tantalum, aluminum, or titanium, or a nitride of titanium, among others. In an example, the material layer 216 may have a thickness of 5nm to 100nm, e.g., 10nm to 70nm,15nm to 65nm, or 20nm to 50 nm.

Although material layer 216 is shown as extending beyond the boundaries of the FET components shown, material layer 216 may extend along the bottom of hole 201 and optionally along the walls of hole 201. The sensor 214 may sense (and generate an associated output signal) the amount of charge 224 present on the material layer 216 opposite the sensor plate 220. The change in the charge 224 can cause a change in current between the source 221 and the drain 222 of the chemFET. Further, chemfets may be used to provide a current-based output signal directly or to provide a voltage-based output signal indirectly with additional circuitry. Reactants, wash solutions, and other reagents may move into and out of the pores through the diffusion mechanism 240.

In embodiments, the reaction performed in the well 201 may be an analytical reaction to identify or determine a characteristic or property of an analyte of interest. Such a reaction may directly or indirectly generate byproducts that affect the amount of charge near the sensor plate 220. If such byproducts are produced in small amounts or decay rapidly or react with other components, multiple copies of the same analyte may be analyzed in well 201 simultaneously to increase the output signal produced. In embodiments, multiple copies of an analyte may be attached to the solid support 212 before or after placement into the well 201. The solid support 212 can be a polymer matrix, such as a hydrophilic polymer matrix, such as a hydrogel matrix or the like. For simplicity and ease of illustration, the solid support 212 is also referred to herein as a polymer matrix.

The aperture 201 may be defined by a wall structure, which may be formed from one or more layers of material. In an example, the wall structure can have a thickness extending from the lower surface to the upper surface of the pores of 0.01 microns to 10 microns, such as 0.05 microns to 10 microns, 0.1 microns to 10 microns, 0.3 microns to 10 microns, or 0.5 microns to 6 microns. In particular, the thickness may be in the range of 0.01 to 1 micron, for example in the range of 0.05 to 0.5 micron, or 0.05 to 0.3 micron. The pores 201 can have a characteristic diameter of 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, no more than 0.8 microns, or no more than 0.6 microns, the diameter defined by 4 times the cross-sectional area (a) divided by the square root of Pi (e.g., sqrt (4 a/Pi)). In an example, the pores 201 may have a characteristic diameter of at least 0.01 microns.

Although fig. 14 shows a single wall structure and a single layer of material 216, the system may include one or more wall structure layers, one or more conductive layers, or one or more layers of material. For example, the wall structure may be formed from one or more layers comprising TEOS or an oxide of silicon or a nitride comprising silicon.

In the embodiment shown in fig. 15, the system 300 comprises a pore wall structure 302 defining an array of pores 304 disposed on or operatively coupled to a sensor pad of a sensor array. The pore wall structure 302 defines an upper surface 306. The lower surface 308 associated with the aperture is placed on a sensor pad of the sensor array. The cell wall structure 302 defines a sidewall 310 between the upper surface 306 and the lower surface 308. As described above, the layer of material in contact with the sensing pads of the sensor array may extend along the lower surface 308 of the wells of the array of wells 304 or along at least a portion of the wall 310 defined by the well wall structure 302. The upper surface 306 may be free of a layer of material. In particular, the polymer matrix may be placed in the wells of the array of wells 304. The upper surface 306 may be substantially free of a polymer matrix. For example, the upper surface 306 may include an area free of the polymer matrix, such as at least 70% of the total area, at least 80% of the total area, at least 90% of the total area, or about 100% of the total area.

Although the wall surface of fig. 14 is shown as extending substantially vertically and outwardly, the wall surface may extend in different directions and may have different shapes. The term "substantially vertically" means extending in a component direction orthogonal to the surface defined by the sensor pad. For example, as shown in fig. 16, the aperture wall 402 may extend vertically, parallel to a normal component 412 of the surface defined by the sensor pad. In another example, the wall surface 404 extends substantially vertically in an outward direction away from the sensor pad, thereby providing a larger aperture opening than the lower surface area of the aperture. As shown in fig. 16, wall surface 404 extends in a perpendicular component direction that is parallel to normal component 412 of surface 414. In an alternative example, the wall surface 406 extends substantially vertically in an inward direction, providing a smaller open area than the lower surface area of the aperture. Wall surface 406 extends in a component direction that is parallel to normal component 412 of surface 414.

Although

surfaces

402, 404, or 406 are shown by straight lines, some semiconductor or CMOS fabrication processes may result in structures having non-linear shapes. In particular, wall surfaces, such as wall surface 408, and upper surfaces, such as upper surface 410, may be arcuate in shape or may have various non-linear forms. Although the structures and devices shown along with them are described as having linear layers, surfaces, or shapes, the actual layers, surfaces, or shapes produced by semiconductor processing may differ to some extent, possibly including non-linear and arcuate variations of the embodiments shown.

Fig. 17 shows an exemplary aperture containing a layer of ion-sensitive material. For example, the aperture structure 502 may define an array of apertures, such as

exemplary apertures

504, 506, or 508. The aperture (504, 506, or 508) may be operably coupled to an underlying sensor (not shown) or connected to such an underlying sensor. The exemplary aperture 504 includes a layer of ion-sensitive material 510 that defines the bottom of the aperture 504 and extends into the structure 502. Although not shown in fig. 17, a conductive layer, such as a gate electrode, for example, the floating gate of an ion sensitive field effect transistor, may be located beneath the layer of ion sensitive material 510.

In another example, as shown by the hole 506, the ion-sensitive material layer 512 may define the bottom of the hole 506 without extending into the structure 502. In other examples, the aperture 508 may include an ion-sensitive layer 514 extending along at least a portion of a sidewall 516 of the aperture 508 defined by the structure 502. As above, the layer of ion-

sensitive material

512 or 514 may be located over a conductive layer or gate of an underlying electronic device.

In fig. 14, the matrix material 212 is conformal to the pore structure. In particular, the matrix material may be cured in situ to conform to the walls and floor of the hole. The upper surface defining the aperture may comprise an area substantially free of matrix material, such as at least 70% of the total area, at least 80% of the total area, at least 90% of the total area, or about 100% of the total area. Depending on the nature of the pore structure, the polymer matrix may be physically fixed to the pore wall structure. In another example, the polymer matrix may be chemically bonded to the pore wall structure. In particular, the polymer matrix may be covalently bound to the pore wall structure. In another example, the polymer matrix may be bound to the pore wall structure by hydrogen or ionic bonding.

The polymer matrix may be formed from matrix precursors, such as fully polymerizable monomers, e.g., vinyl-based monomers. In particular, the monomers may include hydrophilic monomers such as acrylamide, vinyl acetate, hydroxyalkyl methacrylate (hydroxyalkylmethacrylate), variants or derivatives thereof, copolymers thereof, or any combination thereof. In a particular example, the hydrophilic monomer is an acrylamide, such as an acrylamide functionalized to include a hydroxyl group, an amino group, a carboxyl group, a halide group, or a combination thereof. In examples, the hydrophilic monomer is an aminoalkylacrylamide, an acrylamide functionalized with an amine terminated polyallylamine (D, shown below), an propylene piperazine (C, shown below), or a combination thereof. In another example, the acrylamide may be a hydroxyalkyl acrylamide, such as hydroxyethyl acrylamide. Specifically, the hydroxyalkyl acrylamide may include N-tris (hydroxymethyl) methyl) acrylamide (a, shown below), N- (hydroxymethyl) acrylamide (B, shown below), or a combination thereof. In another example, the comonomer may include a halogen-modified acrylate or acrylamide, such as N- (5-bromoacetamidopentyl) acrylamide (BRAPA, E, shown below). In another example, the comonomer may include an oligonucleotide-modified acrylate or acrylamide monomer. In other examples, mixtures of monomers may be used, such as mixtures of hydroxyalkyl acrylamides and amine-functionalized acrylamides or mixtures of acrylamides and amine-functionalized acrylamides. In examples, the amine-functionalized acrylamide may be included in a ratio of hydroxyalkyl acrylamide to amine-functionalized acrylamide or acrylamide to amine-functionalized acrylamide in the range of 100 to 1, such as in the range of 100 to 2, 1 to 1, 50 to 1, or 50. In another example, the amine-functionalized acrylamide may be included in a ratio of hydroxyalkyl acrylamide to bromine-functionalized acrylamide or acrylamide to bromine-functionalized acrylamide in the range of 100 to 1, such as in the range of 100 to 2, 1 to 1, 50.

A

B

C

D

E

In other embodiments, the first and second electrodes may be,may include oligonucleotide-functionalized acrylamide or acrylate monomers, e.g. Acrydite ^TM Monomers to incorporate the oligonucleotides into the polymer matrix.

Another exemplary matrix precursor includes a cross-linking agent. In an example, the crosslinking agent is included in a mass ratio of 15 to 1, e.g. 10. In particular, the crosslinking agent may be a divinyl crosslinking agent. For example, the divinyl crosslinker may include a bisacrylamide, such as N, N '- (ethane-1, 2-diyl) bis (2-hydroxyethyl) acrylamide, N' - (2-hydroxypropane-1, 3-diyl) bisacrylamide, or a combination thereof. In another example, the divinyl crosslinker comprises ethylene glycol dimethacrylate, divinylbenzene, cyclohexane bisacrylamide, trimethylolpropane trimethacrylate, protected derivatives thereof, or combinations thereof.

In one aspect, the polymer network comprises a polyacrylamide gel having a total monomer percentage in the range of 3-20%, more preferably in the range of 5-10%. In one embodiment, the monomer has a crosslinker percentage in the range of 5 to 10%. In a particular embodiment, the polymer network comprises 10% total acrylamide, of which 10% is a bisacrylamide crosslinker.

The polymerization can be initiated by an initiation factor in solution. For example, the initiation factor may be water-based. In another example, the initiator may be a hydrophobic initiator, preferably located in a hydrophobic phase. Exemplary initiation factors include ammonium persulfate or TEMED (tetramethylethylenediamine). TEMED accelerates the rate of formation of free radicals from persulfate salts, thereby catalyzing polymerization. Persulfate radicals, for example, convert acrylamide monomers to radicals, which react with unactivated monomers to initiate a polymerization chain reaction. The extended polymer chains may be randomly cross-linked, resulting in a gel with a characteristic porosity that depends on the polymerization conditions and monomer concentration. Riboflavin (or riboflavin-5' -phosphate) may also be used as a source of free radicals, which is typically used in combination with TEMED and ammonium persulfate. In the presence of light and oxygen, riboflavin is converted into its active, colorless form with the initial polymerization, which is generally referred to as photopolymerization.

In another example, a nitrogen-containing initiator may be used to initiate the polymerization. Exemplary water-soluble nitrogen-containing initiation factors are shown in table 1, and exemplary oil-soluble nitrogen-containing initiation factors are shown in table 2. In particular, the nitrogen-containing initiator may be Azobisisobutyronitrile (AIBN).

TABLE I

Water soluble nitrogenous starter compounds

TABLE II

Oil-soluble nitrogenous starter compound

In other examples, the precursor of the polymer matrix may include surface active additives to enhance bonding to the surface. Exemplary additives include functionalized acrylic monomers or functionalized acrylamide monomers. For example, the acrylic monomer may be functionalized to bind surface materials, such as ceramic materials that form the bottom or sidewalls of the pores. In an example, the additive can include a propenyl-phosphonate, such as a methacrylate phosphonate. In another example, the additive may include dimethylacrylamide or polydimethylacrylamide. In other examples, the additive may include polylysine modified with polymerizable groups (e.g., acrylic groups).

In another example, atom Transfer Radical Polymerization (ATRP) can be used to facilitate polymerization. The ATRP system may include an initiation factor, a transition metal ion, and a ligand. Exemplary transition metal ion complexes include copper-based complexes. Exemplary ligands include 2,2 '-bipyridine, 4' -di-5-nonanyl-2, 2 '-bipyridine, 4',4 "-tris (5-nonanyl) -2,2':6',2" -terpyridine, N, N, N ', N', N "-pentamethyldiethylenetriamine, 1,4,7, 10-hexamethyltriethylenetetramine, tris (2-dimethylaminoethyl) amine, N, N-bis (2-picolyl) octadecylamine, N, N, N ', N' -tetrakis [ (2-pyridylmethyl) ethylenediamine, tris [ 2-pyridylmethyl ] amine, tris (2-aminoethyl) amine, tris (2-bis (3-butoxy-3-oxopropyl) aminoethyl) amine, tris (2-bis (3- (2-ethylhexyloxy) -3-oxopropyl) aminoethyl) amine, tris (2-bis (3-dodecaoxy-3-oxopropyl) aminoethyl) amine, variants and derivatives thereof, or combinations thereof. Exemplary initiation factors include 2-bromopropionitrile, ethyl 2-bromoisobutyrate, ethyl 2-bromopropionate, methyl 2-bromopropionate, 1-phenyl bromoethane, p-toluenesulfonyl chloride, 1-cyano-1-methylethyl diethyldithiocarbamate, ethyl 2- (N, N-diethyldithiocarbamate) -isobutyrate, dimethyl 2, 6-dibromopimelate, variants or derivatives thereof, and any combination thereof. Optionally, BRAPA monomer can be used as a branching agent in the presence of an ATRP system.

In an example, ATRP is initiated at the surface to directly bond the polymer to the surface. For example, acrylic acid monomers, acrylamide monomers, acrydite may be reacted in the presence of a transition metal ion/ligand complex ^TM A monomer, a succinimide acrylate, a bisacrylic acid or bisacrylamide monomer, a derivative thereof, or a combination thereof is applied to the starting surface in solution.

In another, the ATRP system can be used to attach polymers to the surface of pores using modified phosphonate, sulfonate, silicate or zirconate compounds. In particular, an amine or hydroxyl terminated alkyl phosphonate or alkoxy derivative thereof can be applied to a surface and initiated using an initiation factor. The catalyst complex and monomer can be applied to extend the surface compound.

In an exemplary method, an aqueous solution comprising a precursor of the polymer matrix can be applied to the wells of the structure defining the array of wells. The aqueous solution in the wells can be isolated by providing an immiscible fluid over the wells and initiating polymerization of the polymer precursors in solution in the wells.

For example, fig. 18 shows an exemplary aperture structure 602 defining an aperture 604. One or more sensors (not shown) may be operatively coupled or connected to the aperture 604. For example, the one or more sensors may include a gate structure in electrical communication with at least a bottom surface of the aperture 604. An aqueous solution 606 containing, among other components, a polymer precursor is provided over the wells and the solution containing the polymer precursor is dispensed into the wells 604. Exemplary polymer precursors include, among others, monomers, cross-linkers, initiation factors, or surfactants, e.g., as described above. Optionally, pores 604 may be wetted with a hydrophilic solution, such as a solution comprising water, an alcohol, or a mixture thereof, or a solution comprising water and a surfactant, prior to deposition. Exemplary alcohols include isopropanol. In another example, the alcohol comprises ethanol. Although not shown, the bottom surface of the well and optionally the sidewalls of the well can include an ion sensitive material. Such ion sensitive materials may overlie conductive structures of underlying electronic devices such as field effect transistors. The surface active additive may be used to treat one or more surfaces of the pores prior to applying the solution comprising the polymer precursor.

The distribution of the aqueous solution comprising the polymer precursor into the pores 604 may be further enhanced by shaking the structure, for example by rotation or vortexing. In another example, vibration, such as sonic or ultrasonic vibration, may be used to increase the distribution of the aqueous solution within the pores 604. In other examples, the pores may be degassed using a vacuum pump and the solution applied at a negative gauge pressure. In the examples, the aqueous solution was dispensed to the wells at room temperature. In another example, the aqueous solution is dispensed at a temperature below room temperature, particularly when a water-based initiation factor is used. Alternatively, the aqueous solution is dispensed at an elevated temperature.

As shown in fig. 19, an immiscible fluid 708 is applied over the well 604, which pushes the aqueous solution 606 away from the top of the well and isolates the aqueous solution 606 within the well 604, as shown in fig. 20. Exemplary immiscible fluids include mineral oil, silicone oil (e.g., poly (dimethylsiloxane)), heptane, carbonate oils (e.g., diethylhexylcarbonate (Tegosoft)

) Or combinations thereof.

The initiation factor may be administered in an aqueous solution 606. Alternatively, the initiation factor may be provided within immiscible fluid 708. Polymerization can be initiated by changing the temperature of the substrate. Alternatively, the polymerization may occur at room temperature. Specifically, the polymer precursor solution may be held at a temperature of 20 ℃ to 100 ℃, e.g., 25 ℃ to 90 ℃, 25 ℃ to 50 ℃, or 25 ℃ to 45 ℃, for 10 minutes to 5 hours, e.g., 10 minutes to 2 hours or 10 minutes to 1 hour.

Due to polymerization, an array of polymer matrix 912 is formed within the pores 604 defined by the pore structure 602, as shown in fig. 21. Optionally, the array can be washed with NaOH (e.g., 1N NaOH) to remove the polymer from the interstitial regions between the wells.

In an alternative example, an emulsion of an aqueous solution comprising a polymer precursor as the dispersed phase within an immiscible fluid is used to deposit droplets of the aqueous solution within the pores. For example, as shown in fig. 22, the hole structure 1002 defines a hole 1004. The aperture may be operatively coupled or electrically connected to one or more sensors (not shown). As described above, the bottom and optionally side surfaces of the walls of the holes 1004 may be defined by a layer of ion-sensitive material that may overlie conductive members of an underlying electronic device.

Emulsion 1006 may comprise aqueous droplets 1008 comprising polymer precursors dispersed within a continuous immiscible fluid 1010. Droplet 1008 may settle in hole 1004. In particular, aqueous droplets 1008 have a greater density than immiscible fluid 1010. Exemplary immiscible fluids include mineral oil, silicone oil (e.g., poly (dimethylsiloxane)) heptane, carbonate oil (e.g., diethylhexylcarbonate (Tegosoft)

) Or combinations thereof. In other examples, the distribution of aqueous droplets into the wells 1004 can be facilitated by spinning, vortexing, or sonicating the fluid or structure. Optionally, pores 604 may be wetted with a hydrophilic solution, such as a solution comprising water, an alcohol, or a mixture thereof, or a solution comprising water and a surfactant, prior to deposition. The temperature during dispensing of the droplets into the wells may be at room temperature. Alternatively, dispensing may be carried out at an elevated temperature.

As shown in fig. 23, the droplets merge within well 1004 to provide a droplet comprising a polymer precursor1112 of the isolated solution. Optionally, emulsion 1006 may be replaced with an immiscible fluid, such as immiscible fluid 1010 without droplets 1008 or a different immiscible fluid 1116. Exemplary immiscible fluids include mineral oil, silicone oil (e.g., poly (dimethylsiloxane)) heptane, carbonate oil (e.g., diethylhexyl carbonate (Tegosoft)

) Or combinations thereof. Alternatively, the emulsion 1006 may remain in place during polymerization. In this manner, the solution 1112 in the well 1004 is isolated from the solution in the other wells 1004. Polymerization may be initiated resulting in the polymer matrix 1214 within the holes 1004, as shown in fig. 24. As noted above, the polymerization may be thermally initiated. In another example, an oil phase initiation factor can be used to initiate polymerization. Alternatively, an aqueous phase initiation factor may be used to initiate polymerization. In particular, a second emulsion may be applied over the holes 1004. The second emulsion can include a dispersed aqueous phase including an aqueous phase initiation factor.

Polymerization can be initiated by changing the temperature of the substrate. Alternatively, the polymerization may occur at room temperature. Specifically, the polymer precursor solution may be held at a temperature of 20 ℃ to 100 ℃, e.g., 25 ℃ to 90 ℃, 25 ℃ to 50 ℃, or 25 ℃ to 45 ℃, for 10 minutes to 5 hours, e.g., 10 minutes to 2 hours or 10 minutes to 1 hour. Optionally, the array can be washed with NaOH (e.g., 1N NaOH) to remove polymer from the interstitial regions between the wells.

In another example, an array of matrix material can be formed within the wells of the array of wells using an initiation factor immobilized on the surface of the array of wells. For example, as shown in fig. 25, the structure 1302 can define an aperture 1304. The material layer 1306 may define a lower surface of the aperture 1304. An anchor compound 1308, such as an anchor compound for Atom Transfer Radical Polymerization (ATRP), may be secured to the material layer 1306 defining the bottom surface of the pores 1304. Alternatively, the sidewall material defined within the holes or layers of structure 1302 exposed within holes 1304 may anchor a compound such as those described above for ATRP.

In such an example, a solution 1310 comprising a polymer precursor, such as a monomer, a crosslinking agent, and optionally a surfactant additive, can be applied over the structure 1302 and within the pores 1304. The anchor compound 1308 can be initiated to promote polymerization extending from the anchor compound, to sequester the polymerization within the pores 1304, and to fix the polymer to the pores 1304. In an example, the anchor compound has a surface active group and a distal radical forming group. The surface active groups may include phosphonates, silicate sulfonates, zirconates, titanates, or combinations thereof. The remote radical forming group may comprise an amine or hydroxyl group which may undergo, for example, transfer with a halo (e.g., bromo) compound and subsequently form a radical which serves to polymerize the polymer precursor and anchor the resulting polymer to the pore surface. Generally, an ATRP system can be selected to terminate polymerization after a statistically average length or number of monomer additions. In this manner, the total number of aggregates within the pores 1304 can be controlled. In other examples, other actual applications and additions that affect chain extension or termination may be applied and added to the aqueous solution 1310.

In another example shown in fig. 26, the structure 1402 may define an aperture 1404. Aperture 1404 may include a layer of material 1406 extending along sidewalls 1410 of structure 1402 and aperture 1404. Initiation factor 1408 can be secured to material layer 1406 along the bottom of aperture 1404 and along sidewalls 1410. A solution 1412 comprising polymer precursors, cross-linking agents, and other reagents may be applied over structure 1402 and wells 1404.

As shown in fig. 27, the polymer matrix 1512 is formed as a result of initiated polymerization extending from a surface within the pores 1304 (e.g., a surface defined by the material layer 1306).

The polymerization can be initiated by changing the temperature of the substrate. Alternatively, the polymerization may occur at room temperature. Specifically, the polymer precursor solution may be held at a temperature of 20 ℃ to 100 ℃, e.g., 25 ℃ to 90 ℃, 25 ℃ to 50 ℃, or 25 ℃ to 45 ℃ for 10 minutes to 5 hours, e.g., 10 minutes to 2 hours or 10 minutes to 1 hour.

After formation, the polymer matrix can be activated to facilitate conjugation to the target analyte, e.g., a polynucleotide. For example, functional groups on the polymer matrix can be enhanced to allow binding to a target analyte or analyte receptor (e.g., an oligonucleotide primer). In particular examples, the functional groups of the hydrophilic polymer matrix can be modified with reagents capable of converting the hydrophilic polymer functional groups into reactive moieties that can undergo nucleophilic or electrophilic substitution. For example, hydroxyl groups on the polymer matrix can be activated by substituting at least a portion of the hydroxyl groups with sulfonic acid groups or chlorine. Exemplary sulfonic acid groups can be derived from trifluoroethanesulfonyl (tresyl), methanesulfonyl, p-toluenesulfonyl, or p-fluorobenzenesulfonyl (fosyl) chloride, or any combination thereof. The sulfonate ester serves to allow nucleophilic groups to substitute for the sulfonate ester. The sulfonate ester may also react with the liberated chlorine to provide a chlorinated functional group that can be used in the process of conjugating the substrate. In another example, amine groups on the polymer matrix may be activated.

For example, the target analyte or analyte receptor may bind to the hydrophilic polymer through nucleophilic substitution with sulfonic acid groups. In particular examples, target analyte receptors that are blocked with nucleophilic groups such as amines or sulfhydryl groups can undergo nucleophilic substitution to displace sulfonic acid groups in the polymer matrix. Upon activation, a conjugated polymeric matrix may be formed.

In another example, the sulfonated polymer matrix may be further reacted with a mono-or polyfunctional mono-or poly-nucleophile (e.g., maleimide), which may form a linkage to the matrix while retaining nucleophilic activity for the electrophilic group-containing oligonucleotide. Furthermore, the remaining nucleophilic activity can be converted to an electrophilic activity by attachment to a reagent comprising a multi-electrophilic group, which will subsequently be attached to an oligonucleotide comprising a nucleophilic group.

In another example, a monomer comprising a functional group may be added during polymerization. Monomers include, for example, acrylamides that contain carboxylic acid, ester, halogen, or other amine reactive groups. The ester group can be hydrolyzed prior to reaction with an oligoamine (amine oligo).

Other conjugation techniques include the use of amine-containing monomers. The amine group is a nucleophilic group that can be further modified by an amine reactive bifunctional electrophilic reagent that, upon attachment to the polymer matrix, yields a monofunctional electrophilic group. Such electrophilic groups can react with oligonucleotides having nucleophilic groups (e.g., amines or thiols), resulting in attachment of the oligonucleotide (by reaction with the vacated electrophile).

If the polymer matrix is prepared from a combination of amino-and hydroxy-acrylamides, the polymer matrix contains a combination of nucleophilic amino groups and neutral hydroxy groups. The amino group can be modified with a difunctional di-electrophilic moiety such as a diisocyanate or di-NHS ester to produce a hydrophilic polymer matrix that is reactive with nucleophilic groups. Exemplary di-NHS esters include di-succinimidyl C2-C12 alkyl esters, such as di-succinimidyl suberate or di-succinimidyl glutarate.

Other activation chemistries include the incorporation of multiple steps to convert specific functional groups to accommodate specific desired linkages. For example, sulfonate-modified hydroxyls can be converted into nucleophilic groups by several methods. In an example, the reaction of a sulfonate ester with an azide anion produces an azide-substituted hydrophilic polymer. Azides can be used directly for conjugation to acetylene-substituted biomolecules by "CLICK" chemistry, which can be performed with or without copper catalysis. Optionally, the azide can be converted to an amine by, for example, catalytic reduction using hydrogen or reduction using an organophosphine. The resulting amine can then be converted to an electrophilic group using a variety of reagents such as diisocyanates, di-NHS esters, cyanuric chloride, or combinations thereof. In an example, the use of a diisocyanate creates a urea linkage (urea linkage) between the polymer and the linker, which results in a residual isocyanate group that can react with the amino-substituted biomolecule to create a urea linkage between the linker and the biomolecule. In another example, the use of di-NHS esters creates an amide bond between the polymer and the linker and a remaining NHS ester group that can react with the amino-substituted biomolecule to create an amide linkage between the linker and the biomolecule. In other examples, the use of cyanuric chloride results in an amino-triazine bond between the polymer and the linker and two remaining chlorotriazine groups, one of which is capable of reacting with an amino-substituted biomolecule to produce an amino-triazine bond between the linker and the biomolecule. Other nucleophilic groups can be incorporated into the matrix by sulfonyl activation. For example, the reaction of the sulfonyl substrate with the thiobenzoate anion and hydrolysis of the resulting thiobenzoate incorporates a thiol group into the substrate, which can then be reacted with the maleimide-substituted biomolecule to produce a thio-succinimide linkage to the biomolecule.

Sulfhydryl groups may also be reacted with bromo-acetyl or bromo-amido groups. In a specific example, when n- (5-bromoacetamidopentyl) acrylamide (BRAPA) is included as a comonomer, the oligonucleotide may be incorporated by forming a thiobenzamide-oligonucleotide compound for reaction with the bromo-acetyl groups on the polymer, such as shown below.

The thiobenzamide-oligonucleotide compound may be formed by reacting a dithiobenzoic acid-NHS compound described below with an amine-terminated oligonucleotide and activating the dithiobenzamide-oligonucleotide compound to form the thiobenzamide-oligonucleotide compound shown above.

Alternatively, acrydite oligonucleotides can be used for incorporation into oligonucleotides during polymerization. Exemplary acrydite oligonucleotides may include ion-exchanged oligonucleotides.

Covalent attachment of biomolecules on a refractory (refractory) or polymeric material substrate may be produced by using electrophilic moieties coupled to nucleophilic moieties on the biomolecules on the substrate or nucleophilic attachments coupled to electrophilic attachments on the biomolecules on the substrate. Due to the hydrophilic nature of most common biomolecules of interest, the solvent chosen for these couplings is water or water containing some water-soluble organic solvent to disperse the biomolecules on the substrate. In particular, polynucleotides are often coupled to substrates in aqueous systems because they have polyanionic properties. Since water competes with nucleophilic groups for electrophiles by hydrolyzing the electrophiles to an inactive moiety for conjugation, aqueous systems can result in low yields of coupled products, where the yields are based on the electrophile moiety of the conjugate. When high yields of electrophilic moieties of reactive conjugates are desired, high concentrations of nucleophilic groups drive the reaction and slow hydrolysis, resulting in inefficient use of nucleophilic groups. In the case of polynucleic acids, the metal counter ion of the phosphate may be replaced with a lipophilic counter ion to aid in the solubilization of the biomolecule into polar, non-reactive, non-aqueous solvents. These solvents may include amides or ureas such as formamide, N-dimethylformamide, acetamide, N, N-dimethylacetamide, hexamethylphosphoric acid amide, pyrrolidone, N-methylpyrrolidone, N ' -tetramethylurea, N ' -dimethyl-N, N ' -trimethyleneurea, or a combination thereof; carbonates such as dimethyl carbonate, propylene carbonate, or combinations thereof; esters such as tetrahydrofuran; sulfoxides and sulfones such as dimethyl sulfoxide, dimethyl sulfone, or combinations thereof; hindered alcohols (hindered alcohols) such as t-butyl ethanol; or a combination thereof. The lipophilic cation may include tetraalkylammonium or tetraarylammonium cations such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, tetrapentylammonium, tetrahexylammonium, tetraheptylammonium, tetraoctylammonium, and alkyl and aryl mixtures thereof, tetraarylphosphine cations such as tetraphenylphosphine, tetraalkylarsine or tetraarylarsine such as tetraphenylarsine, and trialkylsulfonium cations such as trimethylsulfonium, or combinations thereof. The conversion of polynucleic acids to organic solvent-soluble materials (by exchanging metal cations for lipophilic cations) can be carried out by a variety of standard cation exchange techniques.

In particular embodiments, the polymeric matrix is exposed to a target polynucleotide having a segment complementary to an oligonucleotide conjugated to the polymeric matrix. The polynucleotide is subjected to amplification, for example by Polymerase Chain Reaction (PCR) or Recombinase Polymerase Amplification (RPA). For example, the target polynucleotides are provided at low concentrations, such that it is possible for a single polynucleotide to be located within a single polymer matrix of an array of polymer matrices. The polymer matrix may be exposed to enzymes, nucleotides, salts, or other components sufficient to facilitate replication of the target polynucleotide.

In particular embodiments, the enzyme, e.g., polymerase, is present in, attached to, or in close proximity to the polymer matrix. A variety of nucleic acid polymerases can be used for the methods described herein. In exemplary embodiments, the polymerase may include an enzyme, fragment, or subunit thereof that may catalyze the replication of a polynucleotide. In another embodiment, the polymerase may be a naturally occurring polymerase, a recombinant polymerase, a mutant polymerase, a variant polymerase, a fused or otherwise engineered polymerase, a chemically modified polymerase, a synthetic molecule, or an analog, derivative, or fragment thereof.

In some embodiments, the methods for dispensing a single target polynucleotide into a reaction chamber and amplifying a single target polynucleotide comprise a nucleic acid amplification reaction. In some embodiments, any type of nucleic acid amplification reaction may be performed including Polymerase Chain Reaction (PCR) (U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis), ligase Chain Reaction (LCR) (Barany 1991 Proceedings National Academy of Science USA 88.

In some embodiments, the amplification reaction comprises Recombinase Polymerase Amplification (RPA). (see, e.g., U.S. Pat. Nos. 5,273,881 and 5,670,316 to Zarling, and U.S. Pat. Nos. 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 to Zarling).

In some embodiments, the methods for dispensing a single target polynucleotide into a reaction chamber and amplifying a single target polynucleotide comprise isothermal amplification conditions. In some embodiments, the nucleic acid amplification reaction can be performed under isothermal conditions. In some embodiments, the isothermal amplification conditions comprise a nucleic acid amplification reaction that undergoes such a temperature change: the temperature change is limited to a limited range during at least some portion of the amplification, including, for example, a temperature change within about 20 ℃, or about 10 ℃, or about 5 ℃, or about 1-5 ℃, or about 0.1-1 ℃, or less than about 0.1 ℃. In some embodiments, the nucleic acid amplification reaction can be performed under isothermal or thermal cycling conditions.

In some embodiments, nucleic acids that have been amplified according to the present teachings can be used in any nucleic acid sequencing workflow, including sequencing by oligonucleotide probe ligation and detection (e.g., SOLiD from Life Technologies ^TM WO 2006/084131), probe-anchor ligation sequencing (e.g.complete Genomics) ^TM Or Polonator ^TM ) Sequencing by synthesis (e.g.genetic Analyzer and HiSeq from Illumina) ^TM ) Pyrophosphate sequencing (e.g., genome sequence FLX from 454Life Sciences), ion sensitive sequencing (e.g., personal Genome Machine (PGM) from Ion Torrent Systems, inc.) ^TM ) And Ion Proton ^TM Sequence) and single molecule sequencing platforms (e.g., from Helicos) ^TM HeliScope (R) ^TM )。

In some embodiments, nucleic acids that have been amplified according to the present teachings can be sequenced by any sequencing method including sequencing by synthesis, ion-based sequencing including detection of sequencing byproducts using field effect transistors (e.g., FETs and ISFETs), chemical degradation sequencing, ligation-based sequencing, hybridization sequencing, pyrophosphate detection sequencing, capillary electrophoresis, gel electrophoresis, next generation, massively parallel sequencing platforms, sequencing platforms that detect hydrogen ions or other sequencing byproducts, and single molecule sequencing platforms. In some embodiments, at least one sequencing primer that can hybridize to any portion of the polynucleotide construct (including the nucleic acid linker or the target polynucleotide) can be used to perform a sequencing reaction.

In some embodiments, a method of detecting one or more by-products of nucleotide incorporation can be used to sequence a nucleic acid amplified according to the present teachings. Polymerase extension detection by detecting physicochemical by-products of the extension reaction may include pyrophosphate, hydrogen ions, charge transfer, heat, and the like, as disclosed, for example, in U.S. Pat. No. 7,948,015 to Rothberg et al and U.S. patent publication No. 2009/0026082 to Rothberg et al (incorporated herein by reference in their entirety). Other examples of methods of detecting polymerase-based extension can be found, for example, in Pourmand et al, proc.natl.acad.sci., 103; purushothaman et al, IEEE ISCAS, IV-169-172; anderson et al, sensors and actors B chem., 129; sakata et al, angew.chem.118:2283-2286 (2006); esfandyapor et al, U.S. patent publication No. 2008/01666727; and Sakurai et al, anal. Chem.64:1996-1997 (1992).

Reactions involving the generation and detection of ions are widely performed. Monitoring the progress of such reactions using direct ion detection methods can simplify many current bioassays. For example, polymerase-mediated template-dependent nucleic acid synthesis can be monitored by detecting hydrogen ions produced as a natural byproduct of nucleotide incorporation catalyzed by the polymerase. Ion sensitive sequencing (also referred to as "pH-based" or "ion-based" nucleic acid sequencing) utilizes direct detection of ionic byproducts such as hydrogen ions (generated as nucleotide-incorporated byproducts). In one exemplary system for ion-based sequencing, nucleic acids to be sequenced can be captured in microwells and the nucleotides flowed through the wells one at a time under nucleotide incorporation conditions. The polymerase incorporates the appropriate nucleotide into the growing strand and the released hydrogen ions can alter the pH of the solution, which can be detected by an ion sensor coupled to the pore. This technique does not require labeling of nucleotides or expensive optical components and allows for much faster completion of sequencing runs. Examples of such Ion-based nucleic acid sequencing methods and platforms include Ion Torrent PGM ^TM Or Proton ^TM Sequencer (Ion Torrent) ^TM Systems,Life Technologies Corporation)。

In some embodiments, target polynucleotides produced using the methods, systems, and kits of the present teachings can be used as substrates for biological or chemical reactions that are detected and/or monitored by sensors, including Field Effect Transistors (FETs). In various embodiments the FET is a chemFET or an ISFET. "chemfets" or chemical field effect transistors are the type of field effect transistors used as chemical sensors. It is a structural analog of a MOSFET transistor in which the charge on the gate electrode is applied by a chemical process. "ISFET" or ion sensitive field effect transistor is used to measure the concentration of ions in a solution; when the ion concentration (e.g., H +) changes, the current through the transistor will change accordingly. A detailed theory of operation of ISFETs is found in "third year of ISFETOLOGY, what has been found in the past 30year and what has been found in the next 30year," P.bergveld, sens.Actuators,88 (2003), pp.1-20.

In some embodiments, the FET may be an array of FETs. As used herein, an "array" is a planar arrangement of elements such as sensors or wells. The array may be one-dimensional or two-dimensional. A one-dimensional array may be an array having one column (or row) of elements in a first dimension and a plurality of columns (or rows) in a second dimension. The number of columns (or rows) in the first and second dimensions may or may not be the same. The FET or array may include 102, 103, 104, 105, 106, 107 or more FETs.

In some embodiments, one or more microfluidic structures may be soldered over the FET sensor array to provide containment and/or containment of biological or chemical reactions. For example, in one instrument, a microfluidic structure may be configured as one or more wells (or microwells or reaction chambers or reaction wells, the terms being used interchangeably herein) disposed over one or more sensors of an array such that the one or more sensors on which a given well is disposed detect and measure the presence, level, and/or concentration of an analyte in the given well. In some embodiments, the correspondence of the FET sensor and the reaction well may be 1.

Microwells or reaction chambers are typically holes or wells of well-defined shape and volume that can be machined into a substrate and welded using conventional micro-welding techniques, such as disclosed in the following references: (ii) teaching and Nishi, editors, handbook of Semiconductor Manufacturing Technology, second Edition (CRCPress, 2007); saliterman, fundamentals of BioMEMS and Medical Microdevices (SPIE Publications, 2006); elwenspoek et al, silicon Micromachining (Cambridge University Press, 2004); and the like. Examples of the structure (e.g., spacing, shape, and volume) of the microwells or reaction chambers are disclosed in U.S. patent publication 2009/0127589 to Rothberg et al; rothberg et al, british patent application GB 24611127.

In some embodiments, a biological or chemical reaction can be performed in a solution or reaction chamber in contact with, operably coupled to, or capacitively coupled to a FET, such as a chemFET or ISFET. The FET (or chemFET or ISFET) and/or the reaction chamber may be an array of FETs or reaction chambers, respectively.

In some embodiments, biological or chemical reactions can be performed in a two-dimensional array of reaction chambers, wherein each reaction chamber can be coupled to a FET, and each reaction chamber does not exceed 10 μm in capacity ³ (i.e., 1 pL). In some embodiments, each reaction chamber does not exceed 0.34pL, 0.096pL or 0.012pL in capacity. The reaction chamber may optionally have a cross-sectional area at the top of no more than 2, 5, 10, 15, 22, 32, 42, 52, 62, 72, 82, 92, or 102 square microns. Preferably, the array has at least 10 ² 、10 ³ 、10 ⁴ 、10 ⁵ 、10 ⁶ 、10 ⁷ 、10 ⁸ 、10 ⁹ Or more reaction chambers. In some embodiments, at least one of the reaction chambers is operatively coupled to at least one of the FETs.

FET arrays for use in accordance with various embodiments of the present disclosure may be soldered in accordance with conventional CMOS soldering techniques as well as modified CMOS soldering techniques and other semiconductor soldering techniques beyond those conventionally used in CMOS soldering. In addition, various lithographic techniques may be used as part of the array soldering process.

Exemplary FET arrays and microwells and fluids suitable for use in the disclosed methods and methods of making the same are disclosed in, for example, U.S. patent publication nos. 20100301398; U.S. patent publication nos. 20100300895; U.S. patent publication nos. 20100300559; U.S. patent publication No. 20100197507, U.S. patent publication No. 20100137143; U.S. patent publication No. 20090127589; and U.S. patent publication No. 20090026082, the above references are incorporated by reference herein in their entirety.

In one aspect, the disclosed methods, compositions, systems, devices, and kits can be used to perform label-free nucleic acid sequencing, particularly ion-based nucleic acid sequencing. The concept of label-free detection of nucleotide incorporation has been described in the literature, including the following (which is incorporated herein by reference): U.S. patent publication 2009/0026082 to Rothberg et al; anderson et al, sensors and actors B chem.,129, 79-86 (2008); and Pourmand et al, proc.natl.acad.sci., 103. Briefly, in nucleic acid sequencing applications, nucleotide incorporation is determined by measuring the natural by-products of polymerase catalyzed extension reactions, including hydrogen ions, polyphosphates, PPi, and Pi (e.g., in the presence of pyrophosphatase). Examples of such Ion-based nucleic acid sequencing methods and platforms include Ion Torrent PGM ^TM Or Proton ^TM Sequencer (Ion Torrent) ^TM Systems,Life Technologies Corporation)。

In some embodiments, the present disclosure generally relates to methods for sequencing nucleic acids that have been amplified by the teachings provided herein. In one exemplary embodiment, the present disclosure generally relates to a method for obtaining sequence information from a polynucleotide, comprising: (a) amplifying a nucleic acid; and (b) performing template-dependent nucleic acid synthesis using at least one of the amplified nucleic acids generated during step (a) as a template. Amplification may optionally be performed according to any of the amplification methods described herein.

In some embodiments, template-dependent synthesis comprises incorporation of one or more nucleotides into a newly synthesized nucleic acid strand in a template-dependent manner.

Optionally, the method may further comprise generating one or more ionic byproducts of such nucleotide incorporation.

In some embodiments, the method may further comprise detecting incorporation of one or more nucleotides into the sequencing primer. Optionally, detecting may include detecting the release of hydrogen ions.

In another embodiment, the present disclosure generally relates to a method for sequencing nucleic acids, comprising: (a) amplifying a nucleic acid according to the methods disclosed herein; (b) The amplified nucleic acids are placed in a plurality of reaction chambers, wherein one or more of the reaction chambers are in contact with a Field Effect Transistor (FET). Optionally, the method further comprises contacting the amplified nucleic acid disposed in one of the reaction chambers with a polymerase to synthesize a new nucleic acid strand by successively incorporating one or more nucleotides into the nucleic acid molecule. Optionally, the method further comprises generating one or more hydrogen ions as a byproduct of such nucleotide incorporation. Optionally, the method further comprises detecting incorporation of one or more nucleotides by detecting production of one or more hydrogen ions using a FET.

In some embodiments, detecting comprises detecting a change in voltage and/or current at least one FET within the array in response to the production of one or more hydrogen ions.

In some embodiments, the FET may be selected from: ion sensitive FETs (isfets) and chemosensitive FETs (chemfets).

One exemplary system that includes sequencing by detecting Ion byproducts of nucleotide incorporation is Ion Torrent PGM ^TM Or Proton ^TM Sequencer (Life Technologies), which is an ion-based sequencing system that sequences nucleic acid templates by detecting hydrogen ions generated as byproducts of nucleotide incorporation. Typically, hydrogen ions are released as nucleotide incorporation byproducts that are present during template-dependent nucleic acid synthesis mediated by a polymerase. Ion Torrent PGM ^TM Or Proton ^TM The sequencer detects nucleotide incorporation by detecting hydrogen ion by-products of nucleotide incorporation. Ion Torrent PGM ^TM Or Proton ^TM The sequencer may comprise a plurality of nucleic acid templates to be sequenced, wherein each template is placed within a respective sequencing reaction well in the array. The wells of the array may each be coupled to at least one ion sensor that can detect H produced as a byproduct of nucleotide incorporation ⁺ Release of ions or change in solution pH. The ion sensor comprises a coupling to an inductive H ⁺ A Field Effect Transistor (FET) of an ion sensitive detection layer for the presence of ions or changes in solution pH. The ion sensor can provide an output signal indicative of nucleotide incorporation, which can be expressed as a change in voltage,the magnitude of the voltage change is related to H in the respective well or reaction chamber ⁺ The ion concentration is relevant. The different nucleotide types may flow continuously into the reaction chamber and may be incorporated into the extension primer (or polymerization site) by the polymerase in an order determined by the sequence of the template. Each nucleotide incorporation can be accompanied by H in the reaction well ⁺ Release of ions and concurrent changes in local pH. H ⁺ The release of ions can be recorded by the FET of the sensor, which generates a signal indicating that nucleotide incorporation occurred. Nucleotides that are not incorporated in a particular nucleotide stream may not produce a signal. The amplitude of the signal from the FET may also be correlated with the number of nucleotides of a particular type incorporated into the extended nucleic acid molecule, allowing for the resolution of homopolymer regions. Thus, during the operation of the sequencer, multiple nucleotide flows into a reaction chamber and incorporation monitoring between a large number of wells or reaction chambers may allow the device to simultaneously resolve the sequences of many nucleic acid templates. PGM for Ion Torrent ^TM Or Proton ^TM Additional details of the composition, design, and operation of sequencers can be found, for example, in U.S. patent application Ser. No. 12/002781, now published as U.S. patent publication No. 2009/0026082; U.S. patent application Ser. No. 12/474897, now published as U.S. patent publication No. 2010/0137143; and U.S. patent application Ser. No. 12/492844, now published as U.S. patent publication No. 2010/0282617, the above references being incorporated herein by reference in their entirety.

FIG. 28 shows a block diagram of components of a system for nucleic acid sequencing according to an exemplary embodiment. The components include a flow cell 101, a reference electrode 108, a plurality of reagents 114 for sequencing, a valve body 116, a wash solution 110, a valve 112, a fluid controller 118, lines 120/122/126, channels 104/109/111, a waste container 106, an array controller 124, and a user interface 128 on an integrated circuit device 100. Integrated circuit device 100 includes microwell array 107 overlying a sensor array that contains a chemical sensor as described herein. Flow cell 101 includes an inlet 102, an outlet 103, and a flow chamber 105 defining a reagent flow path over a microwell array 107.

The reference electrode 108 may be of any suitable type or shape, including a coaxial cylinder with a flow channel or lead inserted into the lumen of the channel 111. The reagent 114 may be driven through the flow path, valves, and flow cell 101 by a pump, air pressure, or other suitable method, and may be discharged into the waste container 106 after exiting the outlet 103 of the flow cell 101. The fluid controller 118 may use appropriate software to control the driving force for the reagent 114, the operation of the valve 112 and the valve body 116.

Microwell array 107 includes an array of reaction zones as described herein, also referred to herein as microwells, that are operatively associated with corresponding chemical sensors in a sensor array. For example, each reaction zone may be coupled to a chemical sensor suitable for detecting the analyte or reaction property of interest within the reaction zone. Microwell array 107 may be integrated into integrated circuit device 100 such that microwell array 107 and the sensor array are part of a single device or chip.

Flow cell 101 may have a variety of structures for controlling the path and flow rate of reagent 114 over microwell array 107. The array controller 124 provides bias voltages and timing and control signals to the integrated circuit device 100 for reading the chemical sensors of the sensor array. Array controller 124 also provides a reference bias voltage to reference electrode 108 to bias reagents 114 flowing through microwell array 107.

During an experiment, the array controller 124 collects and processes output signals from the chemical sensors of the sensor array via an output port on the integrated circuit device 100 over the bus 127. The array controller 124 may be a computer or other computing means. The array controller 124 may include memory and application software for storing data, a processor for accessing data and executing applications, and components to facilitate communication with the various components of the system in FIG. 28.

The value of the output signal of the chemical sensor is indicative of a physical and/or chemical parameter of one or more reactions occurring in the corresponding reaction zone in microwell array 107. For example, in exemplary embodiments, the values of the output signals may be processed using the techniques disclosed in the following references: U.S. patent application Ser. No. 13/339,846, filed 2011 at 12/29, based on U.S. provisional patent application Ser. No. 61/428,743, filed 2010 at 12/30, and U.S. provisional patent application Ser. No. 61/429,328, filed 2011 at 1/3, and U.S. patent application Ser. No. 13/339,753, filed Hubbell at 2011 at 12/29, based on U.S. provisional patent application Ser. No. 61/428,097, filed 2010 at 12/29.

The user interface 128 may display information about the flow cell 101 and the output signals received from the chemical sensors in the sensor array on the integrated circuit device 100. The user interface 128 may also display device settings and controls and allow a user to input or set device settings and controls.

In an exemplary embodiment, the fluid controller 118 may control the delivery of the individual reagents 114 to the flow cell 101 and the integrated circuit device 100 during the experiment in a predetermined sequence, for a predetermined duration, at a predetermined flow rate. The array controller 124 may then collect and analyze the output signals of the chemical sensors indicative of the chemical reactions that occurred in response to the delivery of the reagent 114.

During the experiment, the system may also monitor and control the temperature of the integrated circuit device 100 so that the reaction occurs and measurements are taken at a known, predetermined temperature.

The system may be configured such that a single fluid or reagent contacts the reference electrode 108 throughout a multi-step reaction during operation. Valve 112 may be closed to prevent any wash solution 110 from flowing into channel 109 as reagent 114 flows. Although the flow of wash solution may be prevented, there may still be continuous fluidic and electrical communication between the reference electrode 108, the channel 109, and the microwell array 107. The distance between the reference electrode 108 and the junction between channels 109 and 111 can be selected so that no or very little of the reagent flowing in channel 109 and possibly diffusing into channel 111 reaches the reference electrode 108. In exemplary embodiments, the wash solution 110 may be selected to be in continuous contact with the reference electrode 108, which may be particularly useful for multi-step reactions using frequent washing steps.

Fig. 29 shows a cross-sectional view and an expanded view of a portion of the integrated circuit device 100 and the flow cell 101. During operation, flow cell 105 of flow cell 101 encloses reagent stream 208 of delivered reagent flowing through the open end of the reaction zone in microwell array 107. The volume, shape, aspect ratio (e.g., bottom-to-hole-depth ratio), and other dimensional characteristics of the reaction zone may be selected based on the nature of the reaction taking place and the labeling techniques (if any) or reagents, byproducts used.

The chemical sensors of sensor array 205 sense (and generate output signals) chemical reactions within the associated reaction zones in microwell array 107 to detect the desired analytical or reaction properties. The chemical sensors of sensor array 205 may be, for example, chemically sensitive field effect transistors (chemfets), such as Ion Sensitive Field Effect Transistors (ISFETs). Examples of chemical sensors and array structures that can be used with embodiments are described in U.S. patent application publication nos. 2010/0300559, 2010/0197507, 2010/0301398, 2010/0300895, 2010/0137143, and 2009/0026082, and U.S. patent No. 7,575,865, each of which is incorporated herein by reference.

FIG. 30 shows a cross-sectional view of two representative chemical sensors and their corresponding reaction zones, according to an exemplary embodiment. In fig. 30, two

chemical sensors

350, 351 are shown, representing a small portion of a sensor array that may include millions of chemical sensors. In some embodiments, a sensor array can include at least 1 million chemical sensors and optionally at least 1 million corresponding reaction zones, at least 5 million chemical sensors and optionally at least 5 million corresponding reaction zones or at least 10 million chemical sensors and optionally at least 10 million corresponding reaction zones.

Chemical sensors 350 are coupled to corresponding reaction zones 301 and chemical sensors 351 are coupled to corresponding reaction zones 302. Chemical sensor 350 represents a chemical sensor in a sensor array. In the example shown, the chemical sensor 350 is an ion sensitive field effect transistor. The chemical sensor 350 includes a floating gate structure 318, the floating gate structure 318 having a floating gate conductor (referred to herein as a sensor plate 320) separated from the reaction region 301 by a sensing material 316. As shown in fig. 30, the sensor plate 320 is the uppermost patterned layer of conductive material in the floating gate structure 318 under the reaction region 301.

In the example shown, the floating gate structure 318 includes multiple patterned layers of conductive material in a layer of dielectric material 319. As described in more detail below, the upper surface of the sensing material 316 serves as the sensing surface 317 of the chemical sensor 350.

In the embodiment shown, the sensing material 316 is an ion sensitive material, such that the presence of ions or other charged species in solution in the reaction zone 301 changes the surface potential of the sensing surface 317. The change in surface potential is caused by the protonation or deprotonation of the surface charge groups at the sensing surface by the ions present in the solution. The sensing material 316 may be embedded using a variety of techniques or formed naturally during one or more manufacturing processes used to form the chemical sensor 350. In some embodiments, the sensing material 316 is a metal oxide, such as an oxide of silicon, tantalum, aluminum, lanthanum, titanium, zirconium, hafnium, tungsten, palladium, iridium, and the like.

In some embodiments, the sensing material 316 is an oxide of an upper layer of conductive material of the sensor plate 320. For example, the upper layer of the sensor plate 320 may be titanium nitride and the sensing material 316 may include titanium oxide or titanium oxynitride. More generally, the sensing material 316 may include one or more of a variety of different materials to facilitate sensitivity to particular ions. For example, silicon nitride or silicon oxynitride and metal oxides such as silicon oxide, aluminum oxide or tantalum oxide generally provide sensitivity to hydrogen ions, whereas sensing materials comprising polyvinyl chloride with valinomycin provide sensitivity to potassium ions. Depending on the instrument, materials sensitive to other ions (e.g., sodium, silver, iron, bromine, iodine, calcium, and nitrates) may also be used.

The chemical sensor 350 also includes a source region 321 and a drain region 322 within the semiconductor substrate 354. The source region 321 and the drain region 322 comprise a doped semiconductor material having a conductivity type that is different from the conductivity type of the substrate 354. For example, the source region 321 and the drain region 322 may comprise a doped P-type semiconductor material and the substrate may comprise a doped N-type semiconductor material.

A tub region 323 separates the source region 321 and the drain region 322. The floating gate structure 318 overlies the trench region 323 and is separated from the substrate 354 by a gate dielectric 352. The gate dielectric 352 may be, for example, silicon dioxide. Alternatively, other dielectrics may be used for gate dielectric 352.

As shown in fig. 30, reaction zone 301 extends through fill material 310 on dielectric material 319. Fill material 310 may, for example, comprise one or more layers of dielectric material such as silicon dioxide or silicon nitride.

The dimensions (e.g., width and depth) of the

reaction zones

301, 302, as well as their pitch (center distance between adjacent reaction zones) may vary from instrument to instrument. In some embodiments, the reaction zone can have a characteristic diameter of 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, no more than 0.8 microns, no more than 0.6 microns, no more than 0.4 microns, no more than 0.2 microns, or no more than 0.1 microns, the diameter defined by 4 times the plan view cross-sectional area (a) divided by the square root of Pi (e.g., sqrt (4 a/Pi)).

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

In the embodiment shown, the

reaction zones

301, 302 are separated by a distance equal to their width. Alternatively, the separation distance between adjacent reaction zones may be less than their width. For example, the separation distance may be the minimum feature size for a process (e.g., a lithography process) used to form the

reaction zones

301, 302. In such cases, the separation may be significantly less than the width of the individual reaction zones.

The sensor plate 320, the sensing material 316 and the reaction zone 301 may for example have a circular cross-section. Alternatively, these may be non-circular. For example, the cross-section may be square, rectangular, hexagonal, or irregularly shaped.

Depending on the device and array structure in which the chemical sensors described herein are implemented, the device in fig. 30 may also include additional elements such as array lines (e.g., word lines, bit lines, etc.) for accessing the chemical sensors, additional doped regions in the substrate 354, and other circuitry (e.g., access circuitry, bias circuitry, etc.) for operating the chemical sensors. In some embodiments, devices can be produced, for example, using the techniques described in U.S. patent application publication nos. 2010/0300559, 2010/0197507, 2010/0301398, 2010/0300895, 2010/0137143, and 2009/0026082, and U.S. patent No. 7,575,865 (each of which is incorporated herein by reference).

In operation, reactants, wash solutions, and other reagents may be moved into and out of reaction zone 301 by diffusion mechanism 340. Chemical sensor 350 senses (and generates an associated output signal) the amount of charge 324 present on sensing material 316 opposite sensor plate 320. The change in charge 324 causes a change in the voltage on the floating gate structure 318, which in turn causes a change in the threshold voltage of the transistor. The threshold voltage change can be measured by measuring the current in the tub 323 between the source 321 and drain 322 regions. Thus, the chemical sensor 350 may be used directly to provide a current-based output signal on an array line connected to the source region 321 or the drain region 322, or indirectly with additional circuitry to provide a voltage-based output signal.

In embodiments, the reaction performed in the reaction zone 301 may be an analytical reaction to identify or determine a characteristic or property of an analyte of interest. Such a reaction may directly or indirectly generate byproducts that affect the amount of charge near the sensor plate 320. If such byproducts are produced in small quantities or decay rapidly or react with other components, multiple copies of the same analyte may be analyzed simultaneously in reaction zone 301 to increase the output signal produced. In embodiments, multiple copies of an analyte may be attached to the solid support 312 before or after placement in the reaction zone 301. The solid support 312 can be microparticles, nanoparticles, beads, solid or porous, including gels, and the like. For simplicity and ease of illustration, the solid support 312 is also referred to herein as a particle. For nucleic acid analytes, multiple linked copies can be prepared by Rolling Circle Amplification (RCA), exponential RCA, recombinase Polymerase Amplification (RPA), polymerase chain reaction amplification (PCR), emulsion PCR amplification, or similar techniques to generate amplicons without a solid support.

In various exemplary embodiments, the methods, systems, and computer-readable media described herein may be advantageously used to process and/or analyze data and signals obtained from electron or charge based sequencing of nucleic acids. In electron-or charge-based sequencing (e.g., pH-based sequencing), nucleotide incorporation events can be determined by detecting ions (e.g., hydrogen ions) that are generated as natural byproducts of polymerase-catalyzed nucleotide extension reactions. This can be used to sequence a sample or template nucleic acid, which can be, for example, a fragment of a nucleic acid sequence of interest, and which can be attached directly or indirectly as a clonal population to a solid support such as a particle, microparticle, bead, or the like. The sample or template nucleic acid may be operably associated to the primer and the polymer and may undergo cycles or "flows" (which may be referred to herein as "nucleotide flows" that may result in nucleotide incorporation) of repeated nucleotide additions and washes. The primer may anneal to the sample or template so that the 3' end of the primer may be extended by the polymerase each time a nucleotide complementary to the next base in the template is added. Subsequently, based on the known sequence of the nucleotide streams and on the measured output signal of the chemical sensor indicative of the ion concentration during each nucleotide stream, information can be determined on the type, sequence and number of nucleotides associated with the sample nucleic acid present in the reaction zone coupled to the chemical sensor.

In typical ion-based embodiments of nucleic acid sequencing, nucleotide incorporation can be detected by detecting the presence and/or concentration of hydrogen ions generated by a polymerase-catalyzed extension reaction. In one embodiment, the template, optionally pre-bound to a sequencing primer and/or polymerase, can be loaded into a reaction chamber (e.g., a microwell disclosed in Rothberg et al, cited herein) and then repeated cycles of nucleotide addition and washing can be performed. In some embodiments, such templates may be attached to a solid support, such as a particle, bead, or the like, as a clonal population, and the clonal population loaded into a reaction chamber.

In another embodiment, the template, optionally bound to a polymerase, is partitioned, placed or localized at different sites of the array. The sites of the array comprise primers and the method may comprise hybridizing different templates to the primers in the different sites.

In each addition step of the cycle, the polymerase can extend the primer by incorporating the added nucleotide only when the next base in the template is the complement of the added nucleotide. If there is one complementary base, there is one incorporation, if there are two, there are two incorporations, if there are three, there are three incorporations, and so on. For each such incorporation, there is a released hydrogen ion, and the template population that collectively releases the hydrogen ion changes the local pH of the reaction chamber. The generation of hydrogen ions is monotonically related to the number of consecutive complementary bases in the template (and to the total number of template molecules that the primer and polymer participate in the extension reaction). Thus, when there are many consecutive identical complementary bases (i.e. homopolymer regions) in the template, the amount of hydrogen ions generated, and hence the magnitude of the local pH change, may be proportional to the number of consecutive identical complementary bases. If the next base in the template is not complementary to the added nucleotide, then incorporation does not occur and no hydrogen ions are released. In some embodiments, after each step of adding nucleotides, an additional step may be performed in which an unbuffered wash solution at a predetermined pH is used to remove the nucleotides of the previous step to prevent misincorporation in later cycles. In some embodiments, after each step of adding nucleotides, an additional step may be performed in which the reaction chamber is treated with a nucleotide-destroying reagent, such as apyrase, to remove any residual nucleotides remaining in the chamber that may lead to false extensions in subsequent cycles.

In an exemplary embodiment, different kinds of nucleotides are added to the reaction chamber in succession, so that each reaction can be exposed to different nucleotides one at a time. For example, the nucleotides can be added in the following order: dATP, dCTP, dGTP, dTTP, dATP, dCTP, dGTP, dTTP, and the like; a washing step was performed after each exposure. The cycle may be repeated 50, 100, 200, 300, 400, 500, 750, or more times depending on the length of sequence information desired.

In some embodiments, may be according to PGM ^TM Or Proton ^TM The user manual provided by the sequencer was used to perform sequencing. Example 3 provides for the use of Ion Torrent PGM ^TM Sequencer (Ion Torrent) ^TM Systems, life Technologies, CA) is an exemplary protocol for ion-based sequencing.

In some embodiments, the present disclosure relates generally to methods for sequencing a population of template polynucleotides, comprising: (a) Generating a plurality of amplicons by clonally amplifying a plurality of template polynucleotides on a plurality of surfaces, wherein the amplifying is performed within a single continuous phase of a reaction mixture and wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the amplicons generated are substantially monoclonal in nature. In some embodiments, a sufficient number of substantially monoclonal amplicons are generated in a single amplification reaction to produce a PGM at Ion Torrent ^TM 314. At least 100MB, 200MB, 300MB, 400MB, 500MB, 750MB, 1GB, or 2GB of AQ20 sequencing reads were generated on a 316 or 318 sequencer. As used herein, the term "AQ20" and variants thereof refer to PGM at Ion Torrent ^TM Specific methods for measuring sequencing accuracy in a sequencer. Accuracy can be measured in terms of a Phred-like Q score, which measures accuracy on a logarithmic scale: q10=90%, Q20=99%, Q30=99.9%, Q40=99.99% and Q50=99.999%. For example, in a particular sequencing reaction, an accuracy metric can be calculated by a predictive algorithm or by actual alignment with a known reference genome. The predicted quality score ("Q-score") may be derived from an algorithm that: which takes into account the inherent nature of the input signal and makes a reasonably accurate assessment of whether a given single base contained in a sequencing "read" will align. In some embodiments, such predicted mass fractions can be used to filter and remove low mass reads prior to downstream alignment. In some embodiments, accuracy may be reported as a Phred-like Q scoreThe accuracy was measured on a logarithmic scale: q10=90%, Q17=98%, Q20=99%, Q30=99.9%, Q40=99.99% and Q50=99.999%. In some embodiments, data obtained from a given polymerase reaction may be filtered to measure only polymerase reads that are "N" nucleotides or longer and have a Q score that exceeds a certain threshold, e.g., Q10, Q17, Q100 (referred to herein as an "NQ17" score). For example, a 100Q20 score may indicate the number of reads obtained from a given reaction that are at least 100 nucleotides in length and have a Q20 (99%) or higher Q score. Similarly, a 200Q20 score can indicate the number of reads that are at least 200 nucleotides in length and have a Q20 (99%) or higher Q score.

In some embodiments, accuracy may also be calculated based on an appropriate alignment using the reference genomic sequence, which is referred to herein as "raw" accuracy. This is single pass accuracy (single pass accuracuracy), which includes a measure of the "true" of errors per base associated with a single read, as opposed to shared accuracy, which measures the error rate from a consensus sequence, which is the result of multiple reads. Raw accuracy measurements can be reported as a fraction of "AQ" ("quality of alignment"). In some embodiments, the data obtained from a given polymerase reaction may be filtered to measure only polymerase readings that measure "N" nucleotides or longer and have AQ scores that exceed a certain threshold, e.g., AQ10, AQ17, AQ100 (referred to herein as "NAQ17" scores). For example, a 100AQ20 score may indicate the number of reads obtained from a given polymerase reaction that are at least 100 nucleotides in length and have an AQ score of AQ20 (99%) or higher. Similarly, a 200AQ20 score may indicate the number of reads that are at least 200 nucleotides in length and have an AQ score of AQ20 (99%) or higher.

In some embodiments, the present teachings provide a system for nucleic acid amplification comprising any combination of: a bead having a plurality of first primers attached thereto, a second primer, a third primer, a polynucleotide, a recombinase loading protein, a single-stranded binding protein (SSB), a polymerase, a nucleotide, ATP, phosphocreatine, creatine kinase, a hybridization solution, and/or a wash solution. The system may comprise all or some of these components. In some embodiments, the system for nucleic acid amplification can further comprise any combination of buffers and/or cations (e.g., divalent cations).

In some embodiments, the present teachings provide kits for nucleic acid amplification. In some embodiments, the kit comprises any reagents useful for nucleic acid amplification. In some embodiments, the kit may comprise any combination of: a bead to which a plurality of first primers are linked, a second primer, a third primer, a polynucleotide, a recombinase loading protein, a single-stranded binding protein (SSB), a polymerase, a nucleotide, ATP, phosphocreatine, creatine kinase, a hybridization solution, a wash solution, a buffer, and/or a cation (e.g., a divalent cation). The kit may comprise all or some of these components.

In some embodiments, the present disclosure relates generally to methods, compositions, systems for amplifying different nucleic acid templates in parallel in multiple partitioned reaction volumes (as opposed to amplification within a single continuous liquid phase). For example, nucleic acid templates may be distributed or placed in an array of reaction chambers or an array of reaction volumes such that at least two such chambers or volumes in the array each receive a single nucleic acid template. In some embodiments, multiple separate reaction capacities are formed. The reaction chamber (or reaction volume) may optionally be closed prior to amplification. In another embodiment, the reaction mixture may be divided or separated into a plurality of microreactors dispersed within the continuous phase of the emulsion. The partitioned or separated reaction volumes are optionally not mixed or in communication with each other, or are not capable of being mixed or in communication with each other. In some embodiments, at least some of the reaction chambers (or reaction volumes) comprise a recombinase and optionally a polymerase. The polymerase may be a strand displacing polymerase.

In some embodiments, the present disclosure relates generally to compositions, systems, methods, devices, and kits comprising emulsions for nucleic acid synthesis and/or amplification. As used herein, the term "emulsion" includes any composition comprising a mixture of a first liquid and a second liquid, wherein the first and second liquids are substantially immiscible with each other. Typically, one of the liquids is hydrophilic and the other liquid is hydrophobic. Typically, emulsions comprise a dispersed phase and a continuous phase. For example, a first liquid may form a dispersed phase dispersed in a second liquid, which forms a continuous phase. The dispersed phase optionally consists essentially of the first liquid. The continuous phase optionally consists essentially of the second liquid. In various embodiments, the same two liquids may form different types of emulsions. For example, a mixture comprising oil and water may first form an oil-in-water emulsion, where oil is the dispersed phase and water is the dispersion medium. Secondly, they can form water-in-oil emulsions, in which water is the dispersed phase and oil is the external phase. Multiple emulsions are also possible, including "water-in-oil-in-water" emulsions and "oil-in-water-in-oil" emulsions. In some embodiments, the dispersed phase comprises one or more microreactors in which nucleic acid templates can be individually amplified. One or more microreactors may form partitioned reaction volumes in which separate amplification reactions may occur. One example of a suitable vehicle for nucleic acid amplification includes a water-in-oil emulsion, wherein the water-based phase comprises several aqueous microreactors dispersed within the oil phase of the emulsion. In some embodiments, the emulsion may further comprise an emulsifier or surfactant. Emulsifiers or surfactants can be used to stabilize the emulsion under nucleic acid synthesis conditions.

In some embodiments, the present disclosure generally relates to compositions comprising an emulsion comprising a reaction mixture. The emulsion may comprise an aqueous phase. The aqueous phase may be dispersed in the continuous phase of the emulsion. The aqueous phase may comprise one or more microreactors. In some embodiments, the reaction mixture is contained in a plurality of liquid-phase microreactors within the phase of the emulsion. Optionally, the reaction mixture comprises a recombinase. Optionally, the reaction mixture comprises a plurality of different polynucleotides. Optionally, the reaction mixture comprises a plurality of supports. Optionally, the reaction mixture comprises any combination of a recombinase, a plurality of different polynucleotides, and/or a plurality of supports. Optionally, at least one support can be attached to the substantially monoclonal population of nucleic acids.

In some embodiments, the present disclosure generally relates to compositions comprising a reaction mixture comprising (i) a plurality of supports, (ii) a plurality of different polynucleotides, and (iii) a recombinase, the reaction mixture being comprised in a plurality of liquid-phase microreactors in an emulsion.

In some embodiments, the present disclosure generally relates to a composition comprising a reaction mixture comprising (i) a recombinase enzyme and (ii) a plurality of supports, at least one of which can be attached to a population of substantially monoclonal nucleic acids, wherein the reaction mixture is comprised in a plurality of liquid-phase microreactors in an emulsion.

Optionally, the emulsion comprises a hydrophilic phase.

Optionally, the emulsion comprises a hydrophilic phase dispersed in a hydrophobic phase. For example, the emulsion may comprise a water-in-oil emulsion.

In some embodiments, the hydrophilic phase comprises a plurality of microreactors.

Optionally, the reaction mixture is contained in a single reaction vessel.

Optionally, the sequences of the plurality of different polynucleotides may be the same or different.

Optionally, at least one of the plurality of supports is attached to a plurality of first primers (e.g., forward amplification primers).

Optionally, the reaction mixture further comprises a plurality of second primers (e.g., reverse amplification primers).

In some embodiments, at least one of the plurality of supports further comprises a plurality of second primers.

In some embodiments, at least one of the plurality of supports comprises a plurality of first and second primers.

In some embodiments, the first and second primers comprise the same sequence. In some embodiments, the first and second primers comprise different sequences.

In some embodiments, the support comprises beads, particles, planar surfaces, or the inner walls of a trough or tube.

In some embodiments, the reaction mixture further comprises a polymerase and a plurality of nucleotides.

In some embodiments, the present disclosure generally relates to compositions comprising emulsions. Optionally, the emulsion comprises a hydrophilic phase and a hydrophobic phase. Optionally, the emulsion comprises a hydrophilic phase dispersed in a hydrophobic phase. Optionally, the hydrophilic phase may comprise any combination of multiple polynucleotide templates, multiple supports, and/or a recombinase. Optionally, the hydrophilic phase may comprise a plurality of polynucleotide templates. Optionally, the hydrophilic phase may comprise a plurality of supports. Optionally, the hydrophilic phase may comprise a recombinase.

In some embodiments, the composition comprises an emulsion comprising a hydrophilic phase and a hydrophobic phase, wherein the hydrophilic phase comprises a plurality of polynucleotide templates, a plurality of supports, and a recombinase enzyme.

In some embodiments, the present disclosure relates generally to compositions comprising an emulsion comprising a hydrophilic phase dispersed in a hydrophobic phase. Optionally, the hydrophilic phase comprises a plurality of microreactors. Optionally, at least two microreactors in the plurality comprise different polynucleotide templates. Optionally, the sequences of the different polynucleotide templates are the same or different. Optionally, the first microreactor comprises a first polynucleotide template and the second microreactor comprises a second polynucleotide template. Optionally, the first and second polynucleotide templates comprise the same or different sequences. Optionally, at least two microreactors in the plurality comprise a recombinase.

In some embodiments, the composition comprises an emulsion comprising a hydrophilic phase dispersed in a hydrophobic phase, wherein the hydrophilic phase comprises a plurality of microreactors, at least two microreactors in the plurality comprising different polynucleotide templates and a recombinase enzyme.

In some embodiments, the hydrophilic phase comprises a plurality of aqueous microreactors, at least two of the microreactors each comprising a different polynucleotide template, a support, and a recombinase.

Optionally, the first microreactor comprises a first polynucleotide template and the second microreactor comprises a second polynucleotide template. Optionally, the first and second polynucleotide templates comprise the same or different sequences.

In some embodiments, the first and second primers comprise the same sequence.

In some embodiments, the first and second primers comprise different sequences. In some embodiments, the hydrophilic phase further comprises a polymerase.

In some embodiments, the polymerase includes a strand displacing polymerase. In some embodiments, the hydrophilic phase comprises a nucleotide.

In some embodiments, the present disclosure relates generally to methods (and related compositions and systems) for nucleic acid synthesis, comprising: (a) forming a reaction mixture; and (b) subjecting the reaction mixture to amplification conditions. Optionally, the reaction mixture is contained within the hydrophilic phase of the emulsion. Optionally, the emulsion comprises a hydrophilic phase and a hydrophobic phase. Optionally, the emulsion comprises a hydrophilic phase dispersed in a hydrophobic phase. Optionally, the reaction mixture comprises any combination of multiple supports, multiple different polynucleotides, and/or recombinase enzymes. Optionally, the reaction mixture comprises a plurality of supports. Optionally, the reaction mixture comprises a plurality of different polynucleotides. Optionally, the sequences of the different polynucleotide templates are the same or different. Optionally, the first microreactor comprises a first polynucleotide template and the second microreactor comprises a second polynucleotide template. Optionally, the first and second polynucleotide templates comprise the same or different sequences. Optionally, the reaction mixture comprises a recombinase. Optionally, the amplification conditions comprise isothermal or thermocycling temperature conditions. Optionally, the method further comprises forming at least two supports and subjecting the emulsion to amplification conditions resulting in the formation of a plurality of supports, wherein at least two of the supports are each independently attached to the substantially monoclonal population of nucleic acids.

In some embodiments, the present disclosure relates generally to methods (and related compositions and systems) for nucleic acid synthesis, comprising: (a) Forming a reaction mixture comprising a plurality of supports, a plurality of different polynucleotides, and a recombinase, the reaction mixture being contained within a hydrophilic phase of an emulsion; and (b) subjecting the emulsion comprising the reaction mixture to isothermal amplification conditions, thereby generating a plurality of supports and a population of substantially monoclonal nucleic acids attached thereto.

In some embodiments, the emulsion comprises a water-in-oil emulsion. In some embodiments, the liquid-phase microreactor comprises a hydrophilic phase. In some embodiments, the emulsion comprises a hydrophilic phase dispersed in a hydrophobic phase. In some embodiments, the reaction mixture is formed in a single reaction vessel. Optionally, the sequences of the plurality of different polynucleotide templates are the same or different. Optionally, the first polynucleotide template comprises a first sequence and the second polynucleotide template comprises a second sequence. Optionally, the first and second polynucleotide template sequences are the same or different. Optionally, at least one of the plurality of supports is attached to a plurality of first primers (e.g., forward amplification primers). Optionally, the reaction mixture further comprises a plurality of second primers (e.g., reverse amplification primers). In some embodiments, at least one of the plurality of supports further comprises a plurality of second primers. In some embodiments, at least one of the plurality of supports comprises a plurality of first and second primers. In some embodiments, the first and second primers comprise the same sequence. In some embodiments, the first and second primers comprise different sequences. In some embodiments, the method of nucleic acid synthesis further comprises recovering at least some of the supports attached to the substantially monoclonal population of nucleic acids from the reaction mixture. In some embodiments, the nucleic acid synthesis method further comprises placing at least some of the supports attached to the substantially monoclonal population of nucleic acids on a surface. In some embodiments, the nucleic acid synthesis method further comprises forming an array by placing at least some of the supports attached to the population of substantially monoclonal nucleic acids on a surface. In some embodiments, the nucleic acid synthesis method further comprises sequencing at least one substantially monoclonal nucleic acid population attached to the support. In some embodiments, the support comprises beads, particles, planar surfaces, or the inner walls of a trough or tube. In some embodiments, the reaction mixture further comprises a polymerase and a plurality of nucleotides. In some embodiments, the polymerase includes a strand displacing polymerase.

In some embodiments, the method for nucleic acid synthesis comprises forming an emulsion. Optionally, the emulsion comprises a hydrophilic phase and a hydrophobic phase. Optionally, the emulsion comprises a hydrophilic phase dispersed in a hydrophobic phase. Optionally, the hydrophilic phase comprises a plurality of microreactors. Optionally, at least two microreactors in the plurality comprise an individual polynucleotide template. Optionally, at least two microreactors in the plurality comprise different polynucleotide templates. Optionally, the first microreactor comprises a first polynucleotide template and the second microreactor comprises a second polynucleotide template. Optionally, the first and second polynucleotide templates have the same or different sequences. Optionally, at least two microreactors in the plurality comprise a recombinase.

In some embodiments, the present disclosure relates generally to methods (and related compositions and systems) for nucleic acid synthesis, comprising: forming an emulsion comprising a hydrophilic phase dispersed in a hydrophobic phase, the hydrophilic phase comprising a plurality of microreactors, at least two microreactors in the plurality comprising different polynucleotide templates and a recombinase.

In some embodiments, the emulsion comprises a water-in-oil emulsion. In some embodiments, the hydrophilic phase further comprises a polymerase. In some embodiments, the polymerase is a strand displacing polymerase. In some embodiments, the hydrophilic phase comprises a nucleotide. In some embodiments, the emulsion is formed in a single reaction vessel. Optionally, the sequences of the different polynucleotide templates are the same or different. Optionally, the first microreactor comprises a first polynucleotide template and the second microreactor comprises a second polynucleotide template. Optionally, the first and second polynucleotide templates comprise the same or different sequences. In some embodiments, at least two microreactors in a plurality comprise a plurality of supports. Optionally, at least one of the plurality of supports is attached to a plurality of first primers (e.g., forward amplification primers). Optionally, the reaction mixture further comprises a plurality of second primers (e.g., reverse amplification primers). In some embodiments, at least one of the plurality of supports further comprises a plurality of second primers. In some embodiments, at least one of the plurality of supports comprises a plurality of first and second primers. In some embodiments, the first and second primers comprise the same sequence. In some embodiments, the first and second primers comprise different sequences. In some embodiments, the hydrophilic phase comprises a reaction mixture. In some embodiments, the reaction mixture comprises a plurality of polynucleotide templates, a plurality of supports, and a recombinase. In some embodiments, the method for nucleic acid synthesis further comprises subjecting the emulsion (e.g., comprising a reaction mixture) to isothermal amplification conditions, thereby generating a plurality of substantially monoclonal nucleic acid populations. In some embodiments, a plurality of substantially monoclonal nucleic acid populations are attached to a plurality of supports. In some embodiments, the method of nucleic acid synthesis further comprises recovering at least some of the supports attached to the substantially monoclonal population of nucleic acids from the reaction mixture. In some embodiments, the method of nucleic acid synthesis further comprises placing at least some of the supports attached to the population of substantially monoclonal nucleic acids on a surface. In some embodiments, the nucleic acid synthesis method further comprises forming an array by placing at least some of the supports attached to the population of substantially monoclonal nucleic acids on a surface. In some embodiments, the nucleic acid synthesis method further comprises sequencing at least one substantially monoclonal population of nucleic acids attached to the support. In some embodiments, the support comprises beads, particles, planar surfaces, or the inner wall of a trough or tube. In some embodiments, the reaction mixture further comprises a polymerase and a plurality of nucleotides. In some embodiments, the polymerase includes a strand displacing polymerase.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

All documents and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, treatises, and internet pages, are expressly incorporated by reference in their entirety for any purpose. Where the definitions of terms in the incorporated references appear to differ from those provided in the present teachings, the definitions provided in the present teachings shall control.

It will be understood that there is an implicit "about" preceding the temperature, concentration, number, etc. discussed in the present teachings, and thus slight and insubstantial differences are within the scope of the present teachings.

Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

The terms "comprising," "including," "containing," "having," "with," and "having" are not intended to be limiting.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention.

Unless defined otherwise, scientific and technical terms used in connection with the present teachings described herein will have the meaning commonly understood by one of ordinary skill in the art. Generally, the nomenclature used, and the techniques thereof, in connection with cell and tissue culture, molecular biology, and protein and oligo-or polynucleotide chemistry and hybridization are those well known and commonly employed in the art. Standard techniques are used, for example, for nucleic acid purification and preparation, chemical analysis, recombinant nucleic acid and oligonucleotide synthesis. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions or as commonly practiced in the art or as described herein. The techniques and methods described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., sambrook et al, molecular Cloning: A Laboratory Manual (Third ed., cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y. 2000). The nomenclature used in connection with, and the laboratory procedures and techniques described herein, are those well known and commonly employed in the art.

As used in accordance with the exemplary embodiments provided herein, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

as used herein, the term "amplification" and variants thereof include any process for producing multiple copies or complements of at least some portion of a polynucleotide, which is often referred to as a "template". The template polynucleotide may be single-stranded or double-stranded. Amplification of a given template may result in the production of a population of polynucleotide amplification products, collectively referred to as "amplicons. The polynucleotide of the amplicon may be single stranded or double stranded or a mixture of the two. Typically, the template will comprise the target sequence and the resulting amplicon will comprise a polynucleotide having a sequence that is substantially identical or substantially complementary to the target sequence. In some embodiments, the polynucleotides of a particular amplicon are substantially identical or substantially complementary to each other; alternatively, in some embodiments, the polynucleotides within a given amplicon may have different nucleotide sequences from one another. Amplification can be performed in a linear or exponential manner, and can include repeated and sequential replication of a given template to form two or more amplification products. Some typical amplification reactions involve successive and repeated cycles of template-based nucleic acid synthesis, resulting in the formation of multiple sub-polynucleotides that comprise at least some portion of the nucleotide sequence of the template and share at least some degree of nucleotide sequence identity (or complementarity) with the template. In some embodiments, each nucleic acid synthesis (which may be referred to as a "cycle" of amplification) comprises a primer annealing and primer extension step; optionally, an additional denaturation step may also be included in which the template is partially or fully denatured. In some embodiments, one amplification round comprises a given number of repetitions of a single amplification cycle. For example, an amplification round may include 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100 or more repetitions of a particular cycle. In an exemplary embodiment, amplification includes any reaction in which a particular polynucleotide template undergoes two consecutive cycles of nucleic acid synthesis. Synthesis may include template-dependent nucleic acid synthesis. Each cycle of nucleic acid synthesis optionally includes a single primer annealing step and a single extension step. In some embodiments, the amplifying comprises isothermal amplification.

As used herein, the term "contacting" and variations thereof, when used with respect to any group of components, includes any process in which the components to be contacted are mixed into the same mixture (e.g., added to the same compartment or solution), and does not necessarily require actual physical contact between the components. The components may be contacted in any order or in any combination (or sub-combination), and may include situations where: wherein one or some of said components are subsequently removed from the mixture, optionally before the addition of other of said components. For example, "contacting a with B and C" includes any and all of the following: (ii) (i) mixing a with C, followed by adding B to the mixture; (ii) mixing a and B into the mixture; removing B from the mixture, and subsequently adding C to the mixture; and (iii) adding A to the mixture of B and C. "contacting the template with the reaction mixture" includes any or all of the following situations: (i) Contacting a template with a first component of a reaction mixture to produce a mixture; subsequently adding the other components of the reaction mixture to the mixture in any order or combination; and (ii) the reaction mixture has been completely formed prior to mixing with the template.

As used herein, the term "support" and variants thereof include any solid or semi-solid article on which a reagent, such as a nucleic acid, can be immobilized.

As used herein, the term "isothermal" and variations thereof, when used in reference to any reaction condition, process, or method, includes conditions, processes, and methods that are carried out under substantially isothermal conditions. Substantially isothermal conditions include any condition wherein the temperature is limited to a limited range. In exemplary embodiments, the temperature does not vary by more than 20 ℃, typically by more than 10 ℃, 5 ℃ or 2 ℃. Isothermal amplification includes any amplification reaction in which at least two successive nucleic acid synthesis cycles are carried out under substantially isothermal conditions, and includes amplification reactions in which the temperature does not vary by more than 20 ℃, 10 ℃, 5 ℃, or 2 ℃ during the duration of at least two successive nucleic acid synthesis cycles, although in which the temperature may vary by more than 20 ℃ during other portions of the amplification process, including during other nucleic acid synthesis cycles. Optionally, the temperature is maintained at or about 50 ℃, 55 ℃, 60 ℃, 65 ℃, or 70 ℃ for at least about 10, 15, 20, 30, 45, 60, or 120 minutes in an isothermal reaction (including isothermal amplification). Optionally, any temperature change during one or more amplification cycles (e.g., 1, 5, 10, 20, or all amplification cycles performed) does not exceed 20 ℃, optionally within 10 ℃, e.g., within 5 ℃ or 2 ℃. In some embodiments, isothermal amplification may include thermal cycling, where the temperature variation is within an isothermal range. In an example, the temperature change between the denaturation step and another step, such as annealing and/or extension, is limited. In an example, the difference between the denaturation temperature and the annealing or extension temperature is no more than 20 ℃, optionally within 10 ℃, for example within 5 ℃ or 2 ℃, for one or more amplification cycles. Optionally, the temperature change is limited over at least 5, 10, 15, 20, 30, 35 or substantially all cycles of amplification.

As used herein, the term "sequencing" and variants thereof includes obtaining sequence information from a nucleic acid strand, typically by determining information for at least some nucleotides within the nucleic acid molecule, including its nucleobase component. While in some embodiments, "sequencing" a given region of a nucleic acid molecule includes identifying each nucleotide within the sequenced region, "sequencing" may also include methods in which information for one or more nucleotides is determined while information for some nucleotides remains undetermined or incorrectly determined.

As used herein, the terms "identity" and "identical," and variants thereof, when used with respect to two or more nucleic acid sequences, refer to sequence similarity of two or more sequences (e.g., nucleotide or polypeptide sequences). In the context of two or more homologous sequences, the percent identity or homology of a sequence or subsequence thereof means the ratio of all identical monomer units (e.g., nucleotides or amino acids) (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 95%, or 99% identity). Percent identity can be percent identity over a particular region when comparing and aligning the maximum correspondence over the comparison window or designated region as measured by using the BLAST or BLAST2.0 sequence comparison algorithm with default parameters described below or by manual alignment and visual inspection. Sequences are considered "substantially identical" when there is at least 85% identity at the amino acid level or at the nucleotide level. Preferably, there is identity over a region of at least about 25, 50 or 100 residues in length, or over the entire length of at least one of the sequences being compared. Typical algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST2.0 algorithms described in Altschul et al, nuc. Other methods include the algorithms of Smith & Waterman, adv.Appl.Math.2:482 (1981) and Needleman & Wunsch, J.mol.biol.48:443 (1970), among others. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent hybridization conditions.

As used herein with respect to two or more polynucleotides, the term "complementary" and variants thereof refers to a polynucleotide comprising any nucleic acid sequence that can undergo cumulative base pairing (as in a hybridized duplex) in antiparallel directions at two or more individual corresponding sites. Optionally, there may be "complete" or "entire" complementarity between the first and second nucleic acid sequences, where each nucleotide in the first nucleic acid sequence may undergo stable base-pairing interactions with the polynucleotide in the corresponding antiparallel position on the second nucleic acid sequence (however, the term "complementary" may itself include nucleic acid sequences that are not completely complementary over the entire length); "partial" complementarity describes nucleic acid sequences in which at least 20% but less than 100% of the residues of the nucleic acid sequence are complementary to residues in other nucleic acid sequences. In some embodiments, at least 50% but less than 100% of the residues of the nucleic acid sequence are complementary to residues in other nucleic acid sequences. In some embodiments, at least 70%, 80%, 90%, or 95% but less than 100% of the residues of the nucleic acid sequence are complementary to residues in other nucleic acid sequences. Sequences are considered "substantially complementary" when at least 85% of the residues of one nucleic acid sequence are complementary to residues in other nucleic acid sequences. "non-complementary" describes nucleic acid sequences in which less than 20% of the residues of the nucleic acid sequence are complementary to residues in other nucleic acid sequences. A "mismatch" occurs at any position where two opposing nucleotides are not complementary. Complementary nucleotides include nucleotides that are effectively incorporated opposite each other by a DNA polymerase during DNA replication under physiological conditions. In typical embodiments, complementary nucleotides can form base pairs with each other, such as A-T/U and G-C base pairs formed by hydrogen bonding of a particular Watson-Crick type between nucleotides and/or nucleobases of a polynucleotide in positions that are anti-parallel to each other. Other artificial base pair complementarity may be based on other types of hydrogen bonding and/or hydrophobic phases of bases and/or shape complementarity between bases.

As used herein with respect to any polynucleotide or nucleic acid molecule, the term "double-stranded" and variants thereof refer to any polynucleotide or nucleic acid molecule having one or more strands and including regions (e.g., as in a nucleic acid duplex) comprising nucleotide residues that base-pair with the nucleotide residues. Optionally, a double-stranded polynucleotide (or nucleic acid molecule) may be "entirely" or "entirely" double-stranded, such that each nucleotide residue in the polynucleotide (or nucleic acid molecule) base pairs with another corresponding nucleotide residue. In some embodiments, a double-stranded polynucleotide comprises one or more single-stranded regions comprising nucleotide residues that do not base pair with any other nucleotide residues. In some embodiments, at least 51%, 75%, 85%, 95%, 97%, or 99% of the nucleotide residues in a double-stranded polynucleotide (or nucleic acid molecule) are base paired with other nucleotide residues. In some embodiments, a double-stranded polynucleotide (or nucleic acid molecule) comprises two strands that are not covalently linked to each other; alternatively, a double-stranded polynucleotide (or nucleic acid molecule) comprises a single strand that base pairs with itself (e.g., as in a hairpin oligonucleotide) over at least some portion of its length. A polynucleotide is considered "substantially double-stranded" when at least 85% of its nucleotide residues are base-paired with corresponding nucleotide residues. Two nucleic acid sequences are considered "double-stranded" when residues from one nucleic acid sequence base-pair with corresponding residues in the other nucleic acid sequence. In some embodiments, base pairing can occur according to some conventional pairing paradigm, such as A-T/U and G-C base pairs formed by hydrogen bonding of a particular Watson-Crick type between nucleobases at nucleotide and/or polynucleotide positions that are antiparallel to each other; in other embodiments, base pairing can occur by any other paradigm in which base pairing occurs according to established and predictable rules.

As used herein, the term "single-stranded" and variants thereof, when used with respect to any polynucleotide or nucleic acid molecule, refers to any polynucleotide or nucleic acid molecule that includes a region of nucleotide residues that are not base-paired with any nucleotide residue. Optionally, a single-stranded polynucleotide (or nucleic acid molecule) may be "entirely" or "entirely" single-stranded, such that each nucleotide residue in the polynucleotide (or nucleic acid molecule) is not base-paired with any other nucleotide residue. In some embodiments, the single-stranded polynucleotide comprises one or more double-stranded regions comprising nucleotide residues that base-pair with nucleotide residues. In some embodiments, at least 51%, 75%, 85%, 95%, 97%, or 99% of the nucleotide residues in the single-stranded polynucleotide (or nucleic acid molecule) are not base paired with other nucleotide residues. A polynucleotide is considered "substantially single-stranded" when at least 85% of its nucleotide residues are not base-paired with nucleotide residues.

As used herein, the term "denature" and variants thereof, when used with respect to any double-stranded polynucleotide molecule or double-stranded polynucleotide sequence, includes any process in which base pairing between nucleotides within opposite strands of a double-stranded molecule or double-stranded sequence is disrupted. Typically, denaturation involves rendering at least some portion or region of both strands of a double-stranded polynucleotide molecule or sequence single-stranded. In some embodiments, denaturing comprises separating at least some portion or region of both strands of the double-stranded polynucleotide molecule or sequence from each other. Typically, the denatured region or portion is then capable of hybridizing to another polynucleotide molecule or sequence. Optionally, there may be "complete" or "entire" denaturation of the double-stranded polynucleotide molecule or sequence. Fully denaturing conditions are conditions that, for example, result in complete separation of a significant portion (e.g., more than 10%, 20%, 30%, 40%, or 50%) of a plurality of strands from their extended and/or full-length complement. Typically, complete or complete denaturation disrupts all base pairing of the nucleotides of the two strands with one another. Similarly, a nucleic acid sample is considered to be completely denatured, optionally when more than 80% or 90% of the individual molecules of the sample lack any double-stranded properties (or lack any hybridization to a complementary strand).

Alternatively, a double-stranded polynucleotide molecule or sequence may be partially or incompletely denatured. A given nucleic acid molecule is considered to be partially denatured when at least a portion of one strand of the nucleic acid remains hybridized to the complementary strand, while another portion is in an unhybridized state (even in the presence of the complementary strand). The unhybridized portion optionally has a length of at least 5, 7, 8, 10, 12, 15, 17, 20, or 50 nucleotides. The hybridizing portion optionally has a length of at least 5, 7, 8, 10, 12, 15, 17, 20, or 50 nucleotides. Partial denaturation includes situations in which some, but not all, of the nucleotides of one strand or sequence base pair with some of the nucleotides of other strands or sequences within a double-stranded polynucleotide. In some embodiments, at least 20% but less than 100% of the nucleotide residues of one strand of a partially denatured polynucleotide (or sequence) are not base paired with nucleotide residues within the opposite strand. Under exemplary conditions, at least 50% of the nucleotide residues within a double-stranded polynucleotide molecule (or double-stranded polynucleotide sequence) are in single-stranded (or unhybridized) form, but less than 20% or 10% of the residues are double-stranded.

Optionally, a nucleic acid sample can be considered partially denatured when a substantial portion (e.g., more than 20%, 30%, 50%, or 70%) of the individual nucleic acid molecules of the sample are in a partially hybridized state. Optionally, less than a substantial amount of the individual nucleic acid molecules in the sample are completely denatured, e.g., no more than 5%, 10%, 20%, 30%, or 50% of the nucleic acid molecules in the sample. Under exemplary conditions, at least 50% of the nucleotide molecules of the sample are partially denatured, but less than 20% or 10% are fully denatured. In other cases, at least 30% of the nucleotide molecules of the sample are partially denatured, but less than 10% or 5% are fully denatured. Similarly, a nucleic acid sample may be considered non-denatured when a minority of the individual nucleic acid molecules in the sample are partially or fully denatured.

In embodiments, the partially denaturing conditions are achieved by maintaining the duplex in an appropriate temperature range. For example, the nucleic acid is maintained at a temperature sufficiently elevated to achieve some thermal denaturation (e.g., greater than 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃ or 70 ℃) but not high enough to achieve complete thermal denaturation (e.g., less than 95 ℃ or 90 ℃ or 85 ℃ or 80 ℃ or 75 ℃)

The temperature of (2). In embodiments, the nucleic acid is partially denatured using substantially isothermal conditions.

Partial denaturation can also be achieved by other methods, such as chemical methods using appropriately adjusted concentrations of chemical denaturants such as urea or formamide or using high or low pH (e.g., pH between 4-6 or 8-9). In embodiments, recombinase-polymerase amplification (RPA) is used

To achieve partial denaturation and amplification. Exemplary RPA methods are described herein.

In some embodiments, complete or partial denaturation is achieved by treating the double-stranded polynucleotide sequence to be denatured with an appropriate denaturing agent. For example, a double-stranded polynucleotide may be subjected to thermal denaturation (also interchangeably referred to as heat denaturation) by raising the temperature to a point where a desired level of denaturation is achieved. In some embodiments, complete thermal denaturation of a double-stranded polynucleotide, the temperature can be adjusted to achieve complete separation of the two strands of the polynucleotide, such that at least 90% of the strands are in single-stranded form throughout their length. In some embodiments, complete thermal denaturation of a polynucleotide molecule (or polynucleotide sequence) is achieved by exposing the polynucleotide molecule (or sequence) to a temperature at least 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25, 30 ℃, 50 ℃ or 100 ℃ above the calculated or predicted melting temperature (Tm) of the polynucleotide molecule or sequence.

Alternatively, chemical denaturation can be achieved by contacting the double-stranded polynucleotide to be denatured with a suitable chemical denaturant, such as a strong base, a strong acid, a chaotropic agent, and the like, and may include, for example, naOH, urea, or a guanidine-containing compound. In some embodiments, partial or complete denaturation is achieved by exposure to appropriately adjusted concentrations of chemical denaturants such as urea or formamide or the use of high or low pH (e.g., pH between 4-6 or 8-9). In embodiments, partial denaturation and amplification is achieved using recombinase-polymerase amplification (RPA). Exemplary RPA methods are described herein.

The terms "melting temperature", "Tm" or "T" when used in reference to a given polynucleotide (or a given target sequence within a polynucleotide) _m "and variants thereof generally refer to a temperature at which 50% of a given polynucleotide (or a given target sequence) is present in double-stranded form and 50% is single-stranded under a defined set of conditions. In some embodiments, the defined set of conditions may include defined parameters indicative of ionic strength and/or pH in the aqueous reaction conditions. The defined conditions can be adjusted by varying the concentration of the salt (e.g. sodium), the temperature, the pH, the buffer and/or the formamide. Generally, the calculated thermal melt temperature may be T _m About 5-30 ℃ or T below _m About 5-25 ℃ or T below _m Below about 5-20 ℃ or T _m About 5-15 ℃ or T below _m About 5-10 ℃ below. For calculating T _m The method of (A) is well known and can be found in Sambrook (1989 in) molecular cloning ^nd edition, volumes 1-3; wetmur 1966, j.mol.biol., 31; wetmur 1991Critical Reviews in Biochemistry and Molecular biology, 26. For calculating T for hybridizing or denaturing nucleic acids _m Other sources of (A) include OligoAnalyze (from Integrated DNA Technologies) and Primer3 (published by the Whitehead Institute for Biomedical Research). In some embodiments, the term "melting temperature"),

"Tm" and "T _m "and variants thereof include the actual Tm (as empirically measured using established conditions) or the predicted or calculated Tm for a given polynucleotide (or target sequence). In some embodiments, can be throughThe Tm of a template is predicted or calculated without using the sequence of the template, assuming that the template contains a proportion of 4 common nucleotides (a, C, G and T) and has a length (or average length in the case of a population of templates). For example, it can be assumed that the template population migrating trailed on the gel contains 25% each of a, C, G or T and has an average length of 200, 300, 400 base pairs.

As used herein with respect to chemical moieties, the term "label" and variations thereof include any composition comprising an optically or non-optically detectable moiety, wherein the detectable moiety has been artificially added, linked or attached to an unlabeled second moiety by chemical treatment. Typically, the user (or upstream supplier) makes the addition of the indicia for the purpose of enhancing the detectability of the second portion. Optically or non-optically detectable components of the composition (e.g., hydrogen ions and amino acids found in typical DNA molecules, RNA molecules, or nucleotides within a natural cell) that are already present in a naturally occurring form of the composition are not labels for purposes of this disclosure. Some typical labels include fluorescent moieties and dyes.

A nucleic acid is considered to be immobilized if it is attached to the support in a manner that is substantially stable at least during selected conditions, e.g., during an amplification reaction. Attachment may be by any mechanism, including but not limited to non-covalent bonding, ionic interaction, covalent attachment. A first nucleic acid may also be considered to be immobilized to a support during amplification if it hybridizes to a second nucleic acid immobilized to the support, if the amplification conditions are such that a substantial amount of the first and second nucleic acids are associated with or linked to each other at any or all of the time during amplification. For example, the first and second nucleic acids may associate together by hybridization including Watson-Crick base pairing or hydrogen bonding. In examples, the amplification conditions are selected to allow at least 50%, 80%, 90%, 95%, or 99% of the first nucleic acid to remain hybridized to the second nucleic acid, or vice versa. A nucleic acid can be considered to be unfixed or non-immobilized if it is not directly or indirectly attached or associated with the support.

A vehicle may be considered flowable under selected conditions if it is a fluid vehicle, at least temporarily, under the selected conditions, which does not substantially or completely inhibit or hinder the transfer or movement of the unfixed molecules. The non-immobilized molecule is not itself immobilized to a solid support or surface or associated with another immobilized molecule. In embodiments, the non-immobilized molecule is a solute (e.g., a nucleic acid) that passes through a flowable vehicle. An exemplary transfer or movement in a vehicle can be by diffusion, convection, turbulence, agitation, brownian motion, advection, current, or other molecular motion within a liquid from any first point in a continuous phase to any other point in a fluid communication or the same continuous phase. For example, in a flowable medium, a significant amount of non-immobilized nucleic acid is transferred from one fixation site to another fixation site within the same continuous phase of the flowable medium or in fluid communication with the first fixation site. Optionally, the rate of transfer or movement of the nucleic acid in the vehicle is comparable to the rate of transfer or movement of the nucleic acid in water. In some examples, the conditions selected are those experienced by the vector during amplification. The conditions selected may or may not allow the flowable medium to remain substantially stationary. Conditions may or may not subject the flowable vehicle to effective mixing, stirring, or shaking. The vehicle is optionally at least temporarily flowable during amplification. For example, the vehicle is flowable under the selected at least one pre-amplification and/or amplification condition. Optionally, the flowable medium does not substantially prevent mixing of different unfixed nucleic acids or transfer of unfixed nucleic acids between different regions of the continuous phase of the flowable medium. The movement or transfer of nucleic acids can be caused, for example, by diffusion or convection. The vehicle is optionally considered to be non-flowable if the non-immobilized nucleic acids cannot propagate or move between different immobilization sites or throughout the continuous phase after amplification. Generally, the flowable vehicle does not substantially confine the non-immobilized nucleic acids (e.g., templates or amplicons) within the effective area of the reaction volume or in a fixed location during the amplification period. Optionally, the flowable medium may be rendered non-flowable by a variety of methods or by changing the conditions of the flowable medium. Optionally, the vehicle is flowable if it is liquid or not semi-solid. The vehicle is considered flowable if its fluidity is comparable to that of pure water. In other embodiments, the vehicle may be considered flowable if it is a fluid that is substantially free of polymers, or if its viscosity coefficient is similar to that of pure water.

As will be understood by those skilled in the art, reference to a template, initiator oligonucleotide, extension probe, primer, etc., may refer to a population or pool of nucleic acid molecules that are substantially identical within the relevant portion rather than a single molecule. For example, "template" may refer to a plurality of substantially identical template molecules; "Probe" can refer to a plurality of substantially identical probe molecules, and the like. In the case of probes that are degenerate at one or more positions, it will be understood that the sequence of the probe molecules comprising a particular probe will differ at degenerate positions, i.e., the sequence of the probe molecules comprising a particular probe may be substantially identical only at non-degenerate positions. These terms within this application are intended to provide support for groups or molecules. When referring to a single nucleic acid molecule (i.e., a molecule), the terms "template molecule", "probe molecule", "primer molecule", and the like, may be used instead. In certain instances, the plural nature of a population of substantially identical nucleic acid molecules will be explicitly indicated.

"template," "oligonucleotide," "probe," "primer," "template," "nucleic acid," and the like are intended to be interchangeable terms herein. These terms refer to polynucleotides, which are not necessarily limited to any length or function. The same nucleic acid can be considered by context as a "template", "probe" or "primer", and can switch between these roles over time. A "polynucleotide," also referred to as a "nucleic acid," is a linear polymer of two or more nucleotides, or variants or functional fragments thereof, linked by covalent internucleoside linkages. In naturally occurring examples of these, the internucleoside linkage is typically a phosphodiester linkage. However, other examples optionally include other internucleoside linkages, such as a phosphorothioate linkage, and may or may not include a phosphate group. Polynucleotides include double-and single-stranded DNA and double-and single-stranded RNA, DNA: RNA hybrids, peptide Nucleic Acids (PNA) and hybrids between PNA and DNA or RNA, and may also include known types of modifications. The polynucleotide may optionally be attached via the 5 'or 3' end to one or more non-nucleotide moieties such as labels and other small molecules, macromolecules such as proteins, lipids, sugars, and solid or semi-solid supports. Labels include any moiety that can be detected using a selected detection method, and thus render a linked nucleotide or polynucleotide similarly detectable using a selected detection method. Optionally, the label emits electromagnetic radiation that is optically detectable or visible. In some cases, the nucleotide or polynucleotide is not linked to a label, but the presence of the nucleotide or polynucleotide is detected directly. "nucleotide" refers to a nucleotide, nucleoside, or analog thereof. Optionally, the nucleotide is an N-or C-glycoside of a purine or pyrimidine base. (e.g., a deoxyribonucleoside comprising 2-deoxy-D-ribose or a ribonucleoside comprising D-ribose). Examples of other analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O-methyl ribonucleotide. Reference to a nucleic acid by any of these terms should not be construed to mean that the nucleic acid has any particular activity, function, or property. For example, the word "template" does not mean that the "template" is being copied by a polymerase or that the template cannot be used as a "primer" or "probe".

It will be appreciated that in certain examples, the nucleic acid reagents involved in amplification, such as templates, probes, primers, etc., may be part of a larger nucleic acid molecule that also comprises another part that does not have the same function. Optionally, the other moiety does not have any template, probe or primer function. In some examples, a nucleic acid that substantially hybridizes to an optionally immobilized primer (e.g., at an immobilization site) is considered a "template". Any one or more nucleic acid reagents (template, immobilized strand, immobilized or non-immobilized primers, etc.) that participate in template walking can be generated from other nucleic acids prior to or during amplification. A nucleic acid agent is optionally generated from (and need not be identical to) the input nucleic acid by one or more modifications to the nucleic acid initially introduced into the template walking vehicle. The input nucleic acid may, for example, be subjected to restriction digestion, ligation, one or more amplification cycles, denaturation, mutation, or the like to produce a nucleic acid that serves as a template, primer, or the like during amplification or further amplification. For example, a double-stranded input nucleic acid can be denatured to produce a first single-stranded nucleic acid, which is optionally used to produce a second complementary strand. If so desired, the first single-stranded nucleic acid may be considered a "template" for the purposes of my party herein. Alternatively, the second complementary strand generated from the first single-stranded nucleic acid may be considered a "template" for the purposes of my party herein. In another example, the template is derived from and not necessarily identical to the input nucleic acid. For example, the template may comprise additional sequences not present in the input nucleic acid. In embodiments, the template may be an amplicon generated from the input nucleic acid using one or more primers having a 5' overhang that is not complementary to the input nucleic acid.

As used herein with respect to two or more polynucleotides, the term "hybridize" and variants thereof refer to any process in which any one or more nucleic acid sequences (each sequence comprising a stretch of contiguous nucleotide residues) within the polynucleotide undergoes base pairing (e.g., as in a hybridized nucleic acid duplex) at two or more individually corresponding positions. Optionally, there may be "complete" or "entire" hybridization between the first and second nucleic acid sequences, wherein each nucleotide residue in the first nucleic acid sequence may undergo base pairing interaction with a corresponding nucleotide in an inverted parallel position on the second nucleic acid sequence. In some embodiments, hybridization may include base pairing between two or more nucleic acid sequences that are not fully complementary or base paired throughout their length. For example, "partial" hybridization occurs when two nucleic acid sequences undergo base pairing in which at least 20% but less than 100% of the residues of one nucleic acid sequence base-pair with residues in the other nucleic acid sequence. In some embodiments, hybridization comprises base pairing between two nucleic acid sequences, wherein at least 50% but less than 100% of the residues of one nucleic acid sequence base pair with corresponding residues in the other nucleic acid sequence. In some embodiments, at least 70%, 80%, 90%, or 95% but less than 100% of the residues of one nucleic acid sequence are base paired with corresponding residues in other nucleic acid sequences. Two nucleic acid sequences are said to be "substantially hybridized" when at least 85% of the residues of one nucleic acid sequence are base-paired with corresponding residues in the other nucleic acid sequence. In cases where one nucleic acid molecule is substantially longer than the other (or where two nucleic acid molecules comprise regions of substantial complementarity and substantial non-complementarity), two nucleic acid molecules may be described as "hybridized" even when portions of one or both nucleic acid molecules may remain unhybridized. "unhybridized" describes nucleic acid sequences in which less than 20% of the residues of one nucleic acid sequence base pair with residues in other nucleic acid sequences. In some embodiments, base pairing can occur according to some conventional pairing paradigm, such as A-T/U and G-C base pairs formed by hydrogen bonding of a particular Watson-Crick type between nucleobases at nucleotide and/or polynucleotide positions that are antiparallel to each other; in other embodiments, base pairing can occur by any other paradigm in which base pairing occurs according to established and predictable rules.

Hybridization of two or more polynucleotides can occur whenever the two or more polynucleotides are contacted under appropriate hybridization conditions. Hybridization conditions include any condition suitable for hybridization of nucleic acids; methods of performing hybridization and suitable conditions for hybridization are well known in the art. The stringency of hybridization can be affected by a number of parameters, including the degree of identity and/or complementarity between the polynucleotides (or any target sequence within a polynucleotide) to be hybridized; melting temperature of the polynucleotide and/or target sequence to be hybridized, referred to as "T _m "; parameters such as salt, buffer, pH, temperature, GC% content of the polynucleotide and primer and/or time. Generally, hybridization is favored at lower temperatures and/or increased salt concentrations and reduced organic solvent concentrations. High stringency hybridization conditions will generally require that between two target sequencesA higher degree of complementarity to allow hybridization to occur, while low stringency hybridization conditions will promote hybridization even when the two polynucleotides to be hybridized exhibit a low level of complementarity. Hybridization conditions may be applied during the hybridization step or the optional and subsequent washing steps or the hybridization and optional washing steps.

Examples of high stringency hybridization conditions include any one or more of the following: a salt (e.g., naCl) concentration of about 0.0165 to about 0.0330; melting temperature (T) of target sequence (or polynucleotide) to be hybridized _m ) A temperature of from about 5 ℃ to about 10 ℃; and/or formamide concentrations of about 50% or more. Generally, high stringency hybridization conditions allow for binding between sequences with high homology, e.g., > 95% identity or complementarity. In an exemplary embodiment of high stringency hybridization conditions, at about 42 ℃ comprises 25mM KPO ₄ (pH 7.4), 5 XSSC, 5 XDenhardt's solution, 50. Mu.g/mL denatured sonicated salmon sperm DNA, 50% formamide, 10% dextran sulfate, and 1-15ng/mL of a double stranded polynucleotide (or double stranded target sequence) in a hybridization solution, and washing at about 65 ℃ with a wash solution comprising 0.2 XSSC and 0.1% sodium dodecyl sulfate.

Examples of moderately stringent hybridization conditions include any one or more of the following: a salt (e.g., naCl) concentration of about 0.165 to about 0.330; melting temperature (T) of target sequence to be hybridized _m ) A temperature of from about 20 ℃ to about 29 ℃; and/or a formamide concentration of about 35% or less. Typically, such moderately stringent hybridization conditions allow for binding between sequences having high or moderate homology, e.g., > 80% identity or complementarity. In an exemplary embodiment of moderately stringent hybridization conditions, 25mM KPO is included at about 42 deg.C ₄ (pH 7.4), 5 XSSC, 5 XDenhart solution, 50. Mu.g/mL denatured sonicated salmon sperm DNA, 50% formamide, 10% dextran sulfate, and 1-15ng/mL of a hybridization solution of a double stranded polynucleotide (or double stranded target sequence), and washed at about 50 ℃ with a wash solution comprising 2 XSSC and 0.1% sodium lauryl sulfate.

Examples of low stringency hybridization conditions include any one or more of the following: salts of about 0.330 to about 0.825 (e.g., naC)l) concentration; melting temperature (T) of the target sequence to be hybridized _m ) A temperature of from about 40 ℃ to about 48 ℃; and/or formamide concentrations of about 25% or less. Typically, such low stringency conditions allow for binding between sequences with low homology, e.g., > 50% identity or complementarity.

Some exemplary conditions suitable for hybridization include incubating the polynucleotide to be hybridized in a solution with a sodium salt, such as NaCl, sodium citrate, and/or sodium phosphate. In some embodiments, the hybridization or wash solution may comprise about 10-75% formamide and/or about 0.01-0.7% Sodium Dodecyl Sulfate (SDS). In some embodiments, the hybridization solution can be a stringent hybridization solution, which can include any combination of 50% formamide, 5X SSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt's solution, 0.1% sds, and/or 10% dextran sulfate. In some embodiments, the hybridization or wash solution may comprise BSA (which may be bovine serum albumin). In some embodiments, hybridization or washing may be performed at a temperature in the range of about 20-25 ℃, or about 25-30 ℃, or about 30-35 ℃, or about 35-40 ℃, or about 40-45 ℃, or about 45-50 ℃, or about 50-55 ℃ or higher.

In some embodiments, hybridization or washing may be performed for a time period ranging from about 1 minute to about 10 minutes, or from about 10 minutes to about 20 minutes, or from about 20 minutes to about 30 minutes, or from about 30 minutes to about 40 minutes, or from about 40 minutes to about 50 minutes, or from about 50 minutes to about 60 minutes, or longer.

In some embodiments, hybridization or washing may be performed at a pH in the range of about 5 to 10, or about pH6 to 9, or about pH6.5 to 8, or about pH6.5 to 7.

In some embodiments, the term "monoclonal" and variants thereof are used to describe a population of polynucleotides in which a substantial portion (e.g., at least about 50%, typically at least 75%, 80%, 85%, 90%, 95%, or 99%) of the population members share at least 80% identity at the nucleotide sequence level. Typically, at least about 90%, typically at least about 95%, more typically at least about 99%, 99.5%, or 99.9%) of the population is produced by amplification or template-dependent replication of a particular polynucleotide sequence present in a substantial portion of the members of the population of monoclonal polynucleotides. All members of a monoclonal population need not be identical or complementary to each other. For example, different portions of a polynucleotide template may be amplified or replicated to produce members of the resulting monoclonal population; similarly, a certain number of "errors" and/or incomplete extensions may occur during amplification of the original template, thereby generating a monoclonal population whose individual members may exhibit sequence diversity among themselves. In some embodiments, at least 50% of the members of a monoclonal population are at least 80% identical to a reference nucleic acid sequence (i.e., a nucleic acid having a defined sequence used as a basis for sequence comparison). In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more members of the population comprise a sequence that is at least 80%, 85%, 90%, 95%, 97%, or 99% identical (or complementary) to a reference nucleic acid sequence. In some embodiments, low or insubstantial levels of mixing of non-homologous polynucleotides can occur during the nucleic acid amplification reactions described herein, and thus a substantially monoclonal population can comprise a small number of different polynucleotides (e.g., less than 30%, less than 20%, less than 10%, less than 5%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.001% of the different polynucleotides). As used herein, the phrase "substantially monoclonal" and variants thereof, when used with respect to one or more polynucleotide populations, refers to one or more polynucleotide populations comprising polynucleotides at least 80% identical to an original single template used as a basis for clonal amplification to produce a substantially monoclonal population.

In some embodiments, at least 80% of the members of the amplicon, typically at least 90%, more typically at least 95%, more typically at least 99% of the members of the amplicon, will share greater than 90% identity, typically greater than 95% identity, more typically greater than 97% and more typically greater than 99% identity with the polynucleotide template. Alternatively, the members of the amplicon may have greater than 90% complementarity to the original template, typically greater than 95% complementarity, more typically greater than 97% complementarity, more typically greater than 99% complementarity. In some embodiments, members of a substantially monoclonal population of nucleic acids can hybridize to each other under stringent hybridization conditions.

In some embodiments, an amplicon is referred to as "monoclonal" or "substantially monoclonal" if it contains sufficiently few polyclonal contaminants to produce a detectable signal in any method of nucleic acid analysis affected by the template sequence. For example, a "monoclonal" population of polynucleotides may include any population that produces signals (e.g., sequencing signals, nucleotide incorporation signals, etc.) that can be detected using a particular sequencing system. Optionally, the signal may be subsequently analyzed to correctly determine the sequence and/or base information of any one or more nucleotides present within any polynucleotide of the population. Examples of suitable sequencing systems for detecting and/or analyzing such signals include Ion Torrent sequencing systems, such as Ion Torrent PGM ^TM Sequencing systems, including 314, 316 and 318 systems, and Ion Torrent Proton ^TM Sequencing systems, including Proton I, proton II, and Proton III (Life Technologies, carlsbad, calif.). In some embodiments, the monoclonal amplicons allow for accurate sequencing of at least 5 consecutive nucleotide residues on an Ion Torrent sequencing system.

As used herein, the term "clonal amplification" and variants thereof refer to any process in which a population of substantially monoclonal polynucleotides is generated by amplification of a polynucleotide template. In some embodiments of clonal amplification, two or more polynucleotide templates are amplified to produce at least two substantially monoclonal polynucleotide populations.

As used herein, the term "linker" includes polynucleotides or oligonucleotides comprising DNA, RNA, chimeric RNA/DNA molecules, analogs thereof and generally means added or foreign sequences that are linked or bound to a target polynucleotide (e.g., template) of interest during the course of a manipulation. Ligation of the adaptor to the template may optionally occur before or after amplification of the template. In some embodiments, an adapter may comprise a primer binding sequence that is substantially identical or substantially complementary to a sequence within a corresponding primer. In some embodiments, a first adaptor comprising a first primer binding site is ligated to one end of a linear double stranded template, and a second adaptor comprising a second primer binding site is ligated to the other end.

As used herein, the term "binding partner" includes two molecules, or portions thereof, that have a particular binding affinity for each other and will typically bind to each other in preference to binding to other molecules. Typically, but not necessarily, some or all of the structure of one member of a particular binding pair is complementary to some or all of the structure possessed by the other member, wherein the two members are capable of binding together, particularly by way of a bond between the complementary structures, optionally through multiple non-covalent attractive forces.

In some embodiments, molecules that function as binding partners include: biotin (and its derivatives) and its binding partner avidin moieties, streptavidin moieties (and their derivatives); his-tag binding nickel, cobalt or copper; cysteine, histidine or histidine tract (histidine patch) binding to Ni-NTA; maltose that binds to Maltose Binding Protein (MBP); a lectin-carbohydrate binding partner; calcium-Calcium Binding Protein (CBP); acetylcholine and the receptor acetylcholine; protein a and a binding partner anti-FLAG antibody; GST and the binding partner glutathione; uracil DNA Glycosylase (UDG) and ugi (uracil-DNA glycosylase inhibitor) proteins; an antigen or epitope tag that binds to the antibody or antibody fragment, particularly an antigen such as digoxigenin, fluorescein, dinitrophenol, or bromodeoxyuridine, and their respective antibodies; mouse and goat anti-mouse immunoglobulins; bound IgG and protein a; a receptor-receptor agonist or receptor antagonist; enzyme-enzyme cofactors; enzyme-enzyme inhibitors; and thyroxine-cortisol. Another binding partner for biotin may be a biotin binding protein from chicken (Hytonen et al, BMC Structural Biology 7.

The avidin moiety may include avidin as well as any derivative, analog, and other non-natural form of avidin that can bind the biotin moiety. Other forms of avidin moieties include natural and recombinant avidin and avidin chainsRhzomycins and derived molecules such as unglycosylated avidin, N-acyl avidin and truncated streptavidin. For example, avidin moieties include deglycosylated forms of avidin, bacterial streptavidin produced by Streptomyces (e.g., streptomyces avidinii), truncated streptavidin, recombinant avidin and streptavidin, and derivatives of natural, deglycosylated and recombinant avidin and natural, recombinant and truncated streptavidin, e.g., N-acyl avidin, e.g., N-acetyl, N-phthaloyl, and N-succinyl avidin, and the commercial product Extravidin ^TM 、Captavidin ^TM 、Neutravidin ^TM And Neutralite Avidin ^TM 。

Examples

Embodiments of the present teachings will be further understood in light of the following examples, which should not be construed as in any way limiting the scope of the present teachings.

Example 1

The nucleic acid amplification reaction was performed in a single continuous liquid phase at a total reaction volume of-220. Mu.L in a single reaction vessel.

About 420X10 with water in a 1.5mL tube (tube 1) ⁶ The beads of (a) were washed 1 time (vortexed/spun) followed by 1 time in buffer (vortexed/spun).

The recombinase source is from twist Amp ^TM Basic kit (from TwistDx, cambridge, great Britain). The dehydrated precipitate in the kit contains usvX recombinase, usvY recombinase loading protein, gp32 protein, bsu DNA polymerase, dNTPs, ATP, phosphocreatine, and creatine kinase. Rehydration of rehydrated from TwistAmp in 120. Mu.L of rehydration buffer provided by kit ^TM Four pellets from Basic kit (tube 2). The recombinase solution was vortexed and spun, followed by ice-cooling. Two heating blocks were prepared, one set at about 68-70 ℃ and one set at 40 ℃.

The supernatant was removed from the bead pellet (tube 1), leaving about-20. Mu.L of liquid at the bottom.

Reverse primer (2 μ L of 100 μ M stock) was added to the bead tube (tube 1), followed by vortexing and spinning. Reverse primer sequence: 5' -ATCCTGCGTGTCCCGAC-3.

Biotinylated reverse primer (2. Mu.L of 10. Mu.M stock) was added to the small bead tube (tube 1), followed by vortexing and rotation. Biotinylated reverse primer sequence: 5 'Bio-ATCCTGCGTGTCTCCCGAC-3'.

mu.L of the polynucleotide library (of different concentrations) was added to a small bead tube (tube 1), vortexed/spun, and placed on ice. Library concentrations varied according to the desired DNA-to-bead ratios of 1.

Rehydrated recombinase mix (tube 2, reconstituted in 120 μ L rehydration buffer) was added to the bead tube (tube 1), vortexed, spun; and placed on ice.

65 μ L of an exemplary sieving agent of the present disclosure was added to a small bead tube, vortexed and rotated, and placed on ice.

11 μ L of ice-cold 280mM Mg-acetate was added to the bead tube (in the middle), then vortexed at maximum setting for 3 seconds and placed back on ice for 10 seconds, and incubated on a heat block at 40 ℃ for 20 minutes.

The reaction was heat inactivated in a heating block at 68 ℃ to 70 ℃ for 10 minutes.

Fill the reaction tube with TE buffer, vortex and spin at maximum setting (. About.20 KG) for 3 minutes, remove the solution from the beads, leaving 100. Mu.L. The washing step was repeated twice.

The beads were washed once with the recovery solution.

Fill the reaction tube with wash buffer, vortex and spin at maximum setting (. About.20 KG) for 3 minutes, remove the solution from the beads, leaving 100. Mu.L. The washing step was repeated twice.

After the final spin, the solution was reduced to 100 μ L (wash solution).

By conjugating biotinylated polynucleotide to streptavidin-conjugated paramagnetic beads (MyOne from Dynabeads) ^TM Bead) bound to enrich the beads.

Will enrichThe beads were loaded onto an Ion Torrent Ion sensitive chip and subjected to standard sequencing reactions. A significant portion of the enriched beads are determined to comprise a substantially monoclonal population of amplified polynucleotides, as by Ion Torrent PGM ^TM As evidenced by observation of detectable sequencing signals from such beads on a sequencer. Sequencing signals were analyzed to determine the sequence present within the amplicon of each such bead.

Example 2

Will be about 240x10 ⁶ Beads (with forward primer bound) were washed once in 2mL tubes in annealing buffer (from Ion Sequencing kit, e.g., PN 4482006). Remove (except-50. Mu.L) and discard the supernatant. Resuspend the beads in 100. Mu.L of annealing buffer.

Will have 300bp or 400bp inserts (about 120-240X10 ⁶ Copy) was prehybridized with washed beads. The library contains an insertion sequence that is ligated at one end to an adaptor that hybridizes to the forward primer and at the other end to an adaptor that hybridizes to the reverse primer. The template/bead ratios tested included 1, 0.75, and 0.5. The final volume was adjusted to 200. Mu.L with annealing buffer. By swirling and rotating the mixing tube. The tubes were incubated at 95-100 ℃ for 3 minutes and at 37 ℃ for 5 minutes. Add 1mL of annealing buffer, vortex and rotate tube for 3.5 minutes above 16,000xG, and discard the supernatant. 1mL of 10mM potassium acetate was added, vortexed at above 16,000xG and the tube rotated for 3.5 minutes, and the supernatant discarded. The potassium acetate wash was repeated once. Resuspend the beads in 480. Mu.L of potassium acetate (tube 1).

The recombinase source is from twist Amp ^TM Basic kit (from TwistDx, cambridge, great Britain). A list of components in the dehydrated pellet from the Basic kit can be found in example 1 above. Rehydrating from TwistAmp in 2.88mL of rehydration buffer provided by the kit in 15mL tubes (tube 2) ^TM 96 pellets from Basic kit.

48 μ L of 100 μ M reverse primer (non-immobilized primer) was added to the washed/prehybridized beads (tube 1). mu.L of 10. Mu.M biotinylated reverse primer (non-immobilized primer) was added to the washed/prehybridized beads and the tube was vortexed (tube 1). The contents of tube 1 (containing library, beads and reverse primer) were added to tube 2 (containing rehydrated pellet), tube 2 was vortexed for 5 seconds and placed on ice. 144 μ L of T4gp32 protein (15 μ g/. Mu.L) was added, vortexed and placed back on ice. Add 1.56mL of the exemplary sizing agent of the present disclosure, vortex the tube and place back on ice. After the reaction was kept on ice for more than 5 minutes, 264 μ L of magnesium acetate was added, vortexing the tube 3 times for 3 seconds each. Aliquots of 50 μ L samples were aliquoted into ice-chilled 96-well plates. The 96-well plate was incubated at 40 ℃ for 25 minutes on a thermal cycler (temperature maintained at 40 ℃).

To stop the reaction, 150 μ L of 100mM EDTA was added to each well. All reactions were pooled and centrifuged for 3.5 minutes above 16,000xg. The supernatant was discarded. Add 1mL Tris/1% SDS, vortex tube. The beads were washed twice in 1mL of OneTouch wash solution. The beads were resuspended in 100. Mu.L.

By mixing with paramagnetic streptavidin beads (MyOne from Dynabeads) ^TM Beads) to enrich the beads for the copy of the grafted library. The enriched beads were loaded onto Ion Torrent PGM Ion sensitive chips.

PGM according to Ion ^TM Standard Sequencing reactions were performed according to the manufacturer's instructions in the Sequencing 400Kit (User Guide PN 4474246B). A significant portion of the enriched beads loaded onto the chip are determined to comprise a substantially monoclonal population of amplified polynucleotides, as determined by Ion Torrent PGM ^TM As evidenced by observation of detectable sequencing signals from these beads on the sequencer. Sequencing signals were analyzed by Torrent Suite Software to determine the sequences present within the amplicons of these beads.

The sequencing data yielded an average read length of 305bp (FIG. 9), and the quality measures of the alignments were 1.16G (AQ 17) and 1.07G (AQ 20).

Example 3

Will be about 250x10 ⁶ The beads (with forward primer bound) were washed once in 1.5mL of annealing buffer (from Ion Sequencing kit, e.g., PN 4482006), vortexed at 15,000xG and spun for 6 minutes. Is discarded onClear solution, leaving about 50 μ Ι _ in the tube.

Will have a 140bp insert (about 50X 10) ⁶ Copy) was prehybridized with washed beads. The library contains an insertion sequence that is ligated at one end to an adaptor that hybridizes to the forward primer and at the other end to an adaptor that hybridizes to the reverse primer. The library (0.81 μ L of 62M stock) and 0.1mL of annealing buffer were added to the washed beads and mixed by pipetting up and down. Bead/template ratio was about 5. The tubes were incubated at 92-95 ℃ for 7 minutes and at 37 ℃ for 10 minutes. Add 1mL of annealing buffer, vortex and rotate tube for 6 min at above 15,000xG, discard supernatant. 1mL of 10mM potassium acetate was added, vortexed and spun at above 15,000XG for 6 minutes, and the supernatant discarded. The potassium acetate wash was repeated once and the tubes were placed on ice. About 60. Mu.L of liquid remained in the tube (tube 1).

The recombinase source is from twist Amp ^TM Basic kit (from TwistDx, cambridge, great Britain). A list of components in the dehydrated pellet from the Basic kit can be found in example 1 above. Rehydration in approximately 240 μ L of rehydration buffer provided by the kit from the TwistAmp ^TM 8 precipitates from Basic kit. The pellet and rehydration buffer were vortexed, spun and ice-cooled.

mu.L of 100. Mu.M reverse primer (non-immobilized primer) and 1. Mu.L of 10. Mu.M biotinylated reverse primer (non-immobilized primer) were added to the washed/prehybridized beads (tube 1), and the tube was vortexed and spun (tube 1). The contents of tube 1 (containing library, beads and reverse primer) were added to tube 2 (containing rehydrated pellet), tube 2 was vortexed and placed on ice. Add 130 μ Ι _ of an exemplary sizing agent of the present disclosure, vortex the tube and place back on ice. Add 24. Mu.L of ice cold 280mM magnesium acetate, vortex and spin tube. The total reaction volume was about 332. Mu.L. The tubes were incubated at 40 ℃ for 60 minutes.

1ml of 100mM EDTA was added to stop the reaction. The tube was vortexed and spun at 15,000xg for 6 minutes. The supernatant was discarded and about 20. Mu.L was left in the tube. The EDTA stop reaction step, vortexing and spinning steps were repeated. Add 1mL of Tris/1% SDS, vortex and rotate the tube at 15,000xG for 6 minutes. The supernatant was discarded and about 50. Mu.L was left in the tube. The beads were washed in 1ml of lonetouch wash solution by vortexing and spinning, leaving about 100 μ Ι _ in the tube. All reactions were pooled and spun at 15,000xg for 6 minutes, the supernatant discarded, leaving approximately 100 μ Ι _ in the tube.

By mixing with paramagnetic streptavidin beads (MyOne from Dynabeads) ^TM Beads) to enrich for beads that have attached copies of the library. Loading the enriched beads into Ion Torrent Proton I ^TM An ion sensitive chip.

According to Ion PI ^TM Standard Sequencing reactions were performed according to the manufacturer's instructions in the Sequencing 200Kit (User Guide PN MAN 0007491). A significant portion of the enriched beads loaded onto the chip are determined to comprise a substantially monoclonal population of amplified polynucleotides, as determined by Ion Torrent Proton ^TM As evidenced by observation of detectable sequencing signals from these beads on the sequencer. Sequencing signals were analyzed by Torrent Suite Software to determine the sequences present within the amplicons of these beads.

Two sequencing runs were performed. The sequencing data yielded an average read length of 96bp on the first run (FIG. 10) and 94bp on the second run (FIG. 11). The quality measurements of the comparisons were 1.76G (AQ 17) and 1.43G (AQ 20) in the first run and 1.48G (AQ 17) and 1.17G (AQ 20) in the second run.

Example 4:

20 μ L of beads (with forward primer bound) (103X 10) ⁶ /. Mu.L) was mixed with 440. Mu.L of 10mM potassium acetate and 3. Mu.L of 1M Tris (pH 8). The beads were mixed by vortexing and spinning.

16 μ L of insert with 200bp (approx.199X 10) were denatured by mixing with 2 μ L of NaOH ⁶ Copy), vortexed and spun, and allowed to stand for 1 minute. The reaction was neutralized by adding 440. Mu.L of 10mM potassium acetate and 3. Mu.L of 1M Tris pH 8. The library comprises an insertion sequence linked at one end to an adaptor that hybridizes to the forward primer and at the other end to an adaptor that hybridizes to the reverse primer.

Beads were added to the denatured library. The bead/template ratio was about 10 (2000 hundred million beads: 2 hundred million library). The tube was vortexed and allowed to stand at room temperature for 5 minutes (tube 1).

The recombinase source is from twist Amp ^TM Basic kit (from TwistDx, cambridge, great Britain). The list of components in the dehydrated pellet from the Basic kit can be seen above in example 1. Rehydrating from TwistAmp in 3mL of rehydration buffer provided by the kit in 15mL tubes (tube 2) ^TM 96 pellets from Basic kit (tube 2).

mu.L of 100. Mu.M reverse primer (non-immobilized primer) and 2. Mu.L of 100. Mu.M biotinylated reverse primer (non-immobilized primer) were added to the washed/prehybridized beads, vortexed tubes (tube 1) and iced tubes. 1.6mL of an exemplary sieving agent of the present disclosure was added to tube 2, the tube was vortexed for 5 seconds, manually inverted/rotated for 10 seconds, vortexed for 5 seconds, and placed on ice. Add 260 μ L of 280mM magnesium acetate to tube 2, vortex the tube for 5 seconds, manually tumble/rotate for 10 seconds (vortex and tumble/rotate 3 times), and place on ice. Aliquots of 50 μ L samples were aliquoted into ice-chilled 96-well plates. The 96-well plate was incubated at 40 ℃ for 60 minutes.

100 μ L of 200mM EDTA was added to each well to stop the reaction. All reactions were pooled and centrifuged at maximum speed for 7 minutes. The supernatant was discarded. Resuspend pellet in 1mL recovery buffer with 1% sds, vortex for 30 seconds, and rotate at highest speed for 6 minutes. After each rotation, the tube was reduced in half by combining the contents of both tubes. Resuspend pellet in 1mL recovery buffer with 1% sds, vortex for 30 seconds, and spin at 1550rpm for 7 minutes.

By mixing with paramagnetic streptavidin beads (MyOne from Dynabeads) ^TM Beads) to enrich the beads for the copy of the grafted library. During the enrichment step, the ES-wash buffer was replaced with a recovery buffer having 0.1% SDS. Finally the beads were resuspended in 1mL of water and reduced to 100. Mu.L. Loading the enriched beads into Ion Torrent Proton I ^TM An ion sensitive chip.

According to Ion PI ^TM The manufacturer's instructions in the Sequencing 200Kit (User Guide PN MAN 0007491) were standardizedAnd (5) sequencing reaction. A significant portion of the enriched beads loaded onto the chip are determined to comprise a substantially monoclonal population of amplified polynucleotides, as determined by Ion Torrent Proton ^TM As evidenced by observation of detectable sequencing signals from these beads on the sequencer. Sequencing signals were analyzed by Torrent Suite Software to determine the sequences present within the amplicons of these beads.

Sequencing data yielded an average read length of 144bp (figure 12) and the quality measure of the alignment was 4G (AQ 17).

Example 5

At dH ₂ 375x10 in O by vortex and spin wash ⁶ (iii) beads (with forward primer bound). The supernatant was removed (except-50. Mu.L). DNA library (about 75X 10) ⁶ Molecules) were added to the washed beads. Approximately 4 μ L of reverse primer (non-biotinylated) and 0.4 μ L of biotinylated reverse primer were added to the beads. Approximately 0.8 μ Ι _ of fusion forward primer was added to the beads. 40 μ L of magnesium acetate was added to the beads (final concentration 14 mM). Will dH ₂ O was added to the beads to a final total volume of 320 μ L.

The recombinase source is from twist Amp ^TM Basic kit (from TwistDx, cambridge, great Britain). The list of components in the dehydrated pellet from the Basic kit can be seen above in example 1. Rehydrating from TwistAmp in 488. Mu.L of rehydration buffer provided by the kit in separate tubes ^TM 16 pellets from Basic kit. 0.25mg/ml of T4gp32 protein (0.2 mg final concentration) was added to the 400bp library, and 0.5mg/ml of T4gp32 protein (0.4 mg final concentration) was added to the 600bp library. The tube was swirled to mix and rotate.

The contents of the bead mixture are added to a recombinase tube, vortexed, and spun. The bead/recombinase mixture with pre-cooled oil was transferred to a tube and incubated at Ion Torrent OneTouch ^TM Collected on a 10 micron Sterlitech filter on the device. The emulsion was produced and broken according to the manufacturer's instructions.

By mixing with paramagnetic streptavidin beads (MyOne from Dynabeads) ^TM Small ball)Beads bound to enrich for copies of the attached library (or omitting the enrichment step). The enriched beads were loaded onto an Ion Torrent Ion sensitive chip and subjected to standard sequencing reactions. A significant portion of the enriched beads are determined to comprise a substantially monoclonal population of amplified polynucleotides, such as by Ion Torrent PGM ^TM As evidenced by observation of detectable sequencing signals from these beads on the sequencer. Sequencing signals were analyzed to determine the sequences present within the amplicons of such beads.

Example 6

About 120X10 with annealing buffer ⁶ Beads (with forward primer bound) were washed once. The supernatant was removed (except-50. Mu.L). Resuspend the washed beads in 100. Mu.L of annealing buffer. About 60x10 of DNA library ⁶ Molecules are added to the beads. The final volume was adjusted to 200. Mu.L with annealing buffer. Beads and library were mixed by vortexing and spinning. The bead/library mixture was heated to 95-100 ℃ for 3 minutes followed by incubation at 37 ℃ for 5 minutes.

1mL of 10mM potassium acetate was added to the bead/library mixture, followed by vortexing and spinning. The supernatant was discarded. The potassium acetate wash was repeated once. Resuspend beads/DNA in 120. Mu.L of potassium acetate.

The recombinase source is from twist Amp ^TM Basic kit (from TwistDx, cambridge, great Britain). The list of components in the dehydrated pellet from the Basic kit can be seen above in example 1. Rehydrating from TwistAmp in 720. Mu.L of rehydration buffer provided by the kit in a separate tube ^TM 24 precipitates from Basic kit. An additional 54 μ L of dNTP mix (containing 10mM of each dNTP) was added to the twist Amp ^TM And (3) mixing.

In the third tube, 3. Mu.L of streptavidin (500. Mu.M) was mixed with 12. Mu.L of biotinylated reverse primer (100. Mu.M) and subsequently transferred to a bead/library tube. The recombinase mixture was added to the bead/reservoir tube, followed by vortexing to mix and ice-cooling. 27 μ L of T4gp32 protein (15/. Mu.g/. Mu.L) was added to the beads/library tube and vortexed to mix. Add 390 μ L of an exemplary sieving agent to the smallBead/reservoir tube, inverted tube and vortex to mix, ice cold for at least 5 minutes. Add 80 μ Ι _ of magnesium acetate to the bead/library tube and vortex, spin and ice cool the tube for at least 10 seconds. The beads/library tubes were incubated at 40 ℃ for 40 minutes. The reaction was stopped by the addition of 500. Mu.L of EDTA (250 mM). The tube was rotated above 18,000xg for 3 minutes. The supernatant was discarded and the pellet was resuspended in 1mL of TE with 1% SDS. The beads were washed twice in 1ml of lonetouch wash solution. The beads were resuspended in 100. Mu.L. Resuspend pellet by pipetting up and down and add 2mL of Ion Torrent OneTouch ^TM Washing with a washing solution. The washing step was repeated once. The pellet was resuspended in 300. Mu.L of melt-off solution (melt-off solution) and incubated for 5 minutes with shaking.

By mixing with paramagnetic streptavidin beads (MyOne from Dynabeads) ^TM Beads) to enrich for beads that have a copy of the library grafted (or to omit the enrichment step). The enriched beads were loaded onto an Ion Torrent Ion sensitive chip and subjected to standard sequencing reactions. A significant portion of the enriched beads are determined to comprise a substantially monoclonal population of amplified polynucleotides, as by Ion Torrent PGM ^TM As evidenced by observation of detectable sequencing signals from these beads on the sequencer. Sequencing signals were analyzed to determine the sequences present within the amplicons of such beads.

Example 7

Nucleic acid amplification was performed on an Ion sequencing chip. The template walking amplification reaction is first performed on the chip followed by a recombinase-mediated amplification reaction.

Preparation of Ion Torrent PGM ^TM Sequencing chips to include low T _M The single-stranded primer of (3), said primer being attached at its 5' end to the bottom of the well. The immobilized primer comprises a polyA (30) sequence.

The double stranded DNA template comprises a single stranded terminal overhang sequence having a polyT (30) sequence.

Treating Ion Torrent PGM with a Polymer ^TM The chip was sequenced to create a matrix at the bottom of the well. The capture primer is attached to a substrate.

Pre-washing Ion To with TE-containing bufferrrent PGM ^TM The chip was sequenced once and dried in vacuo.

40 microliters of the solution was mixed and loaded onto the chip. The final concentration of the solution contained: 1 × isothermal buffer from New England Biolabs, 1.6mM MgSO ₄ 3mM dNTPs, 1U/uL Bst polymerase (from New England Biolabs), 0.1nM template and nuclease-free water to 40uL volume. The chip was centrifuged for 5 minutes and incubated at 37 ℃ for 30 minutes. The chip was dried in vacuo.

Template walking amplification: 40 microliters of template walking solution was loaded onto the chip. The final concentration of the template walking solution comprises: 1 × isothermal buffer from New England Biolabs, 3.6mM MgSO ₄ 5mM dNTPs, 2uM soluble single-stranded primer, 6U/uL Bst polymerase (from New England Biolabs) and nuclease free water to 40uL volume. The chips were centrifuged and incubated at 60 ℃ for 30 minutes. The chip was washed once with 1 XTE containing buffer and dried in vacuo.

Recombinase-mediated amplification: the recombinase source of this example is from twist Amp ^TM Basic kit (from TwistDx, cambridge, great Britain). A list of components in the dehydrated pellet from the Basic kit can be found in example 1 above. 50 microliter of the amplification reaction mixture (containing the recombinase) was loaded onto the chip. The amplification reaction mixture comprises: from twist Amp ^TM One precipitate from Basic kit (from TwistDx, cambridge, great Britain), twist Amp ^TM 30uL of rehydration buffer from Basic kit, 2uM of soluble primer hybridized to one linker of DNA template and nuclease free water to a total volume of 50 uL. The chip was centrifuged for 2 minutes. 2 microliters of magnesium acetate (280 mM stock) was added to the chip. The chip was centrifuged for 2 minutes and then incubated at 40 ℃ for 1 hour.

The chips were washed successively with 0.5M EDTA (pH 8), TE-containing buffer, 1% SDS, and 2X with the washing solution.

A color-coded alignment map of the chip is used to determine that a majority of the wells contain a substantially monoclonal population of amplified polynucleotides.

According to Ion PI ^TM Manufactured in Sequencing 200Kit (User Guide PN MAN 0007491)The manufacturer's instructions perform standard sequencing reactions. Sequencing signals were analyzed by Torrent Suite Software to determine the sequences present within the amplicons of these beads. Sequencing data yielded an average read length of 151 bp.

Example 8

Direct Ion Torrent PGM in the presence of recombinase ^TM Nucleic acid amplification is performed on the sequencing chip under isothermal conditions. Placement of the polymerized hydrogel on PGM ^TM In the wells of the chip.

The recombinase source of this example is from a twist Amp ^TM Basic kit (from TwistDx, cambridge, great Britain). The list of components in the dehydrated pellet from the Basic kit can be seen above in example 1.

The polynucleotide template is denatured using either a thermal denaturation method or a recombinase method (as described above) followed by a nucleic acid amplification step.

(A) The heat denaturation method comprises the following steps: the library of polynucleotide templates was diluted in annealing buffer to a final volume of 60. Mu.L. Dilution is intended to place approximately 5 copies of the template into Ion Torrent Proton ^TM Sequencing chip (about 600-650x 10) ⁶ Holes) in each of the wells. The chip was washed once with annealing buffer and placed on a thermal cycler set at 40 ℃. 100 μ L aliquots of 1. The template library was denatured by placing on a chip and incubating at 95 ℃ for 2 minutes. The buffer/water mixture (preheated to 95 ℃) was pipetted into the flow cell. The chip was transferred to a 40 ℃ thermal cycler and incubated for 5 minutes. The chip was transferred to a bench (about 25 ℃). The chip was washed with 100. Mu.L of annealing buffer. The chip was placed on a 4 ℃ thermal cycler. The following steps are optional: rehydration of rehydrated cells from TwistAmp in 20. Mu.L of water and 30. Mu.L of rehydration buffer provided by the kit ^TM 1 pellet from Basic kit. The mixture was vortexed vigorously to dissolve the precipitate. 50 μ L of the precipitation mixture was loaded onto the chip. The chip is incubated at room temperature for at least 1 minute to allow the recombinase to bind to the primers preloaded into the wells of the chip.

(B) Recombinase denaturation method: wash Ion Torrent Proton with 150. Mu.L of annealing/water mixture (1 ratio of annealing buffer to water) ^TM Sequencing chip (about 600-650X 10) ⁶ A hole). The chip was placed on a 40 ℃ thermal cycler. Rehydration of TwistAmp from 60. Mu.L of rehydration buffer provided by kit ^TM 2 pellets from Basic kit. The library of polynucleotide templates was diluted in annealing buffer to a final volume of 50. Mu.L. Dilution is intended to place approximately 5 copies of the template into Ion Torrent Proton ^TM In each well of the sequencing chip. The total volume of diluted template was added to the rehydrated pellet mixture. The volume was adjusted to 100. Mu.L with water. Vortex the pellet/template to mix and rotate. The precipitation/template mixture was loaded into Ion Torrent Proton ^TM The sequencing chip (placed on a 40 ℃ thermal cycler) and incubated for 20 minutes. The chip was removed from the thermal cycler and allowed to stand at room temperature.

Nucleic acid amplification was performed as follows: all reagents were kept on ice. Rehydration of the DNA from TwistAmp in 30. Mu.L of rehydration buffer provided by the kit, 16. Mu.L of nuclease-free water and 1. Mu.L of 100. Mu.M reverse amplification primer ^TM 1 pellet from Basic kit. The precipitate was dissolved by vortexing and spinning. Immediately prior to loading onto the chip, 3 μ L of 280 μ M magnesium acetate was added to the precipitation mixture. The entire precipitation mixture was loaded onto the chip and incubated at 40 ℃ for 1 hour.

The amplification reaction was stopped by washing the chip with 0.1M EDTA (pH 8). The chip is washed with a chip washing solution. Wash the chip with 1% SDS. The chip was washed twice with TEX wash solution.

Chips were prepared for sequencing: the chip was washed using the melt-out solution. The chip was washed 3 times with annealing buffer and placed on a 40 ℃ thermocycler. Sequencing primers were prepared in separate tubes: 80 μ L of 50% annealing buffer was mixed with 50% sequencing primer and then pre-heated to 95 ℃. A mixture of 1. The chip was washed with pre-heated annealing/water buffer. 80 μ L of the preheated primer mix was loaded onto the chip. The chip was incubated at 40 ℃ for 5 minutes. The chip was washed once with annealing/water buffer. In a separate tube, 6. Mu.L of sequencing polymerase was mixed with 57. Mu.L of the annealing/water mixture and loaded onto the chip.

Standard sequencing reactions were performed according to the manufacturer's instructions.

Sequence listing

<110> LI, Chieh-Yuan

RUFF, David W.

CHEN, Shiaw-Min

O'NEIL, Jennifer

KASINSKAS, Rachel

ROTHBERG, Jonathan M.

<120> nucleic acid amplification

<130> LT00718 PCT

<140> PCT/US2013/032598

<141> 2013-03-15

<150> 61/635,584

<151> 2012-04-19

<150> 61/699,810

<151> 2012-09-11

<150> 61/767,766

<151> 2013-03-14

<150> 61/792,247

<151> 2013-03-15

<160> 1

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic primer

<400> 1

atccctgcgt gtctccgac 19

Claims

1. A method for non-diagnostic purposes of generating two or more substantially monoclonal populations of template polynucleotides, comprising:

providing a surface comprising two or more reaction sites in fluid communication, wherein each of the two or more reaction sites comprises a plurality of immobilized oligonucleotide primers and a different single-stranded or double-stranded template polynucleotide attached to the immobilized oligonucleotide primers;

contacting two or more reaction sites with a reaction mixture within a continuous liquid phase, the reaction mixture comprising an oligonucleotide primer and a polymerase, a recombinase, in solution, wherein the oligonucleotide primer in solution is complementary to a solution primer binding site of a template polynucleotide strand attached to an immobilized oligonucleotide primer at the two or more reaction sites; and

amplifying the template polynucleotides attached to the immobilized oligonucleotide primers under isothermal nucleic acid amplification conditions, thereby producing two or more separate populations of substantially monoclonal template polynucleotides attached to the immobilized oligonucleotide primers at the two or more reaction sites, wherein the reaction mixture comprises a sizing agent and/or a diffusion reducing agent that limits or slows the migration of polynucleotides through the reaction mixture;

Wherein two or more reaction sites are contacted with a first reaction mixture in a first round of nucleic acid amplification and a second reaction mixture in a second round of nucleic acid amplification, wherein the first round of nucleic acid amplification comprises a first reaction reagent loading round and the second round of nucleic acid amplification comprises a second reaction reagent loading round, wherein the first round of reaction reagent loading round comprises contacting a reaction site with a resistance compound.

2. The method of claim 1, wherein the plurality of immobilized primers at each of the two or more reaction sites are attached to a solid support.

3. The method of claim 2, wherein the solid support is a bead or particle.

4. The method of any one of claims 1-3, wherein the polymerase comprises strand displacement activity and lacks 3 'to 5' exonuclease activity.

5. The method of any of claims 1-3, wherein the sieving agent comprises cellulose and/or a cellulose derivative.

6. The method of claim 1, wherein the resistance compound comprises a receptor moiety.

7. The method of claim 6, wherein the oligonucleotide primer in solution comprises an affinity moiety that interacts with a receptor moiety.

8. The method of claim 7, wherein the affinity moiety comprises biotin and the receptor moiety comprises avidin or an avidin-like moiety.

9. The method of claim 7, wherein the second reaction reagent load round does not comprise contacting the reaction site with a resistance compound.

10. The method of any one of claims 1-3, wherein two or more reaction sites are operably coupled to a sensor capable of detecting the presence of nucleotide incorporation byproducts within the sites.

11. The method of claim 10, wherein the sensor comprises a field effect transistor.

12. The method of claim 10, wherein the sensor comprises an ion sensitive field effect transistor.

13. The method of any one of claims 1-3, further comprising sequencing the substantially monoclonal population of template polynucleotides.

14. The method of claim 13, wherein a population of substantially monoclonal template polynucleotides is sequenced in parallel.