CN102703432A - Method, reagent and kit for constructing nucleic acid library - Google Patents
Method, reagent and kit for constructing nucleic acid library Download PDFInfo
- Publication number
- CN102703432A CN102703432A CN2012101887507A CN201210188750A CN102703432A CN 102703432 A CN102703432 A CN 102703432A CN 2012101887507 A CN2012101887507 A CN 2012101887507A CN 201210188750 A CN201210188750 A CN 201210188750A CN 102703432 A CN102703432 A CN 102703432A
- Authority
- CN
- China
- Prior art keywords
- dna
- label
- transposon
- stranded
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 32
- 102000039446 nucleic acids Human genes 0.000 title abstract description 8
- 108020004707 nucleic acids Proteins 0.000 title abstract description 8
- 150000007523 nucleic acids Chemical class 0.000 title abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 title abstract description 5
- 108020004414 DNA Proteins 0.000 claims abstract description 141
- 239000012634 fragment Substances 0.000 claims abstract description 22
- 102000053602 DNA Human genes 0.000 claims abstract description 20
- 108010020764 Transposases Proteins 0.000 claims abstract description 18
- 102000008579 Transposases Human genes 0.000 claims abstract description 18
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims abstract description 8
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims abstract description 8
- 238000013467 fragmentation Methods 0.000 claims description 45
- 238000006062 fragmentation reaction Methods 0.000 claims description 45
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 12
- 238000012360 testing method Methods 0.000 claims description 7
- 102000012410 DNA Ligases Human genes 0.000 claims description 6
- 108010061982 DNA Ligases Proteins 0.000 claims description 6
- 102000003960 Ligases Human genes 0.000 claims description 6
- 108090000364 Ligases Proteins 0.000 claims description 6
- 108091008146 restriction endonucleases Proteins 0.000 claims description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 108060002716 Exonuclease Proteins 0.000 claims 1
- 102000013165 exonuclease Human genes 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 21
- 101710163270 Nuclease Proteins 0.000 abstract description 7
- 238000012163 sequencing technique Methods 0.000 abstract description 5
- 108091034117 Oligonucleotide Proteins 0.000 abstract description 4
- 238000000338 in vitro Methods 0.000 abstract 1
- 230000017105 transposition Effects 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 15
- 239000000203 mixture Substances 0.000 description 15
- 102000004190 Enzymes Human genes 0.000 description 12
- 108090000790 Enzymes Proteins 0.000 description 12
- 239000000463 material Substances 0.000 description 12
- 230000008569 process Effects 0.000 description 12
- 102000004594 DNA Polymerase I Human genes 0.000 description 11
- 108010017826 DNA Polymerase I Proteins 0.000 description 11
- 239000000523 sample Substances 0.000 description 7
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 241000607598 Vibrio Species 0.000 description 4
- 206010047400 Vibrio infections Diseases 0.000 description 4
- 240000008042 Zea mays Species 0.000 description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 235000005822 corn Nutrition 0.000 description 4
- 238000013016 damping Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 230000012447 hatching Effects 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 230000007023 DNA restriction-modification system Effects 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- HSXUHWZMNJHFRV-UHFFFAOYSA-L disodium;6-oxido-5-phenyldiazenyl-4-sulfonaphthalene-2-sulfonate Chemical compound [Na+].[Na+].OC1=CC=C2C=C(S([O-])(=O)=O)C=C(S([O-])(=O)=O)C2=C1N=NC1=CC=CC=C1 HSXUHWZMNJHFRV-UHFFFAOYSA-L 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 235000019833 protease Nutrition 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 230000004544 DNA amplification Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- SAPGTCDSBGMXCD-UHFFFAOYSA-N (2-chlorophenyl)-(4-fluorophenyl)-pyrimidin-5-ylmethanol Chemical compound C=1N=CN=CC=1C(C=1C(=CC=CC=1)Cl)(O)C1=CC=C(F)C=C1 SAPGTCDSBGMXCD-UHFFFAOYSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 238000012197 amplification kit Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000001963 scanning near-field photolithography Methods 0.000 description 1
- 238000007671 third-generation sequencing Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method, a reagent and a kit for establishing a nucleic acid library with labels, and adopts an in-vitro transposition method. The method comprises the following steps of: randomly breaking target deoxyribonucleic acid (DNA) by using transposase, treating by using nuclease and DNA polymerase, and connecting required oligonucleotide sequences to generate a DNA library with labels. According to the method, DNA fragments of all required labels are allowed to be connected, so that DNA fragments of generated labels can be directly applied to different second-generation sequencing platforms; and the invention comprises, but is not limited to instruments manufactured by companies such as ILLUMINA, Life Technologies (ABI), Roche, HELICOSBiotechnologies and Pacific Biosciences, and is also applicable to sequencers produced by other manufacturers.
Description
Technical field
The present invention provides method, reagent and test kit for setting up nucleic acid library.Transposase, nucleicacidase and ligase enzyme particularly in building the storehouse process, have been used.
Background technology
How effectively with efficiently, set up the library with the DNA and the RNA of various differing materials, this is a challenge to the check order researchist and the operator of (NGS) of the s-generation.Existing technology, comprise through physical means: supersonic method or atomization interrupt double-stranded DNA, and this needs more a large amount of parent materials.This process is very long, is difficult to carry out with high-throughout mode.The scientist of NEB develops recently and uses FRAGMENTASE, in centrifuge tube, carries out enzyme reaction, and this interrupts with physics, and to compare be a good selection, but still a large amount of parent material of needs.EPICENTRE company has developed NEXTERANEXTERA reagent, builds the storehouse process thereby the researchist can be accomplished in 2 hours, and as long as parent material is 50NG.Yet the sequencing primer that it needs and the sequencing primer of existing order-checking platform are incompatible, and this is to let many potential users bring down a bit.
Another challenge that s-generation order-checking Jian Ku field faces is, the development of order-checking platform is very fast, and the research and development of new sequenator only need the time in several years, and it is very fast to update.Third generation sequencing technologies also might be in the near future can throw down the gauntlet or replaces s-generation technology.The fast development of new technology and equipment, for those need carry out very little adjustment, just can with database technology be applied to new instrument and technology platform build the storehouse product brought the technology and the coml competitive power, also brought huge commercial opportunity for those enterprises.
The objective of the invention is to set up the preparation method in a library; Can be used for all new sequenators existing and the few change that is about to release, can be used for high-throughput, and be applicable to machine operation; Only need less parent material, can produce satisfied sequencing result.
Summary of the invention
The invention provides method, component and the test kit of random fracture and while labeled dna fragment.Particularly, the present invention adopts external swivel base, comes the labeling nucleic acid storehouse to use to be fit to order-checking of future generation with nucleicacidase and ligase enzyme.
In some instances, the inventive method is that random fracture and while labeled dna fragment provide method, and suitable dna fragmentation can be the genomic dna of purifying; Double-stranded CDNA; Double-stranded DNA product behind the PCR, or the large fragment DNA of rolling-circle replication generation, the source of these DNA can be the people; Animal, plant or mikrobe.
Comprising: A) (TRANSPOSONE END-TE) mix with double-stranded target dna, under the katalysis of transposase, double-stranded transposon inserts in the target dna sequence at random with transposase and transposon end sequence.Transposase can comprise: (TN5, TN3, TN7, TN8, TN9, TN10, TN11, TN2, TN4, TN6 and their various sudden change versions).In the transposon insertion process, a terminal strand of transposon is held with the 5' that covalent linkage is connected dna segment, and the terminal second chain of transposon does not link to each other with dna segment, but under annealing conditions, still exists with double chain form.The room that 9 bases are arranged between the swivel base end of second chain and dna fragmentation 3' end.Just produced random fracture thus, and by the dna fragmentation of (TE) mark.These fragments start from double-stranded transposon end sequence from 5' to 3', are the single stranded DNA sequence of 9 base pairs then, are double-stranded target dna fragment then, and the single stranded DNA sequence of 9BP and double-stranded transposon are terminal; B) from the dna segment of transposon end sequence (TE) mark, remove double-stranded transposon end sequence (TE), generate unlabelled dna fragmentation; C) dna marker that connection needs on unlabelled dna segment produces a new markd dna fragmentation.
In some instances, transposon can comprise: (TN5).
In some instances, the second chain that transposon is terminal is a free, and the discord target dna sequence is covalently bound.Under hot conditions, the dna fragmentation of transposon end sequence (TE) mark is separated, and target dna partly remains unchanged.After removing the terminal second chain of transposon, can use some specific single stranded DNA nucleicacidases to remove article one chain of remaining transposon and the single stranded DNA of 9 base pairs.
In the other instance, mend flat 5' end with archaeal dna polymerase, add the A tail at the 3' end.The polysaccharase that is applicable to this application not only is confined to archaeal dna polymerase and TAQ archaeal dna polymerase, and large fragment DNA polymerase I (KLENOW fragment) also can use.The KLENOW fragment is to have removed the archaeal dna polymerase I of 3' to the 5' 5 prime excision enzyme activity.And mend flat 5' end with the DNA polysaccharase, and forming flat terminal (rather than A tail), then to use to have the archaeal dna polymerase of 3' to the 5' 5 prime excision enzyme activity, like the T7 archaeal dna polymerase, T4 archaeal dna polymerase, big segment archaeal dna polymerase I etc.
In some embodiments of the present invention, need be coupled with the label of particular requirement by the target dna of random fracture, the dna fragmentation that produces mark is to be applicable to the needs of downstream application.Dna ligase can couple together the DNA of mark and dna segment like the T4 dna ligase.According to the dna fragmentation of downstream application needs, can add required label: like the order-checking label, the BARCODE label, address tag, the restriction enzyme site mark is caught mark, certification mark, amplification label, or transcripting promoter sequence label.The use that also can link together of a plurality of marks.Two ends at unlabelled double chain DNA fragment can connect identical or different label.In some instances, required sequence label is provided by many two generations order-checkings company, like ILLUMINA, and LIFE TECHNOLOGIES, ROCHE, COMPLETE GENOMICS AND PACIFIC BIOSCIENCES etc.
The present invention is employed in external transposon system, and the target dna random fracture is to required segment size, terminal with specific single stranded DNA nucleicacidase equating, and comes labeled dna fragment at the required label of flat terminal connection of DNA.These fragments that are labeled can be used for various application, comprise the analysis of the macro genome DNA of patient's sample, icp gene group analysis, genome sequencing, SNP gene type, in situ hybridization.
The invention provides the dna fragmentation library that a kind of effective means produces the random fracture of mark, adapt to various order-checking platform.An advantage of the invention is that it allows to connect the dna fragmentation of any required mark, make the dna fragmentation of the mark that produces; Can be applied directly to different s-generation order-checking platforms, include but not limited to by ILLUMINA LIFE TECHNOLOGIES (ABI); ROCHE; HELICOSBIOTECHNOLOGIES, the instrument of these manufactured of PACIFIC BIOSCIENCES equally also is applicable to the sequenator of other manufacturers produce.Another advantage of the present invention is that method of the present invention can improve flux at an easy rate, and carries out automated operation by robot.
Technical term " transposon is terminal " or " transposon end sequence " are meant the double-stranded DNA of being made up of specific nucleotide sequence.It can form one at the external swivel base complex body that the swivel base function is arranged with transposase.This is to carrying out the swivel base reaction time to insert double-stranded target DNA be vital external.The transposon end is a double chain DNA sequence; Article one, chain (transferable chain) can be covalently bound with the 5' end of target DNA; Second chain (non-transfer chain) directly is not connected with target DNA in the swivel base process, but to be annealed reaction be connected with the terminal article one chain (transferable chain) of swivel base for this.Specific transposon end sequence can discerned and use to each transposase in the swivel base reaction.
" transposase " is meant the enzyme that can form the function mixture with the swivel base end sequence, and the mixture of formation can be inserted in the target DNA by catalysis transposon end sequence.The provirus intergrase is also included within the transposase.
" swivel base reaction " or " external swivel base reaction " is meant that the transposase mixture can interrupt target DNA, is inserted in the transposon end sequence in the target DNA segment.When the two ends of same target DNA change two transposons over to, the dna segment between those two transposons is separated opening.Therefore the swivel base reaction can interrupt and carries out mark endways the purpose segment at random.The swivel base process can produce the single stranded DNA of 9 bases between target dna and transposon end sequence.
Description of drawings
Fig. 1: the DNA of the same race of identical original bulk is in the difference between molecular weight area after the different buffer solution system cleaved.A uses the interval damping fluid of lower molecular weight; B uses the interval damping fluid of HMW.
Fig. 2: new-generation sequencing technology DNA library construction result: M, 1KB PLUS DNA LADDER (LIFE TECHNOLOGIES, 10787-018); 2, human gene group DNA behind the 50NG purifying; 3, and after the 50NG human gene group DNA adopts rolling-circle replication (ILLUSTRA GENOMIPHI V2 DNA AMPLIFICATION KIT, GE HEALTH CARE LIFE SCIENCES, 25-6600-30); 4,50NG PCR product (fragment that 4KB obtains from human gene group DNA's amplification with particular probe, PHUSION HOT START FLEX 2X MASTER MIX, NEB M0536L).
Fig. 3: M, and 1KB PLUS DNA LADDER (LIFE TECHNOLOGIES, 10787-018); 1, corn gene group DNA behind the 100NG purifying; 2, the double chain CDNA product that 100NG corn MRNA reverse transcription forms
Fig. 4: M, and 1KB PLUS DNA LADDER (LIFE TECHNOLOGIES, 10787-018); 1, hammer vibrios DNA behind the 50NG purifying; 2, the hammer vibrios DNA that interrupts obtains double chain DNA fragment through PCR.
Embodiment
Utilize transposase and transposon end sequence (TE) that target DNA is interrupted at random, make it reach the clip size of our expectation.Usually we place target DNA and transposase and specific swivel base end sequence thereof the damping fluid of swivel base reaction to hatch.Add target DNA (for example human gene group DNA), transposase (EZ-TN5 TRANSPOSASE, EPICENTRE) and the amount of specific swivel base end sequence (0.01NG/UL-1UG/UL) according to different application and different.The composition of damping fluid and concentration (1 * GPS BUFFER, 25MM TRIS-HCL, 2MM DTT), temperature of hatching and time thereof also will decide according to the size of want cutting fragment.As shown in Figure 1: as in two kinds of different buffer solution systems, to be different between the molecular weight area that DNA is ruptured.Adding reaction terminating liquid (10% sucrose, 66 MM EDTA, 20 MM TRIS, 0.1% SDS, 0.9% ORANGE G, AND 100 G/ML Proteinase Ks), 10 minutes afterreactions of 50 ℃ of heating can stop.The size that can on 1% sepharose (V2111 of PROMEGA company) electrophoresis, check post-rift dna fragmentation.
In case the swivel base reaction is accomplished, the dna fragmentation of transposon end sequence (TE) mark is that (ZYMO RESEARCH CORPORATION, IRVINE CA) carries out purifying to available ZYMODNA CLEAN AND CONCENTRATOR KIT.
(S1 NUCLEASE, PROMEGA) (the EXO VII, mixture PROMEGA) is hatched at 37 ℃, up to the terminal equating of the dna fragmentation of strand with excision enzyme for the dna fragmentation of transposon end sequence (TE) mark that purifying is good and single stranded DNA restriction endonuclease.Come purifying not have the DNA of mark with ZYMODNA CLEAN AND CONCENTRATOR KIT.The end sequence of transposon so far is removed, and has produced unlabelled dna fragmentation.
In the unlabelled dna fragmentation of above generation, add the big fragment of archaeal dna polymerase I of no 5 prime excision enzyme activity, promptly the KLENOW fragment was hatched 20 minutes at 37 ℃.Dna fragmentation through this processing can link to each other with oligonucleotide joint.
The dna fragmentation that does not have mark, the label that checks order certainly, the mixture of the T4 dna ligase that the order-checking label of BARCODE label and ligation are used was handled 5 minutes or was spent the night at 16 ℃.Add EDTA or stopped ligation in 20 minutes 72 ℃ of reactions.The dna fragmentation library that has label promptly can be used to downstream analysis.
Parent material with TN5 at 5 minutes (NEXTERA DNA SAMPLE PREPARATION KIT of LMW 500C; EPICENTRE; GA09115); Then 37 degree with S1 NUCLEASE (PROMEGA M5761) is hatched 30 minutes. the dna fragmentation that adds the A tail that obtains with KLENOW from 3' to 5' circumscribed interrupting (NEB, 0212L); 370C hatching 20MINUTES. 160C used fast link test kit link order-checking street corner in 5 minutes. and the tagged DNA that obtains carries out pcr amplification (PHUSION HOT START FLEX 2X MASTER MIX according to the PCR operating process of standard with following system; NEB M0536L). (ZYMO RESEARCH CORPORATION, IRVINE CA) come purifying and enrichment to all samples with ZYMODNA CLEAN AND CONCENTRATOR KIT in the process of at every turn handling with enzyme; The PCR product that obtains is at 1% AGAROSE gel electrophoresis and with SYBR GREEN I NUCLEIC ACID dyeing (LIFE TECHNOLOGIES; S7567), resulting result see the picture 2 that takes with SAFE IMAGER BLUE LIGHT TRANSILLUMINATOR (LIFE TECHNOLOGIES, G6600).
The genomic DNA fragment of employing corn and corn MRNA as material, utilize transposase and transposon end sequence (TE) that target DNA is interrupted at random through reverse transcription synthetic CDNA, make it reach the clip size of our expectation.The buffer system that material adopts is (1 * GPS BUFFER, 25MM TRIS-HCL), (370C under different incubation conditions; 20 minutes) handle sample, add reaction terminating liquid (10% sucrose, 66 MM EDTA then; 20 MM TRIS, 0.1% SDS, 0.9% ORANGE G; AND 100 G/ML Proteinase Ks), 10 minutes afterreactions of 50 ℃ of heating can stop.Can under 1% gel electrophoresis, observe cracked dna fragmentation size.
In case the swivel base reaction is accomplished, the dna fragmentation of transposon end sequence (TE) mark is that (ZYMO RESEARCH CORPORATION, IRVINE CA) carries out purifying to available ZYMODNA CLEAN AND CONCENTRATOR KIT.
(S1 NUCLEASE PROMEGA) is hatched at 37 ℃ with the mixture of excision enzyme, up to the terminal equating of the dna fragmentation of strand for the dna fragmentation of transposon end sequence (TE) mark that purifying is good and single stranded DNA restriction endonuclease.Come purifying not have the DNA of mark with ZYMODNA CLEAN AND CONCENTRATOR KIT.The end sequence of transposon so far is removed, and has produced unlabelled dna fragmentation.
In the unlabelled dna fragmentation of above generation, add the big fragment of archaeal dna polymerase I of no 5 prime excision enzyme activity, promptly the KLENOW fragment was hatched 20 minutes at 37 ℃.Dna fragmentation through this processing can link to each other with oligonucleotide joint.
The dna fragmentation that does not have mark,, catch mark, the mixture of the T4 dna ligase that the order-checking label of certification mark and ligation are used was handled 5 minutes or was spent the night at 16 ℃.Add EDTA or stopped ligation in 20 minutes 72 ℃ of reactions.The dna fragmentation library that has label promptly can be used to downstream analysis.
Parent material with TN5 at 5 minutes (NEXTERA DNA SAMPLE PREPARATION KIT of LMW 500C; EPICENTRE; GA09115), then 37 degree with S1 NUCLEASE (PROMEGA M5761) is hatched 30 minutes. the dna fragmentation that adds the A tail that obtains; With KLENOW circumscribed (NEB that interrupts from 3' to 5'; 0212L), 370C hatching 20MINUTES. 160C used fast link test kit link joint in 5 minutes. the tagged DNA that obtains carries out pcr amplification (PHUSION HOT START FLEX 2X MASTER MIX, NEB M0536L) according to the PCR operating process of standard with following system. all samples in the process of at every turn handling with enzyme with ZYMODNA CLEAN AND CONCENTRATOR KIT (ZYMO RESEARCH CORPORATION; IRVINE; CA) come purifying and enrichment, the PCR product that obtains 1% AGAROSE gel electrophoresis and with SYBR GREEN I NUCLEIC ACID dyeing (LIFE TECHNOLOGIES, S7567); The picture 3 that resulting result takes with SAFE IMAGER BLUE LIGHT TRANSILLUMINATOR (LIFE TECHNOLOGIES, G6600).
Embodiment 3
Adopt the genomic dna of hammer vibrios and with the hammer vibrios DNA that interrupts at round-robin PCR double chain DNA fragment as material, utilize transposase and transposon end sequence (TE) that target DNA is interrupted at random, make it reach the clip size that we expect.The buffer system that material adopts is ((1 * GPS BUFFER, 25MM TRIS-HCL), (250C under different incubation conditions; 2 hours) handle sample, add reaction terminating liquid (10% sucrose, 66 MM EDTA then; 20 MM TRIS, 0.1% SDS, 0.9% ORANGE G; AND 100 G/ML Proteinase Ks), 10 minutes afterreactions of 50 ℃ of heating can stop.Can under 1% gel electrophoresis, observe cracked dna fragmentation size.
In case the swivel base reaction is accomplished, the dna fragmentation of transposon end sequence (TE) mark is that (ZYMO RESEARCH CORPORATION, IRVINE CA) carries out purifying to available ZYMODNA CLEAN AND CONCENTRATOR KIT.
(S1 NUCLEASE PROMEGA) is hatched at 37 ℃ with the mixture of excision enzyme, up to the terminal equating of the dna fragmentation of strand for the dna fragmentation of transposon end sequence (TE) mark that purifying is good and single stranded DNA restriction endonuclease.Come purifying not have the DNA of mark with ZYMODNA CLEAN AND CONCENTRATOR KIT.The end sequence of transposon so far is removed, and has produced unlabelled dna fragmentation.
In the unlabelled dna fragmentation of above generation, add the big fragment of archaeal dna polymerase I of no 5 prime excision enzyme activity, promptly the KLENOW fragment was hatched 20 minutes at 37 ℃.Dna fragmentation through this processing can link to each other with oligonucleotide joint.
The dna fragmentation that does not have mark, BARCODE label, the restriction enzyme site mark, the mixture of the T4 dna ligase that the order-checking label of transcripting promoter sequence label and ligation are used was handled 5 minutes or was spent the night at 16 ℃.Add EDTA or stopped ligation in 20 minutes 72 ℃ of reactions.The dna fragmentation library that has label promptly can be used to downstream analysis.
Parent material with TN5 at 5 minutes (NEXTERA DNA SAMPLE PREPARATION KIT of LMW 500C; EPICENTRE; GA09115); Then 37 degree with S1 NUCLEASE (PROMEGA M5761) is hatched 30 minutes. the dna fragmentation that adds the A tail that obtains with KLENOW from 3 ' to 5 ' circumscribed interrupting (NEB, 0212L); 370C hatching 20MINUTES. 160C used fast link test kit link order-checking street corner in 5 minutes. and the tagged DNA that obtains carries out pcr amplification (PHUSION HOT START FLEX 2X MASTER MIX, NEB M0536L) according to the PCR operating process of standard with following system.All samples in the process of at every turn handling with enzyme with ZYMODNA CLEAN AND CONCENTRATOR KIT (ZYMO RESEARCH CORPORATION; IRVINE; CA) come purifying and enrichment; The PCR product that obtains is at 1% AGAROSE gel electrophoresis and with SYBR GREEN I NUCLEIC ACID dyeing (LIFE TECHNOLOGIES; S7567), resulting result see the picture 4 that takes with SAFE IMAGER BLUE LIGHT TRANSILLUMINATOR (LIFE TECHNOLOGIES, G6600).
*****
The present invention is described on some details, and the variation of various forms and details can not depart from the true scope of invention.All numerals above-mentioned, form, appendix, patent, patented claim and document give at present and include reference in.
Claims (8)
1. the random fracture and the method for labeled dna fragment simultaneously; It is characterized in that; May further comprise the steps: transposase, double-stranded transposon and target dna react after mixing, and are exactly under the katalysis of transposase, and double-stranded transposon end sequence inserts in the target dna sequence at random; Formation is by the dna fragmentation of transposon end sequence mark; These fragments start from double-stranded transposon end sequence from 5' to 3', are the single stranded DNA sequence of 9 base pairs then; Be double-stranded target dna fragment then, the single stranded DNA sequence of 9 base pair BP and double-stranded transposon are terminal; The two strands of the transposon end sequence that is marked on the target dna two ends separated be split into strand. remove strand transposon end sequence; Generating unlabelled dna fragmentation. the DNA label that connection needs on unlabelled dna segment produces a dna fragmentation
that new label is arranged.
2. the method for claim 1 is characterized in that, double-stranded transposon end is to form strand through high temperature.
3. the method for claim 1 is characterized in that, removes the swivel base end sequence of strand with the strand exonuclease.
4. the method for claim 1 is characterized in that, the DNA label that connection needs on unlabelled dna segment is realized with dna ligase.
5. method as claimed in claim 4 is characterized in that, said dna ligase is a T4 DNA ligase enzyme, T7 DNA ligase enzyme, or TAQDNA polysaccharase.
6. the method for claim 1 is characterized in that, described label is for being selected from the order-checking label, the barcode label, and the restriction enzyme site mark is caught mark, certification mark, amplification label, or in the transcripting promoter sequence label one or more.
7. method as claimed in claim 6 is characterized in that, the use that can link together of a plurality of labels.
8. random fracture that is used for of using claim 1-7 method, and form the test kit of markd dna fragmentation, comprising the transposon of purifying, transposon is terminal, specific single stranded DNA nucleicacidase, archaeal dna polymerase, dna ligase and required label.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101887507A CN102703432A (en) | 2012-07-11 | 2012-07-11 | Method, reagent and kit for constructing nucleic acid library |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101887507A CN102703432A (en) | 2012-07-11 | 2012-07-11 | Method, reagent and kit for constructing nucleic acid library |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102703432A true CN102703432A (en) | 2012-10-03 |
Family
ID=46896482
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012101887507A Pending CN102703432A (en) | 2012-07-11 | 2012-07-11 | Method, reagent and kit for constructing nucleic acid library |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102703432A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104153003A (en) * | 2014-08-08 | 2014-11-19 | 上海美吉生物医药科技有限公司 | Method for establishing DNA (Deoxyribose Nucleic Acid) library based on illumina sequencing platform |
| WO2016037394A1 (en) * | 2014-09-12 | 2016-03-17 | 深圳华大基因科技有限公司 | Method for constructing nucleic acid single-stranded cyclic library and reagents thereof |
| CN107557450A (en) * | 2017-10-09 | 2018-01-09 | 湖南大地同年生物科技有限公司 | A kind of reagent and method and its application of rapid build high-throughput sequencing library |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102264914A (en) * | 2008-10-24 | 2011-11-30 | 阿霹震中科技公司 | Transposon end compositions and methods for modifying nucleic acids |
| CN102703426A (en) * | 2012-05-21 | 2012-10-03 | 吴江汇杰生物科技有限公司 | Method for constructing a nucleic acid library, reagent and kit |
-
2012
- 2012-07-11 CN CN2012101887507A patent/CN102703432A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102264914A (en) * | 2008-10-24 | 2011-11-30 | 阿霹震中科技公司 | Transposon end compositions and methods for modifying nucleic acids |
| CN102703426A (en) * | 2012-05-21 | 2012-10-03 | 吴江汇杰生物科技有限公司 | Method for constructing a nucleic acid library, reagent and kit |
Non-Patent Citations (1)
| Title |
|---|
| ELAINE R. MARDIS: "Next-Generation DNA Sequencing Methods", 《ANNU. REV. GENOMICS HUM. GENET.》, vol. 9, 24 June 2008 (2008-06-24), pages 387 - 402, XP002512993, DOI: doi:10.1146/ANNUREV.GENOM.9.081307.164359 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104153003A (en) * | 2014-08-08 | 2014-11-19 | 上海美吉生物医药科技有限公司 | Method for establishing DNA (Deoxyribose Nucleic Acid) library based on illumina sequencing platform |
| WO2016037394A1 (en) * | 2014-09-12 | 2016-03-17 | 深圳华大基因科技有限公司 | Method for constructing nucleic acid single-stranded cyclic library and reagents thereof |
| CN107075731A (en) * | 2014-09-12 | 2017-08-18 | 深圳华大基因科技有限公司 | Method and reagent for constructing nucleic acid single-stranded circular library |
| US10023906B2 (en) | 2014-09-12 | 2018-07-17 | Mgi Tech Co., Ltd. | Method for constructing nucleic acid single-stranded cyclic library and reagents thereof |
| CN107075731B (en) * | 2014-09-12 | 2019-11-08 | 深圳华大智造科技有限公司 | Construction method and reagent of nucleic acid single-stranded circular library |
| CN107557450A (en) * | 2017-10-09 | 2018-01-09 | 湖南大地同年生物科技有限公司 | A kind of reagent and method and its application of rapid build high-throughput sequencing library |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7550816B2 (en) | Genome-wide, unbiased identification of DSBs assessed by sequencing (GUIDE-Seq) | |
| US20250109426A1 (en) | Compositions and methods for targeted depletion, enrichment, and partitioning of nucleic acids using crispr/cas system proteins | |
| US11584959B2 (en) | Compositions and methods for selection of nucleic acids | |
| CN102703426A (en) | Method for constructing a nucleic acid library, reagent and kit | |
| JP6998404B2 (en) | Method for enriching and determining the target nucleotide sequence | |
| CN112639094B (en) | Single-tube bead-based DNA co-barcoding for sequencing, haplotype typing, and assembly | |
| CA2955382C (en) | Polynucleotide enrichment using crispr-cas systems | |
| CN108138364B (en) | A kind of construction method and reagent of nucleic acid single-stranded circular library | |
| US20240209427A1 (en) | Rare nucleic acid detection | |
| WO2018013558A1 (en) | Compositions and methods for detecting nucleic acid regions | |
| CN113811618B (en) | Sequencing library construction based on methylated DNA target region, system and application | |
| CA2989976C (en) | Reagents, kits and methods for molecular barcoding | |
| WO2017218979A1 (en) | Unbiased detection of nucleic acid modifications | |
| WO2006127423A2 (en) | Methods of producing polynucleotide libraries using scarless ligation | |
| US12305218B2 (en) | Optimization of in vitro isolation of nucleic acids using site-specific nucleases | |
| JP2023513606A (en) | Methods and Materials for Assessing Nucleic Acids | |
| EP3768855B1 (en) | Methods and compositions for recombinase-mediated selective cleavage of nucleic acids | |
| CN102703432A (en) | Method, reagent and kit for constructing nucleic acid library | |
| US20250230493A1 (en) | Method for combined genome methylation and variation analyses | |
| KR20230154078A (en) | Genomic library construction and targeted epigenetic assay using CAS-gRNA ribonucleoprotein | |
| WO2025083338A1 (en) | An in vitro method for fragmenting polynucleotides for next generation sequencing | |
| CA2953362C (en) | Genomewide unbiased identification of dsbs evaluated by sequencing (guide-seq) | |
| HK1214843B (en) | Compositions and methods for detecting rare sequence variants |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20121003 |