CN105803534A

CN105803534A - Integrated sample preparation system and stable enzyme mixture

Info

Publication number: CN105803534A
Application number: CN201610058616.3A
Authority: CN
Inventors: M.W.埃舒; J.皮库里
Original assignee: Aibis Biological Science Co Ltd
Current assignee: Aibis Biological Science Co Ltd
Priority date: 2010-05-06
Filing date: 2011-05-06
Publication date: 2016-07-27

Abstract

Provided are an integrated sample preparation system and a stable enzyme mixture. Specifically, provided is a microfluid card which is used to process samples and generate a DNA library suitable for a sequencing method (for example, next generation sequencing method) or other proper nucleic acid-based methods. Also provided is a stable enzyme mixture containing enzyme (for example, enzyme used in whole genome amplification), BSA, and sugar. The enzyme mixture can be frozen in a dried manner, and the enzyme mixture does not obviously lose enzymatic activity when being stored at indoor temperature for a plurality of months.

Description

Integrated sample preparation system and stable enzymatic mixture

The application be international filing date be that on May 6th, 2011, national applications number are for 201180033729.3(international application Number be PCT/US2011/035597), the application of invention entitled " integrated sample preparation system and stable enzymatic mixture " point Case application.

The present invention advocates the U.S. Patent Application No. 13/102,520 (with submitting to the most simultaneously) submitted on May 6th, 2011 The priority of U.S. Provisional Application 61/331,910 submitted to on May 6th, 2010, each of which entirely through quoting all It is expressly incorporated herein.

Statement of Government Interest

The present invention completes under the U.S. Government support that contract number is HDTRA-1-07-C-0096.U.S. government in the present invention There is certain right.

Invention field

The present invention relates to the preparation of integrated sample and sequencing system (integrated sample preparation and Sequencing system) and stable enzymatic mixture.Specifically, the present invention provides and is configured to process sample and produce The microfluidic card (microfluidic card) of DNA library, described DNA library is applicable to sequence measurement (order-checking such as of future generation Method) or other suitable method for nucleic acid analysis, wherein can integrate the output from these cards with DNA sequencing system, this gives Sequencing system provides automatization and integrated sample.The present invention also provides and (such as uses in whole genome amplification containing enzyme Enzyme), BSA and the stable enzymatic mixture of sugar.Described enzymatic mixture lyophilizing can be stored at room temperature the several months and lives without significant enzyme Property loss.

Background

DNA sequencing needs a large amount of DNA extracted to be used for preparing sequencing library.Cultivate cell, cell lysis, extraction DNA, make DNA Fragmentation, jointing and the process of purification sequencing template, be possible to be taken days in fact by skilled investigative technique personnel The multi-step process executed.The challenge of detection based on order-checking is existing genome sequencing system such as Roche 454, needs number The preparation of it sample and a couple of days check order.It is tediously long that Fig. 1 shows that demonstration is currently used for relating in the sample preparation of sequencing technologies of future generation The flow chart of process such as Roche 454 method.As shown in the drawing, it carries for such as sample pretreatment, cell cracking, nucleic acid Take and can spend the time with whole genome amplification (WGA).Then can spend 1-5 days and generate DNA library, this relates to following step Rapid: DNA fragmentation；DNA end is repaired；Adapter connects；Fragment is fixed；Breach is repaired；Single stranded DNA separates；Drip with emulsion-based PCR Fixed.Finally, the sample prepared over 1-4 days for order-checking, order-checking use following steps: body emulsion-based PCR (bulk itself can be spent emulsion PCR)；Breakdown of emulsion PCR (break emulsion PCR)；Purification PCR positive beadlet；Preparation order-checking beadlet；And enforcement Sequencing reaction.

Need the method preparing DNA sequencing library of more rapid and easier enforcement and need to integrate these sides with sequenator Method.

Invention summary

The present invention provides integrated sample to prepare/nucleic acid sequencing system and stable enzymatic mixture.Specifically, the present invention provides micro- Fluid card, it is configured to process sample and generation is applicable to sequence measurement (sequence measurement such as of future generation) or other is suitable The DNA library of method for nucleic acid analysis.The present invention also provide containing enzyme (such as in whole genome amplification use enzyme), BSA and The stable enzymatic mixture of sugar.By described enzymatic mixture lyophilizing, and room temperature can be stored in reach the several months and damage without significant enzymatic activity Lose.

In certain embodiments, the present invention provides and comprises following microfluidic card: a) is configured to introduce biology and imitates The load port (loading port) of product；B) following fragmentation branch road (sub-circuit): i is comprised) it is configured to digestion Nucleic acid (nucleic acid such as expanded) is to produce the reagent mixture of the nucleic acid of fragmentation；And/or ii) be configured to make nucleic acid machinery Upper fragmentation is to produce the fragmentation component of the nucleic acid of fragmentation；And the c) joint that is operably connected with described fragmentation branch road Connecting branch road, wherein said joint connects branch road and comprises: i) be configured to the nucleic acid linker of sequence measurement or other method；With Ii) the ligase mixing allowing described nucleic acid linker be connected to produce nucleic acid sequencing library it is configured to the nucleic acid of described fragmentation Thing.

In certain embodiments, the present invention provides and comprises following microfluidic card: a) is configured to introduce biology and imitates The load port of product；B) cell cracking branch road；C) nucleic acid extraction branch road；D) nucleic acid amplification branch road, wherein said amplification branch road bag Contain: i) target sequence specific amplification, such as PCR；Or ii) total nucleic acid amplification branch road, such as pass through the complete of multiple displacement amplification Genome amplification；E) following fragmentation branch road is comprised: i) be configured to digesting nucleic acid (nucleic acid such as expanded) to produce sheet The reagent mixture of sectionization nucleic acid；And/or ii) be configured to make nucleic acid physically fragmentation to produce the fragment of fragmented nucleic acids Change component；F) blunt end of fragmented nucleic acids；G) joint being operably connected with described fragmentation branch road is connected branch road, its Described in joint connect branch road comprise: i) be configured to the nucleic acid linker of sequence measurement or other method；And ii) be configured to allow Described nucleic acid linker is connected with described fragmented nucleic acids to produce the ligase mixture in nucleic acid sequencing library；H) for removing not The method of the nucleic acid that connect or part connects: i) enzymatic of this available such as exonuclease is implemented and or ii) pass through In conjunction with and eluting extract or iii) by by affinity tag as the biotin labeled affine separation of connection product；I) enzyme is passed through Promote or combine eluting or affine or size exclusion etc. and carry out final purified library；J) integrate with sequencing system.

In specific embodiments, microfluidic card comprises at least one the other branch road selected from following group further: 1) Comprise following cracking branch road (being such as operably connected): i) mixing chamber and ii with described load port) lysis buffer (example As in packing)；2) nucleic acid extraction branch road is (such as with both described cracking branch road and waste compartment (waste chamber) It is operably connected), wherein said nucleic acid extraction branch road comprises: i) configuration is to be combined in nucleic acid present in the sample of cracking Nucleic acid extraction component；Ii) lavation buffer solution (such as in packing)；Iii) elution buffer (is such as packing In)；And iv) pump assembly (is such as configured to be pumped into elution buffer nucleic acid extraction component and mixes to produce the nucleic acid extracted Compound)；3) the amplification branch road (being such as operably connected with nucleic acid extraction branch road) comprising stable enzymatic mixture, wherein said Stable enzymatic mixture comprises at least one the amplification phase that can be used for that the nucleic acid extracted expands the nucleic acid to produce amplification The enzyme closed；With 4) waste compartment；

In certain embodiments, the present invention provides and comprises following microfluidic card: a) be configured to introduce biological sample Load port；B) the amplification branch road (being such as operably connected with nucleic acid extraction branch road) comprising stable enzymatic mixture, wherein Described stable enzymatic mixture comprises at least one of the nucleic acid that can be used for carrying out nucleic acid whole genome amplification to produce amplification The enzyme that amplification is relevant.

In some embodiments, the present invention provides and comprises following microfluidic card: a) is configured to introduce biology and imitates The load port of product；B) waste compartment；C) the cell cracking branch road being operably connected with load port, wherein said cell cracks Branch road comprises: i) mixing chamber and ii) pack containing the first of lysis buffer；Wherein configure described cracking branch road with mixed Close the sample producing cracking in room with lysis buffer cracking biological sample；D) can with both cell cracking branch road and waste compartment The nucleic acid extraction branch road being operatively connected, wherein said nucleic acid extraction branch road comprises: i) configuration is to be combined in the sample of cracking The nucleic acid extraction component of the nucleic acid existed；Ii) second containing lavation buffer solution packs；Iii) containing elution buffer 3rd packs；And iv) pump assembly, it is configured for elution buffer being pumped to nucleic acid extraction component to produce extraction Mixtures of nucleic acids；E) the amplification branch road being operably connected with nucleic acid extraction branch road, wherein said amplification branch road comprises stable Enzymatic mixture, wherein said stable enzymatic mixture comprise can be used for extract nucleic acid expand with produce amplification nucleic acid The relevant enzyme of at least one amplification；F) the fragmentation branch road being operably connected with amplification branch road, wherein said fragmentation props up Road comprises: i) be configured to the nucleic acid of digest amplification to produce reagent mixture and/or the ii of fragmented nucleic acids) it is configured to make The nucleic acid mechanically fragmentation of amplification is to produce the fragmentation component of fragmented nucleic acids；And g) operationally connect with fragmentation branch road The joint connect connects branch road, and wherein said joint connects branch road and comprises: i) configuration is for the nucleic acid linker (rank of sequence measurement Connect son) and ii) be configured to allow nucleic acid linker be connected with fragmented nucleic acids to produce the ligase mixture in nucleic acid sequencing library.

In certain embodiments, the enzyme that at least one amplification described is relevant comprises and can be used for implementing whole genome amplification (WGA) (such as multiple displacement amplification) or can be used for implementing PCR or for implementing the enzyme of transcript mediated amplification (TMA).At some In embodiment, the enzyme that at least one amplification described is relevant is selected from: Phi-29 polymerase, e. coli dna polymerase I, inorganic Pyrophosphatase or its combination in any.In certain embodiments, the enzyme that described amplification is correlated with checks order (such as originally with for the next generation Sequence measurement of future generation described in literary composition) joint (adapter) mixing.In specific embodiments, for any amplification step The primer of (such as PCR) comprises and can be used for the joint of sequence measurement of future generation and (be such as used for allowing amplicon with solid carrier even Connect).

In specific embodiments, stable enzymatic mixture farther includes: i) BSA；Ii) sugar；And iii) at least one Other component selected from following: inorganic salt, divalent metal, buffer agent, emulsifying agent and reducing agent.In other embodiment In, stable enzymatic mixture comprises further: i) BSA；Ii) sugar；Iii) inorganic salt；Iv) divalent metal；V) buffering Agent；Vi) emulsifying agent；And vii) reducing agent.

In some embodiments, microfluidic card comprises further: h) be connected with joint that component is operably connected is pure Changing branch road, wherein purification branch road comprises nucleic acid purification component, and wherein nucleic acid purification component comprises and is configured to and nucleic acid sequencing literary composition The locked nucleic acid sequence of the joint hybridization on storehouse.In other embodiments, microfluidic card comprises further and is configured to allow for User is taken out to the outlet in small part nucleic acid sequencing library.In certain embodiments, fragmentation branch road comprises further: configuration At least class of enzymes for polishing fragmented nucleic acids end.In specific embodiments, joint connection branch road comprises further: join Put at least class of enzymes for polishing nucleic acid sequencing library end.In other embodiments, stable enzymatic mixture is to be dried Form exists.In some embodiments, reagent mixture exists in a dry form.

In other embodiments, pump assembly comprises corrugated tube (bellows).In other embodiments, nucleic acid extraction Component comprises film.In further embodiment, nucleic acid extraction component comprises filter.In specific embodiments, microfluid Card comprises multiple valve.In further embodiment, configuration card is to be operably connected process instrumentation being operated by.At other In embodiment, microfluidic card comprise further be configured to multiple with what the pneumatic interface on process instrumentation was operably connected Blow vent.In some embodiments, configuration nucleic acid linker is for selected from following sequence measurement: ABI SOLID, ILLUMINA SOLEXA, ROCHE 454, ION TORRENT, Lifetechnologies STARLITE and PACIFIC BIOSCIENCES SMRT checks order.In further embodiment, microfluidic card comprise further be configured determine to extract mixtures of nucleic acids be The no sensor needing amplification.

Available any kind sequencing system (such as reusable card or one-off card) integrates sample of the present invention Product preparation system.In certain embodiments, with including HiSeq 2000, HiSeq 1000, HiScanSQ, gene element analyzer IIx, MiSeq integrate inventive samples preparation system at interior ILLIMINA SOLEXA sequenator, wherein can configure sample and prepare System is to work together with relevant Illumina software such as PIPELINE and/or CASAVA software kit.Other embodiment party In case, integrate the present invention with ROCHE 454 sequenator including gene order-checking instrument FLX system and GS Junior system Sample preparation system, wherein can configure sample preparation system to work together with following related software: GS Run Browser is soft Part, GS De Novo Assembler software, GS Reference Mapper software, GS Amplicon Variant Analyzer software and GS FLX Titanium Cluster.In some embodiments, with including Ion Personal Genome Machine (PGM) sequenator integrates inventive samples preparation system at interior ION TORRENT sequenator, wherein Configurable sample preparation system is to work together with following related software: DNASTAR SeqMan NGen software, Partek Genomics Suite software, for the NextGENe of Ion PGM platform from SoftGenetics Software or Avadis NGS software.In further embodiment, with the PACIFIC including PacBio RS sequenator BIOSCIENCES sequenator integrates inventive samples preparation system, wherein can configure sample preparation system with relevant soft to following Part works together: RS remote software, RS touch software, Primary Analysis software, SMRT Portal software and SMRT View software.

In some embodiments, the present invention provides and comprises following system: a) microfluidic card described herein；And b) through joining Put to receive and run the process instrumentation of microfluidic card.In certain embodiments, process instrumentation comprise selected from following at least A kind of assembly: pressure accumlator, vacuum reservoir, at least one pump, Duo Gefa, at least one heater, pneumatic interface and defeated Enter-export computer to connect.In further embodiment, process instrumentation comprises: pressure accumlator, vacuum reservoir, at least One pump, Duo Gefa, at least one heater, pneumatic interface and input-output computer connect.

In some embodiments, the present invention provide comprise following or substantially consist of or consist of steady Fixed enzymatic mixture: a) at least class of enzymes；B) bovine serum albumin (BSA)；C) sugar；And d) buffer agent；With optional water.At some In embodiment, stable enzymatic mixture comprises at least one further and selected from following reagent or is substantially selected by least one From following reagent composition or it is made up of selected from following reagent at least one: inorganic salt；Divalent metal；Emulsifying agent； And reducing agent.

In specific embodiments, the present invention provide comprise following or substantially consist of or consist of steady Fixed enzymatic mixture: a) at least class of enzymes；B) bovine serum albumin (BSA)；C) sugar；D) inorganic salt；E) divalent metal； F) buffer agent；G) emulsifying agent；And h) reducing agent.

In certain embodiments, mixture comprises Polyethylene Glycol (PEG) (such as PEG-8000 or other is heavy further Amount).In other embodiments, stable enzymatic mixture is aqueous form or in lyophilized form.In other embodiments, surely Fixed enzymatic mixture allows enzyme to keep its activity (example of at least 70% when 20-25 DEG C of hydration after 20-40 DEG C of storage reaches 2 months Such as 70% ... 75% ... 80% ... 85% ... 90% ... 95% ... 100%).

In other embodiments, BSA in stable enzymatic mixture with 0.05%-3.0% (such as 0.05% ... 0.10% ... 0.5% ... 1.0% ... 2.0% ... 3.0%) concentration exists.In other embodiments, sugar is with surely The concentration of the 5-35% (such as 5% ... 15% ... 25% ... 35%) of fixed enzymatic mixture exists.In other embodiment In, sugar is nonreducing sugar.In other embodiments, sugar is disaccharide.In further embodiment, sugar is trehalose.

In some embodiments, described at least class of enzymes comprises room temperature enzyme, thermal instability enzyme or Zimadzhunt L 340.Additionally Embodiment in, described at least class of enzymes comprises polymerase.In specific embodiments, polymerase is Phi-29 polymerase. In other embodiments, polymerase is escherichia coli (E. coli) polymerase I.In further embodiment, described at least Class of enzymes comprises inorganic pyrophosphatase.In certain embodiments, inorganic pyrophosphatase is saccharomyces cerevisiae (Saccharomyces Cerevisiae) inorganic pyrophosphatase.In further embodiment, described at least class of enzymes comprises: Phi-29 polymerase, big Enterobacteria DNa polymerase I and saccharomyces cerevisiae inorganic pyrophosphatase.

In certain embodiments, inorganic salt in stable enzymatic mixture with 1 mM-25 mM (such as 1 mM ... 10 mM ... 17 mM ... 25 mM) concentration exist.In other embodiments, inorganic salt is (NH₄)₂SO₄.At some In embodiment, divalent metal in stable enzymatic mixture with 1 mM-30 mM (such as 1 mM ... 10 mM ... 20 mM ... or 30 mM) concentration exist.In specific embodiments, divalent metal is MgCl₂.At other In embodiment, buffer agent is with 10 mM-100 mM (such as 10 mM ... 35 mM ... 75 mM ... 100 mM) Concentration exist.In some embodiments, buffer agent is Tris.

In specific embodiments, emulsifying agent in stable enzymatic mixture with 0.01%-0.15% (such as 0.01% ... 0.1% ... 0.15%) concentration exist.In some embodiments, emulsifying agent is polysorbate40, polysorbas20 or Tween 80. In further embodiment, reducing agent is deposited with the concentration of 1 mM-10 mM (such as 1 mM ... 5 mM ... 10 mM) ?.In certain embodiments, reducing agent is dithiothreitol, DTT (DTT).

In some embodiments, the present invention is provided to the compositions of stable enzyme, described compositions comprises following, basic On consist of or consist of: a) bovine serum albumin (BSA)；B) sugar；C) inorganic salt；With optional 1) divalent metal sun Ion；2) buffer agent；3) emulsifying agent；4) reducing agent；With 5) water.

In other embodiments, the present invention provides compositions, and it comprises Escherichia coli polymerase I and bovine serum albumin (BSA) or substantially by Escherichia coli polymerase I and bovine serum albumin (BSA) form or by Escherichia coli polymerase I and cattle Serum albumin (BSA) forms, and wherein compositions is the compositions of lyophilizing.In specific embodiments, the compositions of lyophilizing permits Permitted Escherichia coli polymerase I 20-40 DEG C store up to less 1,2 or 3 months after 20-25 DEG C of hydration time retain its at least 70% Activity (such as 70% ... 90% ... 100%).

In certain embodiments, the present invention provides compositions, and it comprises inorganic phosphate enzyme and bovine serum albumin (BSA) Or be substantially made up of inorganic phosphate enzyme and bovine serum albumin (BSA) or by inorganic phosphate enzyme and bovine serum albumin (BSA) Composition, wherein compositions is the compositions of lyophilizing.In some embodiments, the compositions of lyophilizing allows inorganic phosphate enzyme to exist 20-40 DEG C of storage retains its at least 70% (such as 70% ... 90% ... 100%) when 20-25 DEG C of hydration after reaching 2 months Activity.

In other embodiments, the present invention provides the method for the compositions storing lyophilizing, and described method includes: a) provide Comprise the compositions of lyophilizing that is following or that substantially consist of or consist of: i) at least class of enzymes；Ii) Sanguis Bovis seu Bubali is pure Albumen (BSA)；Iii) sugar；Iv) inorganic salt；V) divalent metal；Vi) buffer agent；Vii) emulsifying agent；And viii) reduction Agent；And under 15-45 DEG C of storage temperature, b) store the compositions of lyophilizing 15 days up to less, so that at least one enzyme is at 20-25 DEG C Its activity of at least 70% is retained during hydration.In other embodiments, within described at least 15 days, it is at least 30 days (for example, at least 30 My god ... 60 days ... 90 days ... 120 days ... or longer).In certain embodiments, storage temperature is 20-25 DEG C. In some embodiments, at least one enzyme described is selected from Escherichia coli polymerase I, Phi-29 polymerase and inorganic pyrophosphate Enzyme.

In specific embodiments, the present invention provides the method producing stable enzymatic compositions, and described method includes: a) carry Supply: i) comprise waterborne compositions that is following or that substantially consist of or consist of: A) at least class of enzymes；B) Ox blood serum Albumin (BSA)；C) sugar；D) inorganic salt；E) divalent metal；F) buffer agent；G) emulsifying agent；And H) reducing agent；And ii) Dialyzer；And b) with dialyzer waterborne compositions carried out dialysis and comprise in solution that is following or that substantially consist of: i) institute State sugar；Ii) described inorganic salt；Iii) described divalent metal；Iv) described buffer agent；V) described emulsifying agent；And vii) institute State reducing agent；C) freezing waterborne compositions is to produce freezing compositions；And d) allow the compositions of freezing suffer fine vacuum with warp Removed water by distillation, thus produce the compositions of lyophilizing.

Accompanying drawing explanation

Fig. 1 shows what demonstration was currently used for relating in the sample preparation of sequencing technologies such as Roche 454 method of future generation The flow chart of lengthy process.

Fig. 2 shows exemplary injection moulding microfluidic card and may make up each layer of card.Fig. 2 also illustrates primary three Road: cell cracking, DNA extraction and whole genome amplification.

Fig. 3 shows the exemplary microfluidic card with various labelling branch road, and described branch road includes that sample inlet, DNA carry Line taking road (circuit) and amplification circuit, it includes the enzyme (enzyme described in such as example 1 below) of lyophilizing.

Fig. 4 step display 1: use the Sample Preparation Procedure of microfluidic card.This figure is explicitly shown sample inlet and mixing chamber.

Fig. 5 step display 2: cleavage step.From blister pack lysis buffer with flow into together with the cell of sample Mixing chamber, in order to cell lysis.

Fig. 6 step display 3, the mixture wherein cracked then leads to waste compartment by capture filter.Then allow from bubble First lavation buffer solution of cover bag is by filter, subsequently into waste compartment.

Fig. 7 step display 4, wherein allows the second cleaning mixture by capture filter, then leads to waste compartment.Then air is dried Filter is to remove lavation buffer solution.

Fig. 8 step display 5, wherein the elution buffer from blister pack utilizes eluting corrugated tube (elution Bellows) nucleic acid of purification removed by inherent filtration device.Elution buffer is slowly pumped into filter by eluting corrugated tube.

Three kinds of reference samples that Fig. 9 uses for assessment cracking and extraction step during being set forth in embodiment of the present invention exploitation (staphylococcus aureus (S. aureus)；Bacillus cereus (B. cereus)；With klebsiella pneumoniae (K. pneumoniae))。

Figure 10 shows and finally verifies cracking with staphylococcus aureus, Bacillus cereus and klebsiella pneumoniae and extract The result of branch road.Shown result inputs and fresh life for Bacillus cereus and the klebsiella pneumoniae of 10,000 CFU Long S. aureus culture input.

Figure 11 shows the production of free of contamination whole genome amplification reagent.

Figure 12 step display 6, wherein allows the nucleic acid samples of purification be contained within reagent by card and (includes stable enzymatic mixture Lyophilizing beadlet) amplification branch road.

Figure 13 shows the exemplary microfluidic card containing the interior reagent (including the lyophilizing beadlet of stable enzymatic mixture) of card And buffer packaging configuration.

Figure 14 shows that the WGA enzymatic mixture of prior art is unstable during storing, and it only stablizes 5 days in room temperature.Figure 14 Also the advantage of the amplification enzymatic mixture of the lyophilizing that example I below provides it is set forth in.

Figure 15 shows the multiple of test (in embodiment 1) and the compatibility of Phi-29, polymerase I and inorganic pyrophosphatase Excipient formulation.

Figure 16 shows the result from embodiment 1, and it shows that BSA is the important component of stable amplification enzyme.

Figure 17 shows the test multiple BSA level (0.13%-1% final concentration) in enzyme stabilized formulations and 0.5% (the denseest Degree) addition of PEG-8000, it is as described in Example 1.

Figure 18 shows that in Ibis formula 33 enzyme of lyophilizing is stored in room temperature and reaches 4 months to show and be equal to fresh enzyme.

Figure 19 shows in Ibis formula 33 that the enzyme of lyophilizing shows after two months at 40 DEG C and is equal to fresh enzyme.

Figure 20 shows exemplary microfluidic card, and it includes moulded barrel (containing refuse pad and liquid reagent), top cover, lamination base plate Gentle washer.

Figure 21 shows the image of exemplary buffer blister pack.Figure 21 illustrates how the molding tip of microfluidic card can be used for Pierce through blister pack and discharge its reagent stored.

Figure 22 shows the schematic diagram of the exemplary process instrumentation for docking and operate microfluidic card with microfluidic card.

Figure 23 shows the system outline of the exemplary expendable microfluidic card worked together with process instrumentation.

Figure 24 shows the exemplary result of the sample fragment making amplification by physics or enzymatic means.

Definition

Phrase used herein " microfluidic card " refers to such device, cylinder (cartridge) or " card ", and its tool is the most internal logical Road, space or other microstructure, other microstructure described has the dimension of at least one about 0.1-500 micron.Available Such as techniques below manufactures microfluidic device from various materials: laser template printing (laser stenciling), embosses, rush Pressure, injection moulding, cover (masking), etching and three-dimensional flexible offset printing.Further with bonding interlayer or passing through adhesive-free Hot adhesion technology (thermal adhesiveless bonding techniques) such as orient poly-third by pressure treatment Alkene manufactures laminated microfluidic device.The micro-architecture of lamination and molding microfluidic device can be different.In certain embodiments, will Microfluidic card of the present invention is designed as and provides control interface and the main frame (host of optional temperature interface and magnetic interface Instrument) interact or " dock (dock) " with it.But, card usually contains all biologys implemented needed for measuring Reagent, it is only necessary to use one or more sample.These cards usually disposable, be intended for single use, generally to clean feature Manufacture so that the risk minimization that contacts with biohazard materials during use and when disposing.

Terms used herein " whole genome amplification " or " WGA " are often referred to such method, and it (removes with non specific manner Non-employing targeting WGA) expand limited DNA sample, in order to produce with primary sample undistinguishable but there is the new of relatively high DNA concentration Sample.Preferably whole genome amplification technology is by sample amplification to microgram level, but keeps original sequence to show.Sample DNA Whole genome or its part can be included.Degenerate oligonucleotide primed PCR (DOP), primer extension round pcr (PEP) and multiple Displacement amplification (MDA) is the example of whole genome amplification method.

Terms used herein " multiple displacement amplification " refers to isothermal method based on non-PCR, and described method is gathered based on random six Thing annealing (or the nonrandom primer in targeted approach) is so that DNA degeneration, and then when constant temperature, strand displacement synthesizes.Already applied In mini-gene group DNA sample, cause synthesizing the high-molecular-weight DNA with finite sequence performance bias.Along with being synthesized by strand displacement DNA, gradually occurs increasing primer to cause (priming) event, and this forms high branched-DNA structures network.Example can be passed through Such as Phi29 archaeal dna polymerase or by Bst archaeal dna polymerase large fragment catalytic reaction.

Describe in detail

The present invention provides integrated sample preparation system and stable enzymatic mixture.Specifically, the present invention provides and is configured to place Reason sample generation are applicable to sequence measurement (sequence measurement such as of future generation) or the DNA literary composition of other suitable method for nucleic acid analysis The microfluidic card in storehouse.It is stable that the present invention also provides containing enzyme (such as in whole genome amplification use enzyme), BSA and sugar Enzymatic mixture.Can enzymatic mixture described in lyophilizing and in the room temperature storage several months without significant loss of enzyme activity.

I. microfluidic card and instrument

Some molecular biology processes/steps are incorporated into single integrated system by offer microfluidic card of the present invention.Then can be by Integrated system is such as used for sampling (such as clinic, biology, environment) cell lysis, extraction nucleic acid, the core of amplification extraction Acid (such as whole genome amplification), make the nucleic acid fragment of amplification, polishing DNA fragmentation end, jointing and purification process mistake The nucleic acid DNA sequencing library of order-checking (be for example suitable for).Integrating process substantially reduces process time, work (labor), and passes through Use automatization and be integrated into the quality control reagents of the system of being used alone to improve the concordance of this process.

Can be by whole process integration to the microfluidic card being intended for single use containing all reagent implemented needed for this process. In some embodiments, in card, process refuse is also contained.Can stable reagent so that card can be in room temperature long-time storage.Cartoon Often containing the port result (the resulting processed) processed can removed, and with various DNA sequencings Technology is used together, described DNA sequencing technology such as Sanger order-checking, ABI SOLID, Illumina Solexa, Roche 454, Ion Torrent, ABI Starlite, PacBio SMRT and other nucleic acid analysis technique.

In certain embodiments, the miniflow being manufactured by Micronics Inc. and illustrating in its patent publications is used Body card is as the part of the present invention.It is illustrated as Micronics ' PanNAT molecular diagnosis platform processing unique cylinder Battery and/or major impetus instrument (main powered instrument) easily, each cylinder is designed to implement list Individual and/or multiple nucleic acid amplification assays.Each mensuration is fully integratible into the disposable cartridges including all required reagent.Only need few Amount biological sample is used for measuring enforcement.Some Micronics card is provided to say in U.S. Patent Publication No. 20090325276 Bright, its entire content is to be incorporated herein by as proposed the most completely.In certain embodiments, in the present invention The microfluidic card used is as described in paragraphs below.

Microfluidic card is integrated into single device or two or many by being formed corresponding to the independent multiple branch roads measuring module The individual device interknited.The most each branch road is made up of microfluidic element or assembly then.These bypass elements can include micro- Fluid passage, threeway, room (chamber), valve, filter, solid-phase capture element, separator-filter, pneumatic manifolds (pneumatic manifolds), blister pack (blister pack) (the such as bag Han reagent), refuse isolation room (waste Sequestration chamber), clean ventilation mouth, bellows chamber (bellows chamber), bellowspump, optical window Mouth, testing cushion and the microchannel deposit of dehydrated reagent, described dehydrated reagent optionally includes buffer agent, solubilizing agent and passivator. Branch road is typically made of a plastic, and can be by lamination, by molding with by offset printing or incompatible by these technology groups Manufacture.

Card device is usually single entrance, implies that and is introducing after one or more samples, and sealing device is so that any potential Biohazard be for good and all embedded in card to dispose.But, in certain embodiments, card has for removing gained DNA The outlet in library.Block the most self-contained, because any reagent required for Ce Dinging all is provided with this device by manufacturer.Should This understanding, microfluidic device optionally can include RFID, microchip, bar code and the label of help Treatment Analysis data, and block The main frame of docking is optionally intelligent instrument, patient data and test result can be passed to network.

The microfluidic channel of the most named " microchannel " is to have the fluid passage of variable-length, but one-dimensional in cross section Less than 500 um.In microfluidic channel, the fluid flow behavior of microfluid is the most nonideal laminar flow, may be compared with from this end Pressure drop or cross-sectional area to that end rely more on the wettability of wall, roughness, liquid viscosity, adhesiveness and cohesiveness.Miniflow The flow pattern of body is the most relevant with the existence of " dummy fluid body wall (virtual liquid wall) " in passage.Microfluid leads to Road is connected to each other on fluid by three links: link of trade, travel and post or is connected with other process component.In microfluidic channel formed valve, its can till Returning valve, pneumatic check-valves, folder valve (pinch valve), surface tension valve etc., it uses by convention.

Card device usually contains for controlling and the pneumatic manifolds of fluid actuated overlying, but also can use electrical activation valve. Blow vent is connected with pneumatic manifolds, generally activates bellowspump.When valve is by pneumatic unlatching, also involve blow vent.Sometimes provide There is the blow vent of hydrophobic separator-filter (the most any liquid impermeable, the filter membrane of infiltration gas), wherein flow in device Body seepage is to be not intended to and unsafe.Vent is not generally directly connected with pneumatic manifolds, but rises and balance its interior pressure Effect.

Reative cell provides on microfluidic card, and can be any suitable shape, such as rectangular chamber, circular cell, taper Room, spirality channel and for implement reaction various solids.These rooms can have the window of scope of examination thing, and it is as in inspection Survey room.Refuse spacing container is typically provided on microfluidic card.Waste canister is optionally with cleaning hydrophobic membrane ventilation.

Fig. 2 shows exemplary injection molding microfluidic card and may make up each layer of card.Fig. 2 also illustrates primary three Road: cell cracking, DNA extraction and whole genome amplification.

Fig. 3 shows the exemplary microfluidic card having various labelling branch roads, including sample inlet, DNA extraction circuit and amplification Circuit, it includes the enzyme (enzyme described in such as example 1 below) of lyophilizing.

Fig. 4 shows the step 1 of the Sample Preparation Procedure utilizing microfluidic card.This figure is explicitly shown sample inlet and mixing Room.

Fig. 5 step display 2 cleavage step.Lysis buffer from blister pack flows to mix with the cell from sample Room, so that cell cracking.

Fig. 6 step display 3, wherein allows the mixture of cracking then lead to waste compartment by capture filter.Then allow from First lavation buffer solution of blister pack passes through filter, then leads to waste compartment.

Fig. 8 step display 5, wherein the elution buffer from blister pack removes purification with eluting corrugated tube from filter Nucleic acid.Elution buffer is slowly pumped across filter by eluting corrugated tube.

Fig. 9 illustrates three kinds of reference sample (staphylococcus aureuses；Bacillus cereus；And klebsiella pneumoniae), in order to Assessment cracking and extraction step use them during exploitation embodiment of the present invention.

Figure 10 shows with staphylococcus aureus, Bacillus cereus and klebsiella pneumoniae cracking and extracts branch road Whole the result.Shown in result for the Bacillus cereus of 10,000 CFU and klebsiella pneumoniae input and fresh Growth S. aureus culture inputs.

Figure 11 produces free of contamination whole genome amplification reagent.In certain embodiments, the reagent for WGA is allowed to pass through DNA absorptive unit and 0.2uM sterilizing filter.Result is the reagent of purification, even if wherein negative control is the most not Produce DNA.

Figure 12 step display 6, wherein allows the nucleic acid samples of purification by amplification branch road.In this route, certain district available Whole genome amplification is implemented in territory, and sample and amplification buffer (it can be as elution buffer) at this zone refining mix, Being heated to 95 DEG C, then move on to conversion zone, herein, it mixes at 30 DEG C with amplification enzyme.Preferred enzyme is the amplification enzyme of lyophilizing, It is the part of stable enzymatic mixture, and it is as hereinafter described in Example 1.

Figure 13 shows have the exemplary micro-of reagent (including the lyophilizing beadlet of stable enzymatic mixture) dry in card Fluid card and buffer packaging arrangement.

Figure 14 illustrates that prior art WGA enzymatic mixture is unstable during storing, and only stablizes 5 days in room temperature.Figure 14 is also The advantage illustrating the amplification enzymatic mixture of lyophilizing, it provides in example 1 below.

Figure 21 shows the image of exemplary buffer blister pack.Figure 21 illustrates the molding tip (molded on microfluidic card Sharps) can how to be used for piercing through blister pack to discharge its reagent stored.

Figure 22 shows the schematic diagram of the exemplary process instrumentation for docking and operate microfluidic card with microfluidic card.Such as this Figure is illustrated, process instrumentation includes the processor running instrument；The input-output that USB or Ethernet connect can be included；For user The LCD screen of offer state；With the touch screen controlled for user.Process instrumentation also can include pneumatic system, its contain air pump, Actuator, manifold, pressure/vacuum bin；And valve.Process instrumentation also can include the card/cylinder interface of carrying jig, pneumatic manifolds and Heater.

Figure 23 shows the illustrative system general picture of the consumptive microfluidic card with process instrumentation work.

Figure 24 shows the exemplary result of the sample fragment making WGA expand with physics or enzymatic.Described fragmentation Can be in step 7 in the fragmentation branch road enforcement of microfluidic card.The sample of amplification can be moved on to fragmentation branch road, then with machinery Shearing (such as by the aperture in fragmentation branch road) maybe can stand Restriction Enzyme and carry out fragmentation.Adoptable fragmentation methods bag Include but be not limited to such as with Restriction Enzyme or cutting primer cutting.It is that this area is common by the method for Restriction Enzyme and cutting primer Known to technical staff.Then can be with the end of enzyme such as Klenow fragment polishing fragment.

In certain embodiments, next branch road in microfluidic card is that joint connects branch road.At this branch road, joint and sheet Sectionization nucleic acid end connects.Preferably joint is the joint used in sequence measurement, such as at ABI SOLID, ILLUMINA SOLEXA, ROCHE 454, ION TORRENT, ABI STARLITE and PACIFIC BIOSCIENCES SMRT order-checking use Joint.Explanation to these sequencing technologies and associated adapter is being provided further below.

II. whole genome amplification method

In certain embodiments, microfluidic card has that to implement whole genome amplification (WGA) institute in amplification branch road required, enough Or useful reagent.It should be noted that the invention is not restricted to WGA as amplification technique, because can use any other type of The amplification technique (and corresponding reagent) being suitable for, such as PCR or TMA, both of which is for it is well known that.

The most non-target WGA

In a lot of research fields, such as genetic diagnosis, cancer research or prudence, lacking genomic DNA can be can be real to sample The genetic test type executed and the serious restriction factor of quantity.The method being designed to overcome this problem is that full-length genome expands Increase (WGA).Target is with the non specific manner limited DNA sample of amplification, to produce with primary sample undistinguishable and tool relatively The fresh sample of high DNA concentration.The target of typical whole genome amplification technology should be amplification sample to microgram level, maintain simultaneously Original series shows.

Whole genome amplification method is elaborated first, its principle based on polymerase chain reaction in 1992.Zhang and with Thing (Zhang, L. etc., Proc. Natl. Acad. Sci. USA, 1992,89:5847-5851, it is incorporated by reference into Develop herein) primer extension round pcr (PEP), Telenius and partner (Telenius etc., Genomics. 1992, 13 (3): 718-25, be incorporated herein by) devise degenerate oligonucleotide primed PCR method (DOP-PCR) Zhang etc., 1992)。

DOP-PCR is for utilizing Taq polymerase and half degenerate oligonucleotide (such as CGACTCGAGNNNNATGTGG (SEQ ID NO:1), wherein N=A, T, C or G) method, described half degenerate oligonucleotide in human genome about 100 Ten thousand sites combine with low temperature thermal oxidation.First circulation followed by with a lot of circulations of higher anneal temperature, this allow to In first step, tagged fragment expands.

Multiple displacement amplification (MDA, also known as strand displacement amplification；SDA) be isothermal method based on non-PCR, its based on Machine six aggressiveness is annealed into the DNA of degeneration, then at constant temperature chain-displacement synthesis (Blanco etc., 1989, J. Biol. Chem. 264:8935-40, is incorporated herein by).It has been applied to mini-gene group DNA sample, causes synthesis to have finite ordered High-molecular-weight DNA (Lizardi etc., the Nature Genetics 1998,19,225-232 of the existing bias of list；Dean etc., Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5261-5266；Both of which is incorporated herein by).With By strand displacement synthetic DNA, increasing primer firing event gradually occurs, this forms super-branched DNA structure network.Can By Phi29 archaeal dna polymerase or by the large fragment catalytic reaction of Bst archaeal dna polymerase.Phi29 archaeal dna polymerase has school Positive activity, this causes error rate lower 100 times than Taq polymerase.

In some embodiments, the reactant mixture used in multiple displacement amplification includes multiple polymerase.At some In embodiment, catalysis activity includes that 5'-3'DNA polymerase activity, 3'-5' exonuclease proofreading activity and DNA repair and lives Property, such as, 5'-3' excises repairing activity.Various polymerase examples include but not limited to following: Phi29, Klenow fragment, T4 polymerase, T7 polymerase, BstE polymerase, escherichia coli Pol I, Vent, Deep Vent, Vent exo-, Deep Vent exo-、KOD HiFi、Pfu ultra、Pfu turbo、Pfu native、Pfu exo-、Pfu exo- Cx、Pfu cloned、 PROOFSTART (Qiagen), rTth, Tgo and Tfu Qbio.These polymerases be it is known that and major part commercially available.

In other embodiments, other non-polymeric enzyme or auxilin, such as spiral is included at MDA reactant mixture Enzyme, gyrase, T4G32 and SSBP.In some embodiments, MDA reactant mixture includes pyrophosphatase, and it is for by Jiao's phosphorus Hydrochlorate is converted into phosphate.Result as amplified reaction is gathered pyrophosphate and (each is mixed from add in the reactive mixture Enter triphosphate deoxy-nucleotide and produce the pyrophosphate of monovalent, it is known that its suppression of amplification reacts).In some embodiments, The pyrophosphate of about 0.004 unit is added in reactant mixture.

B. targeting WGA

In certain embodiments, use targeting whole genome amplification (TWGA) as part of the present invention.Targeting WGA is set forth in example In U.S. Patent Publication No. 20100035232, it is incorporated herein by.

For designing the target gene group of targeting whole genome amplification primer

In some preferred embodiments, one or more target gene groups are selected.The selection of target gene group is by analyzing target Determine.Such as, if the phase of targeting whole genome amplification process needs result to be that acquisition represents biological warfare biology such as anthrax bacillus The nucleic acid of the genome of (Bacillus anthracis) (in its doubtful pedotheque being present in biological warfare attack scene), Then people can select to select anthrax bacillus genome as unique target gene group.On the other hand, if targeting full genome The phase of group amplification procedure needs result to be that acquisition represents antibacterial group (the most potential biological warfare medium (biowarfare agents) Group) nucleic acid, then may select more than one target gene group, such as, comprise any one of following antibacterial or whole groups: Anthrax bacillus, soil draw hot Francisella (Francisella tularensis), Yersinia pestis (Yersinia Pestis), Brucella (Brucella sp.), Burkholderia mallei (Burkholderia mallei), Pu Shi Rickettsia (Rickettsia prowazekii) and colon bacillus 0157.Equally, optional different genes group or genome Group is as target gene group for other purposes.Such as, human genome or mitochondrial DNA can be that pedotheque maybe can occur The target of the total genome found in other sample environment of crime.Therefore, current method and composition can be used, in background Selective amplification human genome (target) in genome.Other example can include the genome of following biology: causes respiratory disease Sick virus groups, the pathogen causing sepsis or the fungus group of known pollution household wares.

For designing the background genes group of targeting whole genome amplification primer

Existence probability based on some organism nucleic acid can select background genes group.Such as, it is contemplated that the soil processed by people Earth sample contains and represents the nucleic acid of genome including but not limited to following organism: people (Homo sapiens), jungle fowl (Gallus gallus), Lan Yinzao (Guillardia theta), Oryza sativa L. (Oryza sativa), arabidopsis (Arabidopsis thaliana), Yarrowia lipolytica (Yarrowia lipolytica), saccharomyces cerevisiae, Han Xunde Bali Yeast (Debaryomyces hansenii), Kluyveromyces lactis (Kluyveromyces lactis), schizosaccharomyces pombe (Schizosaccharmyces pom), Aspergillus fumigatus (Aspergillus fumigatus), Cryptococcus histolyticus (Cryptococcus neoformans), rabbit appendix B ly-mphocyte (Encephalitozoon cuniculi), Eremothecium Gossypii, smooth candida mycoderma (Candida glabrata), Apis mellifera (Apis mellifera), Drosophila melanogaster (Drosophila melanogaster), red flour beetle (Tribolium castaneum), malarial mosquito (Anopheles And beautiful hidden bar line bacterium (Caenorhabditis elegans) gambiae).These genomes any one or all be suitable for Background genes group as sample is assessed.Actually the organism in any specific sample is by source based on each sample And/or environment and change.Therefore, identity based on the organism actually existed in sample background genes group can be selected. Sample composition can be measured by any one of multiple technologies known to those of ordinary skill in the art.In another embodiment In, can based on the actual qualification biological to one or more backgrounds in sample and based on any one of sample or multiple additionally The biological probability of background design primer.

Differentiate that unique genome sequence section is as primer hybridization sites

Once it is determined that the target of sample and background genes group, next step is exactly the target identified and can be used as primer hybridization sites Genome sequence section in genome.Specific targeting whole genome amplification efficiency depends on effective utilization of primer.In order to produce The raw amplified production representing full-length genome, in the range of genome length, primer hybridization sites should have appropriate intervals.Preferably Average distance between primer hybridization sites is about 1000 core bases or less.More preferably average gap length is about 800 Individual core base or less.Even more preferably average gap length is about 600 core bases or less.Most preferably primer hybridization sites Between average gap length be about 500 core bases or less.

It will be appreciated by those of ordinary skill in the art that and effectively cause whole genome amplification to depend on a number of factors, such as, draw The fidelity of the polymerase used by thing extension and processivity.If polymerase has high processivity, then primer is miscellaneous Longer average distance between site is handed over to become more acceptable.This shows that polymerase is combined closely with nucleic acid-templated.This is target The characteristic needed to whole genome amplification institute's phase because it makes polymerase can keep and the combination of template nucleic acid, continuation extension just by The complementary nucleic acid chain of synthesis.The polymerase example with high processivity includes but not limited to that Phi29 polymerase and Taq gather Synthase.Such as, by making polymerase covalently bound with DNA-associated proteins, protein engineering Reconstruc-tion policy is used for producing height and holds (Wang etc., Nucl. Acids Res., 2004,32 (3) 1197-1207, by quoting also for the polymerase of continuous synthesis capability Enter herein).Owing to the polymerase of the processivity with improvement can be obtained, for targeting whole genome amplification, longer Average distance, even substantially exceeds 1000 core bases and is probably acceptable.

Hybridization sensitivity and selectivity

For the purpose of targeting whole genome amplification, primer hybridization sites (genome sequence section) length and the phase being hybrid with it Answer the selection of primer length, preferably will balance following two factor: (1) sensitivity, it shows specific primer and target gene group Combination frequency；(2) selectivity, it shows that specific primer is to hybridize higher frequency and target gene than with background genes group The degree of group hybridization.Generally the shortest primer tends to that selectivity is higher and sensitivity is relatively low, the most anti-for shorter primer As the same.The primer of about 5-about 13 core bases of preferred length can be used for targeting whole genome amplification；It is also possible, however, to use Fall at this extraneous primer length.It should be understood that this scope comprises has 5,6,7,8,9,10,11,12 and 13 The primer of core bases longs.Primer size affects the balance between Primer selection and primer sensitivity.Notice that this balance is with really The optimal primer length of fixed each sample.If can the selectivity of best maintained sample and sensitivity, then length is less than 5 core bases Or it is the most useful more than the primer of 13 core bases.The multiple primer with different length is selected to provide between target gene group sequence Extensively cause, the most also provide primer to combine (relative to background genes group sequence) with the preferential of target gene group sequence.

Select threshold values standard

In some embodiments, in order to reduce the total primer number in targeting whole genome amplification group, in order to reduce and spend and draw The complexity of thing group, preferably determines the suitable subgroup of total genome sequence section of uniqueness.In some embodiments, determine solely The suitable subgroup of special genome sequence section must select to show the sensitivity of specific gene group sequence section and/or selective Useful and one or more threshold values standards of the cut-off point of reality.Described standard instance includes but not limited to selected event Threshold frequency (frequency of event threshold values) and selected optional ratio's (optional ratio's threshold values).

In some embodiments, it is useful for classifying unique total genome sequence section according to described standard. Such as, according to event frequency to unique total genome sequence section graduation, wherein #1 level shows that event frequency is maximum, and Inferior grade shows that event frequency is minimum.Then the frequency threshold of event can be selected from each grade.The frequency threshold of event is used as Select the demarcation line between subgroup member and the member the most further analyzed for analyzing further.

Design of primers

It is designed as preferably complementary with genome sequence section 100% with the primer of selected genome sequence section hybridization.Real at other Execute in scheme, be designed as the primer with the hybridization of selected genome sequence section for genome sequence section at least about 70%-about 100% (or any integer therebetween or mark) is complementary.It is said that in general, for the design with the primer of selected nucleic acid array hybridizing It is well known to those skilled in the art, and can be assisted by commercial computer program.Generally preferably design specific primer so that Its with analyze and to be chosen as the genome sequence section length of primer hybridization sites identical.But, in some cases, relatively Changing primer length in primer hybridization sites may be favourable.Such as, if analyzing primer and finding that it has disadvantageous fusion temperature, Then may benefit from extending, at 5' or 3' end, the primer improved with the affinity produced with target gene group sequence.Can be increased or decreased Primer length.Those of ordinary skill it should be understood that the change of primer length also changes primer hybridization sites so that it is no longer with just The genome sequence section beginning to select is just the same.In some cases, primer miscellaneous changed corresponding to length-specific is analyzed The genome sequence section handing over site is probably useful.By inspection, this analysis can include but not limited to that data below is come Implement: event frequency and optional ratio, and also can be implemented by the primer that actual vitro detection length changes.

In some embodiments, its corresponding genome sequence section primer less than 100% complementation is designed wherein In the case of possibility is favourable, to supposing that genome sequence section recalculates selection standard (such as event frequency and selectivity Ratio) complementarity carry out checking also favourable, described supposition genome sequence section is complementary with such primer 100%, described in draw Thing its corresponding initial gene group sequence section has the complementation less than 100%.If this selection standard is unfavorable, it is considered to design The alternative primer sequence of the selection standard with improvement will be favourable.

III. sequencing technologies

As it has been described above, the DNA library that embodiment of the present invention relates to producing with microfluidic card checks order.The present invention is not by institute The restriction of the sequence measurement type used.Exemplary sequence measurement is as described below.

The illustrating property limiting examples of Nucleic acid sequencing techniques includes but not limited to that chain terminating agent (Sanger) checks order and contaminates Material terminator order-checking.Chain terminating agent order-checking uses modified nucleotides substrate to utilize the sequence-specific of synthesis reaction of DNA eventually Only.By using the short radioactive label complementary with template in this region or the oligonucleotide primers of other labelling, in template The specific position of DNA starts and extends.With a chain of archaeal dna polymerase, four kinds of Deoxydization nucleotide bases of standard and low concentration eventually Only nucleotide (acid of the most frequently used di-deoxynucleoside) extends oligonucleotide primers.This reaction is repeated in four independent pipes, each Plant base alternately as two-Deoxydization nucleotide.Produced a series of relevant by archaeal dna polymerase limited incorporation chain termination nucleotide DNA fragmentation, described fragment uses the site of specific two-Deoxydization nucleotide to terminate the most wherein.For each reaction tube, by Electrophoresis in the capillary tube of slab polyacrylamide gel or filling sticky polymers carrys out isolated fragment by size.When from gel top When portion scans bottom, produce from labeled primer and manifest labelling by reading any bar swimming lane and measure sequence.

Dye terminator checks order alternatively labelling terminator.Can by with single fluorochrome label each two-de- Oxygen nucleotide chain-terminator, implements to check order completely in single reaction, and described fluorescent dye fluoresces when different wave length.

Already gone out a prescription method now known as " of future generation order-checking " technology (Voelkerding etc., Clinical Chem., 55: 641-658, 2009；MacLean etc., Nature Rev. Microbiol., 7:287-296；Each is by quoting It is integrally incorporated herein using it) as Sanger and the alternative approach of dye terminator sequence measurement.Nearest method illustrates next For sequencing technologies for full-length genome de novo sequencing to determine the purposes of the one-level nucleotide sequence of organism.It addition, targeting weight-survey Sequence (degree of depth order-checking) makes to carry out sensitizing mutation detection in wild-type sequence colony.Some examples include nearest work, It illustrates to HIV drug resistance variant and for measuring the qualification of the EGFR sudden change of the reaction to anti-TK medicine.Illustrate The nearest publication of the purposes of bar code primer sequence allows to check order multiple samples at typical case's run duration that checks order simultaneously, institute State publication and include such as: Margulies, M. etc., " Genome Sequencing in Microfabricated High- Density Picolitre Reactors (gene order-checking in the high density Picolitre reactor of micro-manufacture) ", Nature, 437, 376-80 (2005)；Mikkelsen, T. etc., " Genome-Wide Maps of Chromatin State in Pluripotent and Lineage-Committed Cells is (in multipotency and lineage committed cell The genome range collection of illustrative plates of chromatin state) ", Nature, 448,553-60 (2007)；McLaughlin, S. etc., “Whole-Genome Resequencing with Short Reads: Accurate Mutation Discovery with Mate Pairs and Quality Values (redeterminates entirely-genome sequence with short reading: tool matches and quality value The discovery that accurately suddenlys change) ", ASHG Annual Meeting (2007)；Shendure J. etc., " Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome be (bacterial genomes of evolution Accurate multiple Polony checks order) ", Science, 309,1728-32 (2005)；Harris, T. etc., " Single- Molecule DNA Sequencing of a Viral Genome (virus genomic list-molecular dna order-checking) ", Science, 320, 106-9 (2008)；Simen, B. etc., " Prevalence of Low Abundance Drug Resistant Variants by Ultra Deep Sequencing in Chronically HIV-infected Antiretroviral (ARV) Na ve Patients and the Impact on Virologic Outcomes is (logical The ultra-deep order-checking crossed in chronic HIV-infects the inmature patient of antiretroviral (ARV) measures low abundance drug resistance variant Popularity and impact on virus results) ", the 16th international HIV drug resistance seminar, Barbados (2007)； Thomas, R. etc., " Sensitive Mutation Detection in Heterogeneous Cancer Specimens By Massively Parallel Picoliter Reactor Sequencing (is surveyed by large-scale parallel picoliters reactor Sequence sensitizing mutation detection in heterogeneous cancer sample) ", Nature Med., 12,852-855 (2006)；Mitsuya, Y. Deng, " Minority Human Immunodeficiency Virus Type 1 Variants in Antiretroviral- Na ve Persons with Reverse Transcriptase Codon 215 Revertant Mutations (has A few peoples' immunodeficiency virus type 1 variant in the inmature antiretroviral people of reverse transcriptase codon 215 back mutation) ", J. Vir., 82, 10747-10755 (2008)；Binladen, J. etc., " The Use of Coded PCR Primers Enables High-Throughput Sequencing of Multiple Homolog Amplification Products (PCR primer that order-checking use encodes parallel by 454 makes to carry out multiple homology by 454 Parallel Sequencing The high-flux sequence of amplified production) ", PLoS ONE, 2, e197 (2007)；And Hoffmann, C. etc., " DNA Bar Coding and Pyrosequencing to Identify Rare HIV Drug Resistance Mutations(DNA Bar coding and Manganic pyrophosphate complex initiation differentiate rare HIV Drug resistance mutations) ", Nuc. Acids Res., 35, E91 (2007), all lists of references are all incorporated herein by.

Comparing with tradition Sanger order-checking, sequencing technologies of future generation produces a large amount of sequencing data points.Typical case runs and can be easy to Each run produce tens of to hundreds of megabasses, thereby increases and it is possible to day output reach 10 gigabit base scopes.Standard is compared in this explanation A number of level that 96 orifice plates are big, 96 orifice plates can produce hundreds of data points in typically re-running more.Few to a nucleoside The different target amplicon of acid can readily distinguish, even when there is the multiple target from relative species.This carries significantly The high ability of accurate gene type.Can be easy to differentiate new for producing the sequence alignment program program of future generation of consensus sequence Point mutation, described point mutation can produce the new strain relevant to drug resistance.The use of primer barcode encoding also allows Single order-checking carries out multiple order-checking to different Patient Sample A in running.

Order-checking (NGS) method of future generation compares with old sequence measurement, enjoys large-scale parallel, high-throughput strategy and relatively low Spend the common characteristic of target.NGS method can be generally divided into the method needing template amplification He need not template amplification.Need The method of amplification include by Roche as the commercially available Manganic pyrophosphate complex initiation of 454 technology platforms (such as GS 20 and GS FLX), by Solexa platform commercially available for Illumina and being connected by commercially available for Applied Biosystems support oligonucleotide and detection (Supported Oligonucleotide Ligation and Detection, SOLiD) platform.Non-amplification method is also known as For list-molecule order-checking, it is respectively by HeliScope platform commercially available for Helicos BioSciences with by VisiGen and Emerging platform commercially available for Pacific Biosciences carrys out illustration.

At Manganic pyrophosphate complex initiation (Voelkerding etc., Clinical Chem., 55:641-658,2009；MacLean Deng, Nature Rev. Microbiol., 7:287-296；U.S. Patent number 6,210,891；U.S. Patent number 6,258, 568；Each is integrally incorporated herein with it by quoting) in, by template DNA fragmentation, carry out end reparation, be connected to linking Son, and by capturing the next clonal expansion in situ of single template molecule with containing the oligonucleotide complementary with adapter.Will be containing single The beadlet of template type is divided into Water-In-Oil microvesicle region, by the technology clonal expansion template of referred to as emulsion-based PCR.Destroy after amplification Emulsion, allows beadlet deposit in the single hole of skin titer plate (picotitre plate), the conduct during sequencing reaction of described plate Flow cell (flow cell) works.In the presence of Sequenase and luminous reporter such as luciferase, four kinds of dNTP reagent The orderly iteration of each introduces and occurs at flow cell.Suitable dNTP is being added in the event of sequencing primer 3 ' end, is causing Producing ATP, this causes outburst luminescence in hole, and it uses CCD camera record.Reach the reading length more than or equal to 400 bases It is possible, 1 x 10 can be realized⁶Individual sequence is read, and this produces the sequence of up to 500,000,000 base pairs (Mb).

Solexa/Illumina platform (Voelkerding etc., Clinical Chem., 55:641-658, 2009；MacLean etc., Nature Rev. Microbiol., 7:287-296；U.S. Patent number 6,833,246；The U.S. is special Profit number 7,115,400；U.S. Patent number 6,969,488；Each is integrally incorporated herein with it by quoting) in, sequencing data Produce with short length reading form.In the method, end repairs single stranded fragmented DNA, to produce the blunt of 5'-phosphorylation End, then the single A base 3' end to fragment is added in Klenow-mediation.A-adds and promotes that adding T-highlights adapter widow's core Thuja acid, it is subsequently used in the template-adapter molecule on the flow cell surface that capture oligonucleotide anchor spreads.Anchor is used as PCR primer, but because template length and with other close adjacent to anchor oligonucleotide, PCR extends and causes " encircleing in (arching Over) " with the molecule of neighbouring anchor oligonucleotide hybridization on, to form bridge construction on flow cell surface.These DNA circles are allowed to become Property also cracks.Then the sequence of forward chain is measured with reversible dye terminator.The nucleotide sequence mixed is by glimmering after mixing The detection of light measures, and removes each fluorescent agent and blocking agent (fluor and before next dNTP adds circulation block).Sequence reading length is between 36 nucleotide-more than 50 nucleotide range, and each to analyze the total output run super Cross 1,000,000,000 nucleotide pairs.

With SOLiD technology (Voelkerding etc., Clinical Chem., 55:641-658,2009；MacLean Deng, Nature Rev. Microbiol., 7:287-296；U.S. Patent number 5,912,148；U.S. Patent number 6,130, 073；Each is integrally incorporated herein by quoting with it) nucleic acid molecules is carried out order-checking it is also related to template segments and few core Thuja acid adapter connects and beadlet adheres to and passes through emulsion-based PCR clonal expansion.Hereafter, the beadlet containing template is fixed to glass The deriving surface of glass flow cell, and make the primer annealing complementary with adapter oligonucleotide.But, it not to utilize this primer to carry out 3' extends, and is to provide 5' phosphate group on the contrary for being connected with exploratory probe (interrogation probe), and described probe contains There are two probe specificity bases, then one of 6 degeneracy bases and four fluorescent labelinies.In SOLiD system, exploratory probe Having 16 kinds of one of four kinds of fluorescent agents at two bases of 3' end of each probe and 5' end may combination.Fluorescence face The identity of color the most each probe is corresponding to the encoding scheme in particular color space.In many wheel (usual 7 take turns) probe anneals, companies Connect with fluoroscopic examination after, then degeneration, then carry out second take turns survey with relative to the primer of initial primers one base of deviation Sequence.In like fashion, template sequence can be reconstructed by calculating, and probe template base twice, this causes accuracy to improve.Sequence 35 nucleotide of reading length average out to, what each order-checking ran always exports more than 4,000,000,000 bases.

In certain embodiments, nano-pore order-checking is used (to see for example Astier etc., J Am Chem Soc. 2006 On February 8, in；128 (5): 1705-10, be incorporated herein by).The theory that nano-pore order-checking is based on is soaked with when nano-pore When entering to conduct fluid and it is applied electromotive force (voltage), event is relevant: under these conditions, can be observed owing to passing through Nano-pore conduction Weak current caused by ion, the magnitude of current is the most sensitive to nano-pore size.If DNA molecular is by (or DNA divides Subdivision is passed through) nano-pore, then this can cause the order of magnitude of the electric current by nano-pore to change, and thereby makes to record DNA and divides The sequence of son.Nano-pore can be the Solid-state hole or nano-pore based on protein, example manufactured on metal and/or nonmetallic surface Such as alpha hemolysin (Clarke etc., Nat. Nanotech., 4,2009 on February 22: 265-270).

The HeliScope of Helicos BioSciences (Voelkerding etc., Clinical Chem., 55: 641-658, 2009；MacLean etc., Nature Rev. Microbiol., 7:287-296；U.S. Patent number 7,169, 560；U.S. Patent number 7,282,337；U.S. Patent number 7,482,120；U.S. Patent number 7,501,245；U.S. Patent number 6, 818,395；U.S. Patent number 6,911,345；U.S. Patent number 7,501,245；Each is integrally incorporated this by quoting with it Literary composition) it is first business-like list-molecule order-checking platform.The method need not clonal expansion.By template DNA fragmentation, at 3' End polyadenylation, and last adenosine contains fluorescent labeling.On flow cell surface by the polyadenylic acid template segments of degeneration It is connected with poly-(dT) oligonucleotide.The initial physical location of the template molecule captured by CCD camera record, then cracking labelling is also Wash off.Order-checking is realized by adding the dNTP reagent of polymerase and order interpolation fluorescence-labelling.Incorporation event produce corresponding to The fluorescence signal of dNTP, was captured signal by CCD camera before each dNTP of wheel adds.Sequence reading length is between 25-50 Nucleotide range, and each analyze run always export more than 1,000,000,000 nucleotide pairs.Other emerging single-molecule sequencing method Including order-checking (Voelkerding etc., Clinical Chem., the 55:641-in real time by synthesizing with VisiGen platform 658, 2009；U.S. Patent number 7,329,492；U.S. Patent Application Serial Number 11/671956；U.S. Patent Application Serial Number 11/ 781166；Each is integrally incorporated herein with it by quoting), wherein with polymerase and the fluorescence acceptor of fluorescent decoration Molecule carries out chain extension to DNA profiling fixing, that cause, and this causes producing detectable fluorescent energy when adding nucleotide Resonance transfer (FRET).Another the real-time Single-molecule Sequencing System (Voelkerding developed by Pacific Biosciences Deng, Clinical Chem., 55:641-658,2009；MacLean etc., Nature Rev. Microbiol., 7: 287-296；U.S. Patent number 7,170,050；U.S. Patent number 7,302,146；U.S. Patent number 7,313,308；United States Patent (USP) Numbers 7,476,503；All it is incorporated herein by) utilize the reacting hole of diameter 50-100 nm, comprise about 20 zeptoliter (10 x 10^-21L) reaction volume.With immobilized template, modified phi29 archaeal dna polymerase and high office The fluorescently-labeled dNTP of portion's concentration implements sequencing reaction.High local concentrations and successive reaction condition make can be by swashing with laser Send out, fiber waveguide and CCD camera carry out fluorescence signal detection and carry out captured in real time and mix event.

In certain embodiments, the use zero mode waveguide (zero-developed by Pacific Biosciences is used Mode waveguide, ZMW) unimolecule in real time (SMRT) DNA sequencing method or the like.By this technology at SMRT core Implementing DNA sequencing on sheet, each chip contains thousands of zero mode waveguide (ZMW).ZMW is the hole of a diameter of tens nanometer, its Deposition 100nm metal film on silicon oxide substrates manufactures.Each ZMW becomes nano-photon and manifests room (nanophotonic visualization chamber), it provides the detection of lucky 20 zeptoliter (10-21 liter) Volume.With this volume, monomolecular activity can detect in the nucleotide background of thousands of labellings.

ZMW provides for observing the window of archaeal dna polymerase along with it implements order-checking by synthesis.In each indoor, allow Single DNA polymerase molecule is attached to lower surface so that it is permanently located in detection volume.Then to promote enzyme acceleration, standard (each class different colours is glimmering for (Phospholinked) nucleotide that really phosphorus is connected by the high concentration of property and processivity Light blob labelling) it is incorporated in reaction solution.Owing to ZMW size is little, even with these high related concentrations biology, detection bodies Amass and the most only occupied by nucleotide within a small amount of time.It addition, the access to detection volume is very fast, only continue several gsec, This is extremely short owing to carrying the distance of nucleotide diffusion.This result is extremely low background.

Along with archaeal dna polymerase mixes complementary nucleotide, each base keeps several ms in detection volume, and it compares nucleoside Acid diffusion long-pending time long number the order of magnitude spent of turnover detection bodies.During this period, the Fluorophore emission color pair being occupied Should be in the fluorescence of Base Identity.Then, the part circulated as natural incorporation, fluorogen is maintained at suitable position by polymerase cracking The key put, dyestuff diffuses out detection volume.After incorporation, signal is immediately returned to baseline, and this process repeats.

In the case of not hindered and be uninterrupted, archaeal dna polymerase continues to mix alkali with the speed of dozens of base per second Base.By this way, in several minutes, produce the most natural long-chain of DNA.On SMRT chip between all thousands of ZMW together Time and be carried out continuously in real time detection.PacBio researcher is it has proved that the method has the energy producing thousands of nucleotide reading lengths Power.

Embodiment

In order to provide some exemplary embodiment of the present invention, presenting following example, described embodiment is not intended to Limit its scope.

Embodiment 1

Stable enzymatic mixture

The present embodiment illustrates the exploitation of the stable enzymatic mixture for methods such as such as whole genome amplifications (WGA).Such as Figure 15 Described, have detected a lot of excipient formulation and Phi-29 polymerase I and the compatibility of the WGA of inorganic pyrophosphatase.Find 16 kinds The enzyme that the stabilized formulations (referred to as excipient) of patent generally uses for successful stabilization in WGA is useless.Be found that BSA be for Stablizing the important component of these enzymes, it is as shown in figure 16.

A kind of preferably excipient developed is referred to as " Ibis formula 33 (Ibis Formula 33) ".How to prepare this Being described as follows of formula.By 8968 u/ml Phi-29 polymerase (Monserate), 180 u/ml polymerase I (Epicentre Biotechnologies), the mixture of 3.6 u/ml inorganic pyrophosphatases (U.S. Biochemical) with containing following thing The solution of matter mixes with 1:1: 20% trehalose, 10 mM (NH₄)₂SO₄、12 MgCl₂、50 mM Tris pH 7.6、0.05% Polysorbate40,4mM DTT and reach the BSA of level of final concentration 0.25% (due to change in volume between dialysis period, in dialysis procedure Middle BSA concentration is higher so that the final volume correct after can reach dialysis of solution).In room temperature with 30 KDa dialyzers by this mixture Carried out dialysis in 4 hours containing 20% trehalose, 10 mM (NH₄)₂SO₄、12 MgCl₂、50 mM Tris pH 7.6、0.05% In the solution of polysorbate40 and 4mM DTT.

Then from the enzymatic mixture of dialyzer removal dialysis, and with containing 20% trehalose, 10 mM (NH₄)₂SO₄、12 MgCl₂, the solution of 50 mM Tris pH 7.6,0.05% polysorbate40s and 4mM DTT reach correct final volume (thoroughly In analysis/dilution, dilution level controls each lyophilizing unit exists how many enzymes).Then this solution is distributed to suitable volumes In, freezing, apply fine vacuum to remove water, to produce the compositions of lyophilizing via distilling.

Multiple BSA level (0.13%-1% final concentration) and 0.5% (final concentration) is detected by above-mentioned same general flow process The addition of PEG-8000.This testing result is shown in Figure 17.

Using the excipient of exploitation in this work, we successfully prove to maintain all three enzyme in freeze-drying process Enzymatic activity, and the stability of these enzymes from room temperature (-25 DEG C) less than within 10 days, being significantly increased at 40 DEG C more than 2 months. When comparing with the enzymatic mixture of fresh (being i.e. stored in-20 DEG C of refrigerators until using), this formula also improves overall reaction productivity. The long-time stability result of room temperature storage is shown in Figure 18.This figure shows that the enzyme of lyophilizing is when being stored in room temperature in Ibis formula 33 Show when reaching 4 months and be equal to fresh enzyme.Figure 19 shows the extended storage stability reaching 2 months with 40 DEG C of accelerated ageings, its It is equal to room temperature storage about 6 months.Figure 19 shows that in Ibis formula 33, the enzyme of lyophilizing shows and after 2 months at 40 DEG C Fresh enzyme is equal to.

Embodiment 2

Sample preparation methods

The present embodiment illustrates the various sample preparation methods that can such as be used for microfluidic device.Described method makes available single Generic buffer, this makes to be retained in single pipe sample to be possibly realized.Described method summary comprises the following steps: cracks and carries Take；Whole genome amplification (or other amplification method)；The fragmentation of DNA；Blunt end；Connect；The not exclusively removal of product；With Final purification (clean-up).Some details multiple in described step set forth below.

I. Exemplary fragments method

Available sample (the such as WGA sample) fragmentation being made amplification by the device formed with lower member: 1) ultrasonoscope assembly: The miniature ultrasonic aerosol apparatus Sonaer 241V of 2.4 megahertzs；2) transducer: golden coated aerosol apparatus crystal Sonaer 24AU；3) Power supply: 24 volts of power supply Sonaer ST624；With 4) lid: the plastics of machining are to produce shell in ultrasonoscope assembly (enclosure).With ultrasonic 10 minutes altogether of implementing of this method, and use within 15 seconds, open, 50% work at interval that 15 seconds close Circulate.

Ii. coupled reaction speed is optimized

Connection speed is optimized, the amount of this end product produced in making can be significantly increased to 30 minutes from initial condition.Optimize Including the buffer composition modified or concentration and the enzyme concentration of modification.Parameter is as shown in table 1 below.Reaction condition is 30 DEG C, 30 points Clock and 65 DEG C, 10 minutes.

Table 1

The important parameter optimized includes: ligase concentration, ATP concentration, PEG concentration, the addition of sodium pyrophosphate after reacting 1 minute. Optimal conditions causes the end product after dramatically increasing 30 minutes.

Iii. the effective polishing of WGA enzyme in amplification buffer

Inspection condition is to test polishing efficiency.Test condition includes: the response time, reaction temperature, dATP concentration, DTT concentration, PEG concentration, spermidine concentration, polylysine concentration.The parameter used is shown in table 2 below.The reaction condition used is: 37 DEG C, 5 minutes；50 DEG C, 2 minutes；With 75 DEG C, 10 minutes.

Table 2

The analysis method used is electronic spraying ionization time of flight mass spectrometry (ESI-TOF MS).(fond) weight found Wanting parameter is that (this is important parameter to reaction temperature, because observe few A-hangover less than 55 DEG C, and observes higher than 55 DEG C Significantly A-hangover), dATP concentration (increase dATP concentration and increase A-hangover).Found that the product of 100% is become suitably Blunt, the end of about 70% is A-hangover.

Iv. exonuclease purifies coupled reaction

Implement coupled reaction with " condition 1 " shown in table 1, with exo III and exo VII digest (have slightly different hair clip/ Insertion sequence).Before hair clip shown below and insert concentration are coupled reaction.During coupled reaction, major part insert converts For " end product ".Condition used is shown in Table 3.Reaction condition be 37 DEG C, 1 hour and 70 DEG C, 10 minutes.

Table 3

It should be noted that WGA amplimer is included in all these reaction, even if not determine that whether it can cause any for they The part of the reaction of suppression (not observing any one).Similarly, dNTP is included in reaction (such as coupled reaction or excision enzyme Digestion) in, even if they need not in the reaction determining if to cause any suppression (not observing any one).Core Acid excision enzyme degraded non-annularity template, this removes any DNA in addition to end-product.

All publications and patents mentioned by this specification is incorporated herein by.Those skilled in the art are shown And be clear to, in the case of without departing from scope and spirit of the present invention, the method for the invention and compositions can be carried out respectively Plant modifications and changes.Although already illustrating the present invention together with concrete preferred embodiment, it is to be understood that claimed this Bright should not be unduly limited to described specific embodiments.It is true that to various equivalent modifications it is readily apparent that use Various amendments in the described mode implementing the present invention, it is intended within the scope of the appended claims.

Claims

1. the method producing nucleic acid sequencing library, comprises:

A) biological sample is introduced the load port of microfluidic card；

B) in the nucleic acid extraction branch road of described microfluidic card, in described biological sample, nucleic acid is extracted；

C) expand described nucleic acid to produce the nucleic acid of amplification, described amplification in the amplification branch road of described microfluidic card Branch road comprises stable enzymatic mixture；

D) in the fragmentation branch road of described microfluidic card, the nucleic acid of described amplification is carried out fragmentation, to produce fragmentation Nucleic acid；

E) in the described fragmentation branch road of described microfluidic card, the end of the nucleic acid of described fragmentation is carried out polishing, to produce The nucleic acid of the fragmentation of the raw end comprising polishing；

F) joint at described microfluidic card connect in branch road by the nucleic acid that comprises the described fragmentation of the end of described polishing with Nucleic acid linker connects, to produce described nucleic acid sequencing library；

G) the described joint at described microfluidic card connects the core in branch road to the described fragmentation being connected with described nucleic acid linker The end of acid carries out polishing, to produce the nucleic acid sequencing library of the end comprising polishing；With

H) in the purification branch road of described microfluidic card, the described nucleic acid sequencing library of the end comprising polishing is purified, To produce the nucleic acid sequencing library of purification.

2. the process of claim 1 wherein that described microfluidic card comprises:

G) the cracking branch road being operably connected with described load port, wherein said cracking branch road comprises:

I) cracking room, and

Ii) lysis buffer.

3. the process of claim 1 wherein that described amplification branch road comprises enzyme, described enzyme is used for implementing whole genome amplification (WGA), Polymerase chain reaction (PCR), or transcript mediated amplification (TMA).

4. the process of claim 1 wherein that described amplification branch road comprises enzyme, described enzyme is selected from: Phi-29 polymerase, escherichia coli DNA polymerase i, inorganic pyrophosphatase or its combination in any.

5. the process of claim 1 wherein that described stable enzymatic mixture comprises: i) BSA；Ii) sugar；And iii) at least one choosing Other component from following: inorganic salt, divalent metal, buffer agent, emulsifying agent and reducing agent.

6. the process of claim 1 wherein that described stable enzymatic mixture comprises: i) BSA；Ii) sugar；Iii) inorganic salt；Iv) two Valency metal cation；V) buffer agent；Vi) emulsifying agent；And reducing agent vii).

7. the method for claim 6, described BSA exists with the concentration of 0.05%-3.0%, and described sugar is deposited with the concentration of 5-35% , described inorganic salt exists with the concentration of 1 mM-25 mM, and described divalent metal is with the concentration of 1 mM-30 mM Existing, described buffer agent exists with the concentration of 10 mM-100 mM, and described emulsifying agent is deposited with the concentration of 0.01%-0.15% , and described reducing agent is with the concentration existence of 1 mM-10 mM.

8. the method for claim 2, wherein said microfluidic card comprises waste compartment, described waste compartment and described cracking branch road, institute State nucleic acid extraction branch road, described amplification branch road, described fragmentation branch road and/or described joint connection branch road and described purification props up One or more in road are operably connected.

9. the process of claim 1 wherein that described microfluidic card comprises the process instrumentation for making buffer and sample flow through.

10. the method for claim 1, comprises offer and one or more packs, described in pack and comprise described cracking and delay Rush liquid, described lavation buffer solution, described elution buffer, described stable enzymatic mixture, be configured to digest described amplification One or more in the described reagent mixture of nucleic acid and described ligase mixture.

The method of 11. claim 2, the described cracking branch road of wherein said microfluidic card, described nucleic acid extraction branch road, described expansion Increase expenditure road, described fragmentation branch road, described joint connects branch road and described purification branch road and is operably connected successively.

The method of 12. claim 2, wherein said extraction branch road comprises lavation buffer solution and elution buffer, and wherein said Lavation buffer solution, described elution buffer and described lysis buffer are single buffer.

13. the process of claim 1 wherein that described joint is nucleic acid adapter.

14. the process of claim 1 wherein that described biological sample is human sample, Patient Sample A, biological warfare sample, environment Sample, pedotheque or comprise target gene group and the sample of background genes group.

15. 1 kinds of methods checking order nucleic acid, comprise:

A) biological sample is introduced the load port of microfluidic card；

F) nucleic acid and the nucleic acid of the described fragmentation of the end of described polishing will be comprised in the joint branch road of described microfluidic card Joint connects, to produce described nucleic acid sequencing library；

G) to the nucleic acid of the described fragmentation being connected with described nucleic acid linker in the described joint branch road of described microfluidic card End carries out polishing, to produce the nucleic acid sequencing library of the end comprising polishing；

H) in the purification branch road of described microfluidic card, the described nucleic acid sequencing library of the end comprising polishing is purified, To produce the nucleic acid sequencing library of purification；

I) the nucleic acid sequencing library of described purification is taken out in the outlet from described microfluidic card；With

J) the nucleic acid sequencing library of described purification is introduced sequencing system.

The method of 16. claim 15, wherein sequencing system comprises chain terminating agent order-checking, dye terminator checks order, based on amplification Order-checking of future generation, Manganic pyrophosphate complex initiation, support oligonucleotide connects and detection (SOLiD) is checked order, based on the nonamplifie next generation Order-checking, single-molecule sequencing, nano-pore are checked order, are checked order and the unimolecule of use zero mode waveguide (ZMWs) by the real-time of synthesis (SMRT) order-checking in real time.