CN108166068A

CN108166068A - A kind of Novel DNA builds library kit and its application

Info

Publication number: CN108166068A
Application number: CN201810000918.4A
Authority: CN
Inventors: 陈昌岳; 王芳; 李静; 李明明; 查洋; 胡秋萍; 张祥林
Original assignee: SHANGHAI MAJORBIO PHARM TECHNOLOGY Co Ltd
Current assignee: SHANGHAI MAJORBIO PHARM TECHNOLOGY Co Ltd
Priority date: 2018-01-02
Filing date: 2018-01-02
Publication date: 2018-06-15

Abstract

The present invention relates to technical field of molecular biology, and library kit and its application are built more particularly to a kind of Novel DNA.DNA provided by the invention builds library kit, including：Cohesive terminus,cohesive termini connector, ligase, DNA extension reagents and DNA cloning reagent.The DNA of the present invention builds the sample that library kit can apply to low initial amount, the connection of common ligase mediated dna and connector can directly be passed through, method before is compared, the joint efficiency of sample greatly improves, retain the diversity of sample to a greater extent, and stability is good, reduces due to library skewed popularity caused by artificial origin.

Description

A kind of Novel DNA builds library kit and its application

Technical field

The present invention relates to technical field of molecular biology, and library kit and its application are built more particularly to a kind of Novel DNA.

Background technology

The function of DNA molecular is that storage determines nearly all protein of species character and all heredity letters of RNA molecule Breath, the deciphering to DNA information studies secrets of life for us, the mechanism etc. of disease has great significance.DNA molecular Exist usually in the form of double-strand in sample, but most precious medicine samples all fix paraffin packet with formalin The form for burying (FFPE) sample exists.

FFPE samples are precious, but but there is the difficult problem of amplification in molecular biology experiment.Usual FFPE all matter Amount is poor, and by fragmentation and on DNA, there are more breakpoints, this is expanded to respective sample there are certain difficulty, because logical Normal polymerase cannot skip these breakpoints, and prevent sample from expanding success.

There are another kind of samples accordingly -- and cfDNA (cell-free circulating DNA) refers generally to be present in blood DNA fragments in the body fluid such as slurry, urine are similarly a kind of sample of fragmentation, usual 140bp~170bp or so.

In order to solve the problems, such as the amplification of the sample of this kind of small fragment, those skilled in the art propose solution below：

Patent " a kind of single stranded DNA two generations sequencing library construction method for cfDNA ", using TS2126RNA ligases (also referred to as Circligase ssDNA ligase) carries out single strand dna and is connected with single-stranded connector, can connect single stranded DNA On the single-stranded DNA molecular of head connection value, the enrichment to fragmentation genomic DNA is realized, is improved to fragmentation sample gene The coverage rate of group.But required enzyme (circligase) is expensive in the program, has centainly to the sequence of junction fragment Selectivity, temporarily the replacement enzyme without equal joint efficiency.

It is influenced by more than limiting factor, the mode of this kind of fragmentation sample generally use pcr is detected wherein at present Indivedual sites；Or the mode of multiplex PCR detects more site simultaneously；Or to FFPE samples by way of double-strand connection The structure of amplification and two generation sequencing libraries after sheet or cfDNA are attached.But PCR amplification mode has segment skewed popularity, generally The preferable region of quality can only be covered, for there is the sample amplification situation of breaking point then very undesirable, can not be efficiently used All DNA information in sample；And in the scheme of double-strand connection, it is imitated by the region connector connection that library fragments are affected Rate is generally poor, also results in the loss of DNA information.

In view of the above problems, being badly in need of a kind of Novel DNA builds library product, to meet the research need to such small fragment sample It asks.

Invention content

In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide a kind of Novel DNAs to build library kit And its application.

In order to achieve the above objects and other related objects, the first aspect of the present invention provides a kind of DNA and builds library kit, It includes at least：Cohesive terminus,cohesive termini connector, ligase, DNA extension reagents and DNA cloning reagent.

Further, well known technology, such as high-temperature denatured rear sudden cold, NaOH solution alkali can be used in those skilled in the art The schemes such as denaturation make sample genomic dna denaturation into single stranded DNA.The high temperature generally refers to 60 DEG C -98 DEG C.Preferably 80 ℃-98℃.More preferably 90 DEG C -98 DEG C.Processing time is generally 30s-10min.The NaOH solution is 0.2~0.6M NaOH, 1~1.5MNaCl.Preferably 0.3~0.5M NaOH, 1~1.5MNaCl.More preferably 0.4M NaOH, 1.5MNaCl。

Further, the cohesive terminus,cohesive termini connector is used to be connected to the single stranded DNA of sample genomic dna denaturation, obtains Obtain the single stranded DNA that one end carries connector.

Further, the ligase for connect cohesive terminus,cohesive termini connector be denaturalized with sample genomic dna it is single-stranded DNA。

Further, the DNA extensions reagent is used to extend single stranded DNA of the described one end with connector, is formed One end carries the double-stranded DNA of connector.

Further, the kit further includes double-stranded adapters, and the double-stranded adapters carry for being connected to described one end The other end of the double-stranded DNA of connector forms the double-stranded DNA that both-end carries connector.

Further, the DNA cloning reagent is used to expand double-stranded DNA of the both-end with connector, obtains Sequencing library.Also referred to as expand sample.

Further, the kit further includes cell pyrolysis liquid or genome DNA extraction reagent.The cell pyrolysis liquid For being cracked to unicellular sample, the genomic DNA of unicellular sample is obtained.The genome DNA extraction reagent is used for more Cell sample extracts, and obtains the genomic DNA of many cells sample.The cell pyrolysis liquid is used to crack unicellular sample, obtains Obtain unicellular sample genomic dna.The genome DNA extraction reagent is used to extract many cells sample, obtains many cells sample This genomic DNA.Sample below 100,000 cells can also be cracked using cell cracking.

Further, the cell pyrolysis liquid, preferably unicellular lysate.Unicellular lysate is straight for trace sample It is than better suited to tap into row following amplification, is conducive to the integrality of sample.

Further, the cohesive terminus,cohesive termini connector is the double-stranded adapters with cohesive terminus,cohesive termini, the cohesive terminus,cohesive termini connector One end is the first cohesive terminus,cohesive termini, and first cohesive terminus,cohesive termini includes at least random sequence.

Further, the length of the random sequence is 2-30nt, more preferably preferably 4-20nt, 5-10nt.

It should be noted that the random sequence is used for the single stranded DNA that sample genomic dna is denaturalized by mutually recruiting To being attached.The sequence of double stranded section in the cohesive terminus,cohesive termini connector can be adapted for corresponding microarray dataset, such as Illumina microarray datasets.

Further, the other end of the cohesive terminus,cohesive termini connector can be flat end or the second cohesive terminus,cohesive termini.When When the other end of the cohesive terminus,cohesive termini connector is the second cohesive terminus,cohesive termini, second cohesive terminus,cohesive termini is or random sequence or A/T/C/ The repetitive sequence of G oligonucleotides, preferably repetitive sequence；Length is preferably 2-10nt, more preferably 2-5nt.

Further, the structure of first chain can be：5 '-the first primer sequence-special sequence complementary series-with Cohesive terminus,cohesive termini is collectively formed with random sequence in machine sequence -3 ', the special sequence complementary series.

Further, first cohesive terminus,cohesive termini includes random sequence and special sequence.

Further, the special sequence is the sequence that sequence determines.

Further, the length of first cohesive terminus,cohesive termini is 2-30nt, more preferably preferably 4-20nt, 5- 10nt。

Further, in the cohesive terminus,cohesive termini connector, the single-stranded length with the first cohesive terminus,cohesive termini is 10-70nt.It is excellent It is selected as 10-30nt.It is further preferably 10-25nt.

Further, in the cohesive terminus,cohesive termini connector, the single-stranded annealing temperature with the first cohesive terminus,cohesive termini is 10-100 ℃.Preferably 20-80 DEG C.It is further preferably 30-60 DEG C.

Further, in the cohesive terminus,cohesive termini connector, 5 ' the single-stranded ends with the first cohesive terminus,cohesive termini carry out phosphorylation and repair Decorations.

Further, in the cohesive terminus,cohesive termini connector, single-stranded 5 ' with the first cohesive terminus,cohesive termini are held into arm in the ranks (spacer) it modifies.Preferably, the single-stranded 5 ' end for carrying the first cohesive terminus,cohesive termini can be used continuous Spacer C12 or The modification of C18Spacer or C3Spacer-CPG or C3Spacer or C9Spacer, Spacer can be optionally combined to increase carbochain Length.

Further, 5 ' the single-stranded ends for carrying the first cohesive terminus,cohesive termini carry out amido modified.

Such as DMS (O) MT-Amino-Modifier-C6, Amino-Modifier-C3-TFA, Amino-Modifier- C12、Amino-Modifier-C6-TFA(trifluoroacetic acid protecting group on amine)、 Amino-dT, Amino-Modifier-5, Amino-Modifier-C2-dT, Amino-Modifier-C6-dT, it is amido modified It can be optionally combined.

Further, 5 ' the single-stranded ends for carrying the first cohesive terminus,cohesive termini were not only modified but also were carried out into arm in the ranks (spacer) Amido modified, arm (spacer) modification can be optionally combined with amido modified.

Further, 3 ' the single-stranded ends for carrying the first cohesive terminus,cohesive termini carry out biotinylation (bioten) modification.

Further, single-stranded 3 ' for carrying the first cohesive terminus,cohesive termini are held into arm in the ranks (spacer).Preferably, it is described Single-stranded 5 ' end with the first cohesive terminus,cohesive termini can be used continuous Spacer C12 or C18Spacer or C3Spacer-CPG or The modification of C3Spacer or C9Spacer, Spacer can be optionally combined to increase carbon chain lengths.

Further, 3 ' the single-stranded ends for carrying the first cohesive terminus,cohesive termini carry out amido modified.Such as DMS (O) MT- Amino-Modifier-C6、Amino-Modifier-C3-TFA、Amino-Modifier-C12、Amino-Modifier-C6- TFA(trifluoroacetic acid protecting group on amine)、Amino-dT、Amino-Modifier- 5th, Amino-Modifier-C2-dT, Amino-Modifier-C6-dT, it is amido modified to be optionally combined.

Further, 5 ' the single-stranded ends for carrying the first cohesive terminus,cohesive termini use the repetition A/T/C/G for the modification that methylates Sequence and the combination of the modification of C3Spacer or C6Spacer or Spacer 12 or Spacer 12 or Spacer C18.It is preferred that Ground uses the repetition A/T/C/G sequences number for the modification that methylates to be preferred for 2-8.

Further, the single-stranded 3 ' end for carrying the first cohesive terminus,cohesive termini not only into arm in the ranks (spacer) but also carries out amino Modification, arm (spacer) and amido modified can be optionally combined.

Further, existing of 3 ' single-stranded end arm (spacer) modification for carrying the first cohesive terminus,cohesive termini carries out ammonia again Base is modified and biotinylation (bioten) modification.Preferably, arm (spacer) modification and amido modified it is located at described the between described Between 3 ' end sequences and biotinylation (bioten) modification of one primer sequence.More preferably, arm (spacer) is repaiied between described It decorations and amido modified is attached between biotin with TEG.

Further, AmC3 can also be used after the random sequence at 3 ' the single-stranded ends for carrying the first cohesive terminus,cohesive termini Or the amido modified or spacer modifications of AmC6 or AmC7.

Further, in the cohesive terminus,cohesive termini connector, without that single-stranded referred to as another list of the first cohesive terminus,cohesive termini Chain.

Further, in the cohesive terminus,cohesive termini connector, another single-stranded length is 10-70nt.Preferably 10-30nt. It is further preferably 10-25nt.

Further, in the cohesive terminus,cohesive termini connector, another single-stranded annealing temperature is 10-100 DEG C.Preferably 20- 80℃.It is further preferably 30-60 DEG C.

Further, in the cohesive terminus,cohesive termini connector, another 5 ' single-stranded ends carry out phosphorylation modification.

Further, it in the cohesive terminus,cohesive termini connector, holds into arm in the ranks (spacer) for another single-stranded 5 ' and modifies.It is preferred that Ground, using C3Spacer or C6Spacer or Spacer C12 or Spacer 12 or Spacer C18 or C3Spacer-CPG Modification, Spacer can be optionally combined to increase carbon chain lengths.More preferably, in the cohesive terminus,cohesive termini connector, another single-stranded 5 ' ends using 1-10 Spacer C12 or 1-10 C18 Spacer or 1-10 C3Spacer or 1-10 C6Spacer or The modification of 1-10 C3Spacer-CPG.More preferably, in the cohesive terminus,cohesive termini connector, another 5 ' single-stranded ends are using methyl Change the repetition A/T/C/G sequences of modification and C3Spacer or C6Spacer or Spacer 12 or Spacer 12 or Spacer C18 Modification combination.Preferably, the repetition A/T/C/G sequences number for the modification that methylates is used to be preferred for 2-8；It is highly preferred that Modification that can also be using AmC3 or AmC6 or AmC7 after random primer or thio-modification.

Further, in the cohesive terminus,cohesive termini connector, another 5 ' single-stranded ends carry out amido modified.

Such as DMS (O) MT-Amino-Modifier-C6, Amino-Modifier-C3-TFA, Amino-Modifier- C12、Amino-Modifier-C6-TFA(trifluoroacetic acid protecting group on amine)、 Amino-dT、Amino-Modifier-5、Amino-Modifier-C2-dT、Amino-Modifier-C6-dT。

Further, in the cohesive terminus,cohesive termini connector, another 3 ' single-stranded ends carry out biotinylation (bioten) and modify.

Further, it in the cohesive terminus,cohesive termini connector, holds into arm in the ranks (spacer) for another single-stranded 3 ' and modifies.It is preferred that Ground, in the cohesive terminus,cohesive termini connector, another 5 ' single-stranded ends can be used continuous Spacer C12 or C18 Spacer or The modification of C3Spacer-CPG or C3Spacer or C9Spacer, Spacer can be optionally combined to increase carbon chain lengths.

Further, in the cohesive terminus,cohesive termini connector, another 3 ' single-stranded ends carry out amido modified.

Further, in the cohesive terminus,cohesive termini connector, another single-stranded 3 ' end not only modified into arm in the ranks (spacer) but also It carries out amido modified, arm (spacer) modification and amido modified can be optionally combined.

Further, in the cohesive terminus,cohesive termini connector, another single-stranded existing arms (spacer) modification in 3 ' end again into Row is amido modified and has biotinylation (bioten) modification.Preferably, arm (spacer) modification and amido modified it is located between described Between 3 ' end terminal sequences and biotinylation (bioten) modification of second primer sequence.More preferably, arm between described (spacer) it and amido modified is attached between biotin with TEG.

Further, the ligase be selected from HiFi Taq DNA ligases, T4 RNA ligases 2,Connection Enzyme, 9 ° of N^TMDNA ligase, Taq DNA ligases, T7 DNA ligases, T3 DNA ligases, electricity turn ligase, flat end/TA Ligase premixed liquid, instantaneous stickiness ligase premixed liquid, T4 DNA ligases, Circligase ssDNA ligase, 5 ' AppDNA/RNA thermostabilization ligase any one or more ofs.

Further, the DNA extensions reagent is selected from archaeal dna polymerase, dNTPs, DNA polymerase buffer liquid, extension primer.

Further, the archaeal dna polymerase be selected from vent archaeal dna polymerases, T7 archaeal dna polymerases, Bsu archaeal dna polymerases, T4 archaeal dna polymerases, Klenow segments, DNA polymerase i (E.coli), Therminator^TMArchaeal dna polymerase, Sulfolobus DNA polymerase i V, phi29 archaeal dna polymerase, Bst2.0Archaeal dna polymerase, 2.0 archaeal dna polymerases of Bst, Bst Archaeal dna polymerase, Bst archaeal dna polymerases, Deep VentR (exo -) archaeal dna polymerase, DeepArchaeal dna polymerase, VentR (exo -) archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Taq archaeal dna polymerases,Heat opens Dynamic Taq archaeal dna polymerases,Taq archaeal dna polymerases, thermal starting Taq archaeal dna polymerases, Taq archaeal dna polymerases are large stretch of Section, Taq archaeal dna polymerases,Thermal starting archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Flex Archaeal dna polymerase,Super fidelity dna polymerase,Thermal starting surpass fidelity dna polymerase,Super fidelity dna gathers The combination of the one or more enzyme such as synthase.

Further, it can also be 10mM can also be other concentration that the dNTPs, which can be 2.5mM,.It is described The best match buffer solution that DNA polymerase buffer liquid is selected for polymerase selected by correspondence.

Further, the sequence of the extension primer and the sequence of the non-stickiness part of any one chain of cohesive terminus,cohesive termini connector Row are all or part of complementary.The non-stickiness part refers to the double stranded section of cohesive terminus,cohesive termini connector.

Further, the DNA cloning reagent is selected from archaeal dna polymerase, dNTPs, DNA polymerase buffer liquid, amplimer.

Further, the archaeal dna polymerase be selected from vent archaeal dna polymerases, T7 archaeal dna polymerases, Bsu archaeal dna polymerases, T4 archaeal dna polymerases, Klenow segments, DNA polymerase i (E.coli), Therminator^TMArchaeal dna polymerase, Sulfolobus DNA polymerase i V, phi29 archaeal dna polymerase, Bst2.0Archaeal dna polymerase, 2.0 archaeal dna polymerases of Bst, Bst Archaeal dna polymerase, Bst archaeal dna polymerases, Deep VentR (exo -) archaeal dna polymerase, Deep VentR^TMArchaeal dna polymerase, VentR (exo -) archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Taq archaeal dna polymerases,Heat opens Dynamic Taq archaeal dna polymerases,Taq archaeal dna polymerases, thermal starting Taq archaeal dna polymerases, Taq archaeal dna polymerases are large stretch of Section, Taq archaeal dna polymerases,Thermal starting archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Flex archaeal dna polymerases,Super fidelity dna polymerase,Thermal starting surpass fidelity dna polymerase,Super fidelity The combination of one or more of enzymes in archaeal dna polymerase etc..

Further, the sequence of the amplimer and the sequence of the non-stickiness part of any one chain of cohesive terminus,cohesive termini connector Row are all or part of complementary.The non-stickiness part refers to the double stranded section of cohesive terminus,cohesive termini connector.

Further, the double-stranded adapters include first chain and Article 2 chain, and the structure of first chain is：5’- Third primer sequence -3 ', the structure of the Article 2 chain are：5 '-the four primer sequences -3 ', the third of first chain are drawn 4th primer sequence all or part complementary pairing of object sequence and the Article 2 chain.

Further, the end of the double-stranded adapters is flat end or cohesive terminus,cohesive termini.

The sequence of the double-stranded adapters can apply to the joint sequence of illumina microarray datasets or other The DNA of duplex structure.

Further, the length of the cohesive terminus,cohesive termini is 2-10nt, preferably 2-5nt.

Further, the cohesive terminus,cohesive termini can be band there are one the cohesive terminus,cohesive termini of T, Y types end or be A or T or C or G Repetitive sequence or other DNA sequence dnas.

For example, one end of the double-stranded adapters can be the cohesive terminus,cohesive termini of flat end or band there are one T.The double-stranded adapters The other end be flat end or Y types end or cohesive terminus,cohesive termini, repetitive sequence that the cohesive terminus,cohesive termini is A or T or C or G or other DNA sequence dna.

Further, the double-stranded adapters include first chain and Article 2 chain.

Further, in the double-stranded adapters, first single-stranded length is 10-70nt.Preferably 10-30nt.It is excellent again It is selected as 10-25nt.

Further, in the double-stranded adapters, first single-stranded annealing temperature is 10-100 DEG C.Preferably 20-80 DEG C. It is further preferably 30-60 DEG C.

Further, in the double-stranded adapters, first 5 ' single-stranded end carries out phosphorylation modification.

Further, it in the double-stranded adapters, holds into arm in the ranks (spacer) for first single-stranded 5 ' and modifies.

Further, in the double-stranded adapters, first 5 ' single-stranded end carries out biotinylation (bioten) and modifies.

Further, in the double-stranded adapters, first single-stranded existing of 5 ' end arm (spacer) modification has life again Object element (bioten) is modified.Preferably, arm (spacer) modification is positioned at 3 ' end terminal sequences of first chain between described Between being modified with biotinylation (bioten).More preferably, it is carried out between arm (spacer) modification and biotin with TEG between described Connection.

Further, in the double-stranded adapters, first 3 ' single-stranded end carries out thio-modification.

Further, in the double-stranded adapters, the single-stranded length of Article 2 is 10-70nt.Preferably 10-30nt.It is excellent again It is selected as 10-25nt.

Further, in the double-stranded adapters, the single-stranded annealing temperature of Article 2 is 10-100 DEG C.Preferably 20-80 ℃.It is further preferably 30-60 DEG C.

Further, in the double-stranded adapters, Article 2 it is single-stranded 3 ' hold into arm in the ranks (spacer) modify.It is preferred that Ground, using the modification of C3Spacer or C6Spacer or Spacer C12 or Spacer 12 or Spacer C18.More preferably, 5 ' single-stranded ends of the Article 2 using 2-10 Spacer C12 or 2-10 C18 Spacer or 2-10 C3Spacer or The modification of 2-10 C6Spacer.More preferably, 5 ' single-stranded ends of the Article 2 use the repetition A/T/C/G for the modification that methylates Sequence and the combination of the modification of C3Spacer or C6Spacer or Spacer 12 or Spacer 12 or Spacer C18.It is preferred that Ground uses the repetition A/T/C/G sequences number for the modification that methylates to be preferred for 2-8.

Further, 3 ' single-stranded ends of the Article 2 carry out biotinylation (bioten) modification.

Further, described two single-stranded existing of 3 ' end arm (spacer) modifications have biotinylation (bioten) to repair again Decorations.

Preferably, arm (spacer) modification can be located at the Article 2 single-stranded 5 ' end terminal sequences and biotin between described Between change (bioten) modification.More preferably, it is attached between arm (spacer) modification and biotin with TEG between described.

Further, 3 ' single-stranded ends of the Article 2 carry out phosphorylation modification.

The second aspect of the present invention provides aforementioned DNA and builds library kit and sets up library product or genome preparing gene Purposes in sequencing products.

The third aspect of the present invention, provide aforementioned DNA build library kit for sample genome dna library establish or Purposes in sequencing.

Further, the DNA builds library kit and is established for the genome dna library of sample.

Method of the library kit to genomic DNA progress library foundation is built using the DNA, is included the following steps：

(1) to make sample genomic dna denaturation into single stranded DNA using well known to a person skilled in the art technologies；

(2) single stranded DNA obtained in step (1) by ligase and cohesive terminus,cohesive termini connector is attached, obtains one end Single stranded DNA with connector；

(3) extend reagent using DNA to extend single stranded DNA of the one end in step (2) with connector, form one end band There is the double-stranded DNA of connector；

(4) other end of double-stranded DNA of the one end with connector obtained by step (3) is connected to using double-stranded adapters, is formed double Double-stranded DNA of the end with connector；

(5) double-stranded DNA of the both-end that step (4) obtains with connector is expanded using DNA cloning reagent, is expanded Increase production object or sequencing library.

Further, the sample genomic dna is cracked to obtain or extracted by many cells sample by unicellular sample and be obtained.

It can be before step (1) is carried out, first unicellular sample be cracked or using genome using cell pyrolysis liquid DNA extraction agents extract many cells sample, so as to obtain the sample genomic dna.It can also use and utilize this by other people Technology known to field cracks the sample genome for obtaining or being obtained by the extracting of many cells sample via unicellular sample DNA。

Further, when the cell number of sample is below 100,000, can Direct Pyrolysis obtain sample genomic dna, Without being stripped.

The cell can be prokaryotic cell or eukaryocyte.The eukaryocyte can be plant cell or zooblast And microorganism.The zooblast be chosen in particular from tissue digestion cell, culture gained cell, embryonic development early stage it is thin Born of the same parents, the cell of cancer early stage, the microbial cell without enrichment culture, the cell of airflow classification acquisition, limiting dilution obtain Any one of cell that the methods of cell, laser capture obtains.

Further, the quality of the sample genomic dna can be more than or equal to 6pg.

In step (1), well known technology, such as high-temperature denatured rear sudden cold, NaOH solution can be used in those skilled in the art The schemes such as alkaline denaturation make sample genomic dna denaturation into single stranded DNA.The high temperature generally refers to 60 DEG C -98 DEG C.Preferably 80 ℃-98℃.More preferably 90 DEG C -98 DEG C.Processing time is generally 30s-10min.The NaOH solution is 0.2~0.6M NaOH, 1~1.5MNaCl.Preferably 0.3~0.5M NaOH, 1~1.5MNaCl.More preferably 0.4M NaOH, 1.5MNaCl。

Further, in other steps, the cohesive terminus,cohesive termini connector, double-stranded adapters, DNA extensions reagent, DNA cloning reagent As described above.

Compared with prior art, the present invention has the advantages that：

(1) present invention provides a kind of novel DNA and builds library kit, is applicable to carry out the genomic DNA of sample single Chain link, amplification or library construction.The program is suitable for unicellular, cfDNA, ctDNA, CHIP DNA, the mitochondria extracted DNA, the genomic DNA of extraction, wherein the genomic DNA extracted can be living cells or tissue extraction or FFPE The second-rate genomic DNA of the extractions such as sample.In addition the program is 6pg to the initial amount requirement of sample.

(2) compared to the scheme of double-strand connection, DNA using the present invention builds the sample that library kit can apply to low initial amount Product (down to 6pg).

(3) compared to random primer amplification, DNA using the present invention builds library kit, can directly be drawn by ligase Enter to carry the double-stranded DNA connector of cohesive terminus,cohesive termini, thus ensuring that the strand of a DNA derives from the same area of template strand, The pattern that more accurate both-end can be used to compare during so as to fulfill subsequently than to arriving reference gene group increase the accuracy of data.

(4) it compared to the amplification mode that single-stranded connection is carried out using circligas (i.e. TS2126 RNA ligases), uses The DNA of the present invention, which builds library kit, to carry out connector connection by such as T4 DNA ligases of more economical practicality.The enzyme price Cheaply, the enzyme cost for each reaction being attached using circligas is about 200 yuan and T4 DNA ligases only need 3 yuan or so. In addition circligas does not connect activity for the segment of more than 500nt, while it is more suitable for the cyclization of intramolecular, In addition the enzyme is for the connection activity G at 5 ends of DNA molecular>A>>>T>>>C, for the connection activity T at 3 ends of DNA molecular>A>G >, there is no connection activity substantially for C (wherein>Expression is better than,>>>Expression is far superior to).And T4 DNA ligases are not above-mentioned Selectivity, therefore the diversity of sample can be retained to a greater extent, reduced due to library skewed popularity caused by artificial origin.

Description of the drawings

Fig. 1：The DNA of the present invention builds the structure diagram of the cohesive terminus,cohesive termini connector in the kit of library, and NNNN is random primer Sequence, 4 N on figure are display structure, and the restriction not to random primer partial-length.

Fig. 2：Show that DNA using the present invention builds the substantially process that library kit to genomic DNA build library, wherein adopting In the step of single stranded DNA being connected with the cohesive terminus,cohesive termini connector in kit, the random primer sequence NNNN in cohesive terminus,cohesive termini connector, Structural representation is only used as, not as restriction；The modifications such as biotin can also be connected on cohesive terminus,cohesive termini connector, it subsequently can be with strepto- The magnetic beads such as Avidin are connected, and are purified；If after cohesive terminus,cohesive termini connector is connected or after extending into double-stranded DNA or flat end fitting Application can be met after connection, then can stop subsequent experimental accordingly, and not necessarily proceed to exponential amplification.

Fig. 3：The cohesive terminus,cohesive termini in the kit of library is built using 3% agarose gel electrophoresis detection single stranded DNA and DNA of the present invention The bonding ratio of connector.

Fig. 4：When building library kit library using DNA of the present invention in the embodiment of the present invention 1 and carrying out building library, the detection of outbound chip Result figure.

Fig. 5：Parallel samples be respectively adopted traditional two chain build library scheme and the solution of the present invention structure text into library, profit With identical experiment condition, the result figure of the content of library molecule is identified using the mode of relative quantification, is passed through as shown in the figure It crosses 3 repetitions to test, the connection mode of single stranded DNA about improves 2 times of molecular amounts than double-stranded DNA connection mode.

Specific embodiment

Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe Embodiment, the protection domain being not intended to be limiting of the invention；In description of the invention and claims, unless in text In addition explicitly point out, singulative "one", " one " and " this " include plural form.

When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and Scientific terminology is identical with the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment, Outside material, according to record of the those skilled in the art to the grasp of the prior art and the present invention, it can also use and this Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real The existing present invention.

Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method using this technology lead Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of related field.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc. MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..

The present invention provides a kind of Novel DNAs to build library kit, including：Cohesive terminus,cohesive termini connector, ligase, DNA extension examinations Agent, DNA cloning reagent.

In one example, those skilled in the art can be used well known technology, for example, it is high-temperature denatured after it is sudden it is cold, NaOH is molten The schemes such as liquid caustic soda denaturation make sample genomic dna denaturation into single stranded DNA.The high temperature generally refers to 60 DEG C -98 DEG C.Preferably 80℃-98℃.More preferably 90 DEG C -98 DEG C.Processing time is generally 30s-10min.The NaOH solution is 0.2~0.6M NaOH, 1~1.5MNaCl.Preferably 0.3~0.5M NaOH, 1~1.5MNaCl.More preferably 0.4M NaOH, 1.5MNaCl.Those skilled in the art can select suitable method to make sample genomic dna denaturation into list according to actual needs Chain DNA, as long as not limiting the purpose of the present invention.

In one example, the cohesive terminus,cohesive termini connector is used to be attached by the ligase and the single stranded DNA, Obtain the single stranded DNA that one end carries connector.The single stranded DNA extends into double-stranded DNA reagent for carrying connector to one end Single stranded DNA is extended, and forms the double-stranded DNA that one end carries connector.

In one example, the kit further includes double-stranded adapters, and the double-stranded adapters are used to be connected to described one end The other end of double-stranded DNA with connector forms the double-stranded DNA that both-end carries connector.The DNA cloning reagent is used for both-end Double-stranded DNA with connector is expanded, and obtains sequencing library.

In one example, the kit further includes cell pyrolysis liquid or genome DNA extraction reagent.The cell is split It solves liquid to be used to crack unicellular sample, obtains unicellular sample genomic dna.The genome DNA extraction reagent for pair Many cells sample extracts, and obtains the genomic DNA of many cells sample.Sample below 100,000 cells can also be used thin Cellular lysate is cracked.

In one example, the cell pyrolysis liquid, preferably unicellular lysate.Unicellular lysate is for micro sample It is than better suited that this, which directly carries out following amplification, is conducive to the integrality of sample.

In one example, the cohesive terminus,cohesive termini connector is the double-stranded adapters with cohesive terminus,cohesive termini, and the cohesive terminus,cohesive termini connects One end of head is the first cohesive terminus,cohesive termini, and first cohesive terminus,cohesive termini includes at least random sequence.Wherein, the random sequence is used for It is attached with the single stranded DNA of sample genomic dna denaturation by complementary pairing.And the double-strand in the cohesive terminus,cohesive termini connector Partial sequence can be adapted for corresponding microarray dataset, such as illumina microarray datasets.If it is required without sequencing, to double-strand Partial sequence is without particular/special requirement, as long as meeting duplex structure.

The length of the random sequence can be adjusted according to the diversity of target sample, if the diversity of target sample is high Then the length of random sequence can relative increase, the length of random primer opposite can be reduced if the diversity of target sample is low.It is logical The length of Chang Suoshu random sequences is 2-30nt, more preferably preferably 4-20nt, 5-10nt.

In one example, the other end of the cohesive terminus,cohesive termini connector can be flat end or the second stickiness end End.When the other end of the cohesive terminus,cohesive termini connector is the second cohesive terminus,cohesive termini, second cohesive terminus,cohesive termini is arbitrary sequence or A/ The repetitive sequence of T/C/G oligonucleotides, preferably repetitive sequence；Length is preferably 2-10nt, more preferably 2-5nt.Ability Field technique personnel can select the sequence and length of the second cohesive terminus,cohesive termini according to actual needs, as long as not limiting the purpose of the present invention .

In one example, first cohesive terminus,cohesive termini includes random sequence and special sequence.Usually, the special sequence It is classified as the sequence that sequence determines.

Such as：When the single stranded DNA that connects is needed to be the single stranded DNA with certain end sequence that is obtained by digestion, institute It can be digestion with restriction enzyme to state digestion, and the restriction enzyme can be MSPI, EcoB, EcoK, EcoRI, Hind Ⅲ、HinfⅢ、XspI、BfaI、MaeI、TaqI、TthHB8I、HpaⅡ、HapⅡ、Apa Ⅰ、BamH Ⅰ、Ban Ⅱ、HgiJ Ⅱ、EcoR Ⅱ、Mva Ⅰ、BstO Ⅰ、Bgl Ⅰ、Bgl Ⅱ、Asu Ⅰ、Cfr13 Ⅰ、Sau96 Ⅰ、Ava Ⅰ、BseJ Ⅰ、Asu IIth, Nsp V, BstB I, Csp45 I, HgiC I, Ban I, BstP I, BstE II, Dra I, Aha III, Dra II etc., with For MSPI digestions so that certain end sequence that single stranded DNA carries is CGG or C, then at this point, first cohesive terminus,cohesive termini In included special sequence be sequence with certain end sequence complementation, that is, GCC or G.

For another example if researcher or experimenter, have certain target sequences the enrichment wish of skewed popularity, with It is specific such as CGCGCGCG for target sequence is CG dinucleotides sequences, then it is at this point, included in first cohesive terminus,cohesive termini Special sequence is the sequence with target sequence complementation, that is, GCGCGCGC.In addition, the number of the CG dinucleotides sequence Can be 1-10, it is therefore preferable to 2-8, more preferably 2-6.

In one example, the length of first cohesive terminus,cohesive termini be 2-30nt, preferably 4-20nt, more preferably 5-10nt。

In one example, in the cohesive terminus,cohesive termini connector, the single-stranded length with the first cohesive terminus,cohesive termini is 10- 70nt.Preferably 10-30nt.It is further preferably 10-25nt.

In one example, in the cohesive terminus,cohesive termini connector, the single-stranded annealing temperature with the first cohesive terminus,cohesive termini is 10- 100℃.Preferably 20-80 DEG C.It is further preferably 30-60 DEG C.

In one example, the length of first chain is 10-70nt.Can be further 10-30nt.Further Can be 10-25nt.

In one example, the annealing temperature of first chain is 10-100 DEG C.It can be further 20-80 DEG C.Again It can be further 30-60 DEG C.

In one example, in the cohesive terminus,cohesive termini connector, single-stranded with the first cohesive terminus,cohesive termini carries out appropriate repair Decorations, to reduce the non-specific amplification of primer.

In one example, in the cohesive terminus,cohesive termini connector, single-stranded 5 ' with the first cohesive terminus,cohesive termini are held into arm in the ranks (spacer) it modifies.Preferably, the single-stranded 5 ' end for carrying the first cohesive terminus,cohesive termini can be used continuous Spacer C12 or The modification of C18 Spacer or C3Spacer-CPG or C3Spacer or C9Spacer, Spacer can be optionally combined to increase carbochain Length.

In one example, 5 ' the single-stranded ends for carrying the first cohesive terminus,cohesive termini carry out amido modified.

In one example, the single-stranded 5 ' end for carrying the first cohesive terminus,cohesive termini is not only into arm spacer in the ranks) modification but also It carries out amido modified, arm (spacer) modification and amido modified can be optionally combined.

In one example, 5 ' the single-stranded ends for carrying the first cohesive terminus,cohesive termini use the repetition A/T/ for the modification that methylates C/G sequences and the combination of the modification of C3Spacer or C6Spacer or Spacer 12 or Spacer 12 or Spacer C18.It is excellent Selection of land uses the repetition A/T/C/G sequences number for the modification that methylates to be preferred for 2-8.

In one example, 3 ' the single-stranded end progress biotinylations (bioten) for carrying the first cohesive terminus,cohesive termini are repaiied Decorations.

In one example, single-stranded 3 ' for carrying the first cohesive terminus,cohesive termini are held into arm in the ranks (spacer) and are modified.It is excellent Selection of land, the single-stranded 5 ' end for carrying the first cohesive terminus,cohesive termini can be used continuous Spacer C12 or C18 Spacer or The modification of C3Spacer-CPG or C3Spacer or C9Spacer, Spacer can be optionally combined to increase carbon chain lengths.

In one example, 3 ' the single-stranded ends for carrying the first cohesive terminus,cohesive termini carry out amido modified.Such as DMS (O) MT- Amino-Modifier-C6、Amino-Modifier-C3-TFA、Amino-Modifier-C12、Amino-Modifier-C6- TFA(trifluoroacetic acid protecting group on amine)、Amino-dT、Amino-Modifier- 5th, Amino-Modifier-C2-dT, Amino-Modifier-C6-dT, it is amido modified to be optionally combined.

In one example, 3 ' the single-stranded ends for carrying the first cohesive terminus,cohesive termini not only into arm in the ranks (spacer) but also carried out It is amido modified, it arm (spacer) and amido modified can be optionally combined.

In one example, existing of single-stranded 3 ' end arms (spacer) modification for carrying the first cohesive terminus,cohesive termini again into Row is amido modified and biotinylation (bioten) is modified.Preferably, arm (spacer) modification and amido modified it is located at institute between described It states between 3 ' end sequences of the first primer sequence and biotinylation (bioten) modification.More preferably, arm between described (spacer) it modification and amido modified is attached between biotin with TEG.

In one example, it can also be used after the random sequence at 3 ' the single-stranded ends for carrying the first cohesive terminus,cohesive termini The amido modified or spacer modifications of AmC3 or AmC6 or AmC7.

In one example, it is single-stranded referred to as another without that of the first cohesive terminus,cohesive termini in the cohesive terminus,cohesive termini connector Item is single-stranded.

In one example, in the cohesive terminus,cohesive termini connector, another single-stranded length is 10-70nt.Preferably 10- 30nt.It is further preferably 10-25nt.

In one example, in the cohesive terminus,cohesive termini connector, another single-stranded annealing temperature is 10-100 DEG C.Preferably 20-80℃.It is further preferably 30-60 DEG C.

In one example, in the cohesive terminus,cohesive termini connector, another 5 ' single-stranded ends carry out phosphorylation modification.

In one example, it in the cohesive terminus,cohesive termini connector, holds into arm in the ranks (spacer) for another single-stranded 5 ' and modifies. Preferably, using C3Spacer or C6Spacer or Spacer C12 or Spacer 12 or Spacer C18 or C3Spacer- The modification of CPG, Spacer can be optionally combined to increase carbon chain lengths.More preferably, in the cohesive terminus,cohesive termini connector, another list 5 ' ends of chain are using 1-10 Spacer C12 or 1-10 C18 Spacer or 1-10 C3Spacer or 1-10 The modification of C6Spacer or 1-10 C3Spacer-CPG.More preferably, in the cohesive terminus,cohesive termini connector, another single-stranded 5 ' End using the modification that methylates repetition A/T/C/G sequences and C3Spacer or C6Spacer or Spacer 12 or Spacer 12 or The combination of the modification of Spacer C18.Preferably, the repetition A/T/C/G sequences number for the modification that methylates is used to be preferred for 2-8； It is highly preferred that modification that can also be using AmC3 or AmC6 or AmC7 after random primer or thio-modification.

In one example, in the cohesive terminus,cohesive termini connector, another 5 ' single-stranded ends carry out amido modified.

In one example, in the cohesive terminus,cohesive termini connector, another 3 ' single-stranded ends carry out biotinylation (bioten) Modification.

In one example, it in the cohesive terminus,cohesive termini connector, holds into arm in the ranks (spacer) for another single-stranded 3 ' and modifies. Preferably, in the cohesive terminus,cohesive termini connector, continuous Spacer C12 or C18 Spacer can be used in another 5 ' single-stranded ends Or the modification of C3Spacer-CPG or C3Spacer or C9Spacer, Spacer can be optionally combined to increase carbon chain lengths.

In one example, in the cohesive terminus,cohesive termini connector, another 3 ' single-stranded ends carry out amido modified.

In one example, in the cohesive terminus,cohesive termini connector, another 3 ' single-stranded ends were both repaiied into arm in the ranks (spacer) Decorations carry out amido modified again, arm (spacer) modification and amido modified can be optionally combined.

In one example, in the cohesive terminus,cohesive termini connector, another single-stranded existing of 3 ' end arm (spacer) modification It carries out not only amido modified but also has biotinylation (bioten) modification.Preferably, arm (spacer) modification and amido modified between described Between 3 ' end terminal sequences and biotinylation (bioten) modification of second primer sequence.More preferably, between described It arm (spacer) and amido modified is attached between biotin with TEG.

The cited joint sequence for applying to illumina microarray datasets, is only used for showing in some embodiments of the invention Meaning, it is not limited to these specific sequences.Cited modification mode is also only used for illustrating in embodiment.It can be according to practical need Carry out other modifications.

In one example, the ligase be selected from HiFi Taq DNA ligases, T4RNA ligases 2,Even Meet enzyme, 9 ° of N^TMDNA ligase, Taq DNA ligases, T7 DNA ligases, T3 DNA ligases, electricity turn ligase, flat end/ TA ligases premixed liquid, instantaneous stickiness ligase premixed liquid, T4 DNA ligases, T4RNA ligases, Circligase ssDNA Ligase, 5 ' AppDNA/RNA thermostabilization ligase any one or more ofs.

In one example, the DNA extensions reagent is selected from archaeal dna polymerase, dNTPs, DNA polymerase buffer liquid, extension Primer.

In one example, the archaeal dna polymerase is selected from vent archaeal dna polymerases, T7 archaeal dna polymerases, Bsu DNA polymerizations Enzyme, T4 archaeal dna polymerases, Klenow segments, DNA polymerase i (E.coli), Therminator^TMArchaeal dna polymerase, Sulfolobus DNA polymerase i V, phi29 archaeal dna polymerase, Bst2.0Archaeal dna polymerase, 2.0 DNA of Bst Polymerase, Bst archaeal dna polymerases, Deep VentR (exo -) archaeal dna polymerase, Deep VentR^TMArchaeal dna polymerase, VentR (exo -) archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Taq archaeal dna polymerases,Heat opens Dynamic Taq archaeal dna polymerases,Taq archaeal dna polymerases, thermal starting Taq archaeal dna polymerases, Taq archaeal dna polymerases are large stretch of Section, Taq archaeal dna polymerases,Thermal starting archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Flex archaeal dna polymerases,Super fidelity dna polymerase,Thermal starting surpass fidelity dna polymerase,Super fidelity The combination of the one or more enzyme such as archaeal dna polymerase.

In one example, the best match buffering that the DNA polymerase buffer liquid is selected for polymerase selected by correspondence Liquid.

In one example, the sequence of the extension primer and the non-stickiness part of any one chain of cohesive terminus,cohesive termini connector Sequence it is all or part of complementary.The non-stickiness part refers to the double stranded section of cohesive terminus,cohesive termini connector.

In one example, the end of the double-stranded adapters is flat end or cohesive terminus,cohesive termini.

In one example, the length of the cohesive terminus,cohesive termini is 2-10nt, preferably 2-5nt.

In one example, the cohesive terminus,cohesive termini can be band there are one the cohesive terminus,cohesive termini of T, Y types end or be A or T or C Or the repetitive sequence or other DNA sequence dnas of G.Those skilled in the art can select the tool of the cohesive terminus,cohesive termini according to actual needs Body sequence, as long as not limiting the purpose of the present invention.

In one example, the double-stranded adapters include first chain and Article 2 chain.

In one example, in the double-stranded adapters, first single-stranded length is 10-70nt.Preferably 10-30nt. It is further preferably 10-25nt.

In one example, in the double-stranded adapters, first single-stranded annealing temperature is 10-100 DEG C.Preferably 20- 80℃.It is further preferably 30-60 DEG C.

In one example, in the double-stranded adapters, first 5 ' single-stranded end carries out phosphorylation modification.

In one example, it in the double-stranded adapters, holds into arm in the ranks (spacer) for first single-stranded 5 ' and modifies.

In one example, in the double-stranded adapters, first 5 ' single-stranded end carries out biotinylation (bioten) and repaiies Decorations.

In one example, in the double-stranded adapters, first single-stranded existing of 5 ' end arm (spacer) modification is again There is biotinylation (bioten) modification.Preferably, arm (spacer) modification is positioned at 3 ' end ends of first chain between described Between sequence and biotinylation (bioten) modification.More preferably, between described TEG is used between arm (spacer) modification and biotin It is attached.

In one example, in the double-stranded adapters, first 3 ' single-stranded end carries out thio-modification.

In one example, in the double-stranded adapters, the single-stranded length of Article 2 is 10-70nt.Preferably 10-30nt. It is further preferably 10-25nt.

In one example, in the double-stranded adapters, the single-stranded annealing temperature of Article 2 is 10-100 DEG C.Preferably 20-80℃.It is further preferably 30-60 DEG C.

In one example, in the double-stranded adapters, Article 2 it is single-stranded 3 ' hold into arm spacer in the ranks modify.It is preferred that Ground, using the modification of C3Spacer or C6Spacer or Spacer C12 or Spacer 12 or Spacer C18.More preferably, 5 ' single-stranded ends of the Article 2 using 1-10 Spacer C12 or 1-10 C18 Spacer or 1-10 C3Spacer or The modification of 1-10 C6Spacer.More preferably, 5 ' single-stranded ends of the Article 2 use the repetition A/T/C/G for the modification that methylates Sequence and the combination of the modification of C3Spacer or C6Spacer or Spacer 12 or Spacer 12 or Spacer C18.It is preferred that Ground uses the repetition A/T/C/G sequences number for the modification that methylates to be preferred for 2-8.

In one example, 3 ' single-stranded ends of the Article 2 carry out biotinylation (bioten) modification.

In one example, described two single-stranded existing of 3 ' end arm (spacer) modifications have biotinylation again (bioten) it modifies.

In one example, between described arm (spacer) modification can be located at 5 ' single-stranded end terminal sequences of the Article 2 with Between biotinylation (bioten) modification.More preferably, connected between arm (spacer) modification and biotin with TEG between described It connects.

In one example, 3 ' single-stranded ends of the Article 2 carry out phosphorylation modification.

In one example, the DNA cloning reagent is selected from archaeal dna polymerase, dNTPs, DNA polymerase buffer liquid, amplification Primer.

In one example, the archaeal dna polymerase is selected from vent archaeal dna polymerases, T7 archaeal dna polymerases, Bsu DNA polymerizations Enzyme, T4 archaeal dna polymerases, Klenow segments, DNA polymerase i (E.coli), Therminator^TMArchaeal dna polymerase, Sulfolobus DNA polymerase i V, phi29 archaeal dna polymerase, Bst2.0Archaeal dna polymerase, 2.0 DNA of Bst Polymerase, Bst archaeal dna polymerases, Deep VentR (exo -) archaeal dna polymerase, Deep VentR^TMArchaeal dna polymerase, VentR (exo -) archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Taq archaeal dna polymerases,Heat opens Dynamic Taq archaeal dna polymerases,Taq archaeal dna polymerases, thermal starting Taq archaeal dna polymerases, Taq archaeal dna polymerases are large stretch of Section, Taq archaeal dna polymerases,Thermal starting archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Flex archaeal dna polymerases,Super fidelity dna polymerase,Thermal starting surpass fidelity dna polymerase,Super fidelity The combination of one or more of enzymes in archaeal dna polymerase etc..It can also be 10mM can also be that the dNTPs, which can be 2.5mM, Other concentration.Those skilled in the art can select appropriate concentration according to actual needs, as long as not limiting the mesh of the present invention .

In one example, the DNA polymerase buffer liquid delays for the best match that archaeal dna polymerase selected by correspondence is selected Fliud flushing.

In one example, the sequence of the amplimer and the non-stickiness part of any one chain of cohesive terminus,cohesive termini connector Sequence it is all or part of complementary.The non-stickiness part refers to the double stranded section of cohesive terminus,cohesive termini connector.

The DNA of the present invention builds library kit and can be used for preparing gene establishment library product or gene order-checking product.

The DNA of the present invention builds the genome dna library foundation that library kit can be also used for sample.

When the use DNA builds library kit, and genomic DNA progress library is established, it may include following steps：

(1) to make sample genomic dna using well known to a person skilled in the art for example high-temperature denatured rear sudden cold schemes of technology It is denaturalized into single stranded DNA；

(3) double-stranded DNA is extended into using single stranded DNA to carry out single stranded DNA of the one end in step (2) with connector with reagent Extension forms the double-stranded DNA that one end carries connector；

(5) double-stranded DNA of the both-end that step (4) obtains with connector is expanded using DNA cloning reagent, is surveyed Preface library.

In one example, the sample genomic dna is cracked by unicellular sample obtains or is extracted by many cells sample It obtains.

It can be before step (1) is carried out, first unicellular sample is cracked or many cells sample is extracted, so as to obtain The sample genomic dna.It can also use and cracked via unicellular sample using technology known in the art by other people The sample genomic dna for obtaining or being obtained by the extracting of many cells sample.

Unicellular sample is cracked using cell pyrolysis liquid or many cells sample is taken out using genome DNA extraction reagent It carries, so as to obtain the sample genomic dna.It can also use by other people using technology known in the art via slender The sample genomic dna that the cracking of born of the same parents' sample is obtained or obtained by the extracting of many cells sample.The initial amount of individual cells is generally 6pg。

The minimum 5ng of initial amount of existing DNA sequencing scheme.Compared with prior art, Novel DNA provided by the invention is built The sample initial amount of library kit reduces 3 orders of magnitude.Exploration for cell heterogeneity, the research for treasuring sample have pole Its important meaning.

In one example, the quality of the genomic DNA of the sample is more than or equal to 6pg.

In one example, the cell is specially prokaryotic cell or eukaryocyte.

In one example, the eukaryocyte is specially plant cell or zooblast.

In one example, the zooblast is chosen in particular from the cell of tissue digestion, the cell of culture gained, embryo's hair Educate cell that the cell of early stage, the cell of cancer early stage, the microbial cell without enrichment culture, airflow classification obtain, limited Any one of cell that the methods of diluting the cell obtained, laser capture obtains.

Usually, in step (1), those skilled in the art can be used well known technology, for example, it is high-temperature denatured after it is sudden it is cold, The schemes such as NaOH solution alkaline denaturation make sample genomic dna denaturation into single stranded DNA.The high temperature generally refers to 60 DEG C -98 DEG C.It is excellent Selection of land is 80 DEG C -98 DEG C.More preferably 90 DEG C -98 DEG C.Processing time is generally 30s-10min.The NaOH solution is 0.2 ~0.6M NaOH, 1~1.5MNaCl.Preferably 0.3~0.5M NaOH, 1~1.5MNaCl.More preferably 0.4M NaOH, 1.5MNaCl.Those skilled in the art can select suitable method to be denaturalized sample genomic dna according to actual needs Into single stranded DNA, as long as not limiting the purpose of the present invention.

In addition, the cohesive terminus,cohesive termini connector, double-stranded adapters, DNA extension reagent, DNA cloning reagent are as described above.

Random primer amplification in compared with the prior art, Novel DNA using the present invention build library kit and carry out DNA Library is built, can cohesive terminus,cohesive termini joint sequence directly be introduced using ligase, therefore, can ensure that the strand of a DNA derives from mould The same area of plate chain, so as to fulfill it is follow-up than to arriving reference gene group when more accurate both-end can be used to compare pattern, increasing The accuracy of addend evidence.

The middle amplification mode that single-stranded connection is carried out using circligase compared with the prior art, it is using the present invention novel DNA builds library kit progress DNA and builds library, and the generic connectivity enzyme such as T4 DNA ligases that more economical practicality can be used carry out connector Connection.These common ligases are cheap, and can retain sample to a greater extent without selectivity to target DNA fragment Diversity, reduce due to library skewed popularity caused by artificial origin.

Novel DNA using the present invention builds library kit progress DNA and builds library, and operation is flexible, can be as needed using special Property cohesive terminus,cohesive termini connector be directly connected to single stranded DNA, the amplicon obtained by amplification is suitable for a series of subsequent applications, such as may be used For building generation sequencing library, two generation sequencing libraries of structure are can be used for, three generations's sequencing library can also be built, also may be used Foundation for qPCR templates, the clone that can be also used for short segment DNA etc..User can be according to the sequencing library of structure Classification, expanded using corresponding PCR, to build different sequencing libraries.

It should be noted that Novel DNA using the present invention builds library kit and carries out DNA and builds the original samples in library and can be The genome of one cell, is illustrative only and not limiting.It is easily understood that the original samples can also be multiple cells Genome.

Novel DNA using the present invention builds library kit and carries out DNA and build library that full-length genome can be covered, including being rich in CpG islands Promoter region, be scattered CpG sites, CHG CHH sites；In addition to genomic promoter region further includes enhancer, transcription Initiation site, noncoding region (UTR), extron, intergenic region section (intergenic), genosome (gene body) etc., to complete Most sites of genome carry out augmentation detection.

The present invention furthermore provides a kind of gene order-checking product, and library kit is built including aforementioned DNA.

Further, the sequencing can be generation sequencing, the sequencing of two generations or three generations's sequencing.

Further, the platform of the sequencing can be illumina microarray datasets.

Hereafter the technical solution of the embodiment of the present invention is further illustrated with specific embodiment 1.

Embodiment 1

First, DNA builds the preparation of library kit

(1), DNA builds cohesive terminus,cohesive termini connector in the kit of library and synthesizes：

The cohesive terminus,cohesive termini connector is the double-stranded adapters with cohesive terminus,cohesive termini, and one end of the cohesive terminus,cohesive termini connector is first Cohesive terminus,cohesive termini, first cohesive terminus,cohesive termini include at least random sequence.

1st, as an example, synthesizing cohesive terminus,cohesive termini connector using Primer1 and Primer2,

Primer1：5’-AGATCGGAAG-3’(SEQ ID NO.1)；

Primer2：5’-CTTCCGATCTNNNNNN-3’(SEQ ID NO.2)；Wherein, NNNNNN represents random sequence, The length of the usual random sequence is 2-30nt, more preferably preferably 4-20nt, 5-10nt.

2nd, according to the form below 1 adds in configuration reaction solution：

Table 1

Reagent	Volume (ul)
		Primer1(10μM)	1
Primer2(10μM)	1
		TE buffer	7
5M NaCl	1
		Total volume	10

The TE buffer used in wherein reacting can also be had by water, Tris-Hcl (0.05mM, PH7.0-8.0) etc. The buffer solution of identity function is substituted.

3rd, according to the form below 2 is reacted：

Table 2

Temperature	Time	Recurring number
			95℃	60s	1
95℃-16℃	0.5℃/s	1
			16℃	forever	1

Obtain cohesive terminus,cohesive termini connector：

5’-CTTCCGATCT-NNNNNN-3’

bioten-3’-GAAGGCTAGA-5’.Can unloading in -20 DEG C preservation.

Also that is, illustratively, Primer2 and Primer1 together form cohesive terminus,cohesive termini connector, the cohesive terminus,cohesive termini connector To carry the double-stranded adapters of cohesive terminus,cohesive termini, one end of the cohesive terminus,cohesive termini connector is the first cohesive terminus,cohesive termini, the first stickiness end End includes random sequence NNNNNN, and the length of the usual random sequence NNNNNN is 2-30nt, preferably 4-20nt, more excellent Choosing is 5-10nt.If by single-stranded referred to as first chain with the first cohesive terminus,cohesive termini, without the single-stranded of the first cohesive terminus,cohesive termini Referred to as Article 2 chain, then the 3 ' of Article 2 chain carried out bioten modifications.In this example, the cohesive terminus,cohesive termini connector is another It holds as flat end.

Reaction condition in table 2 is intended only as example, similar scheme can also be used to carry out, and the overall principle is first high temperature Slowly annealing forms complementary DNA double chain after denaturation.

(2), DNA builds the preparation of library kit double center chain connector

The end of the double-stranded adapters is flat end or cohesive terminus,cohesive termini.

1st, it is a cohesive terminus,cohesive termini as an example, double-stranded adapters are prepared by Primer3 and Primer4.

Primer3：5’-CTCTTCCGATC-3’(SEQ ID NO.3)；

Primer4：5’-GGAAGAGCGTCGTGTAGGGAAAGA-3’(SEQ ID NO.4)；

At 3 ' ends, preferably by the C of phosphorylation modification, bi-deoxyribose nucleic acid more preferably may be used in Primer3 C carries out the synthesis of primer；Primer4 can select to carry out thio-modification or without thio-modification, it is therefore preferable to carry out thio 3 bases that modification, more preferably thio-modification 3 ' are held；5 ' the ends of primer4 are preferably by phosphorylation modification.

2nd, according to the form below 3 adds in configuration reaction solution：

Table 3

3rd, according to the form below 4 is reacted：

Table 4

Obtain double-stranded adapters can unloading in -20 DEG C preservation.

Reaction condition in upper table 5 is intended only as example, similar scheme can also be used to carry out, and the overall principle is first high Slowly annealing is complementary DNA double chain structure after temperature denaturation.

(3) ligase, DNA extensions reagent, the DNA cloning reagent that DNA is built in the kit of library can be by commercially available approach It obtains.

Specifically, the DNA extensions reagent includes archaeal dna polymerase, dNTPs, DNA polymerase buffer liquid, extension primer.

The sequence of the non-stickiness part of the sequence of the extension primer and any one chain of cohesive terminus,cohesive termini connector all or Partial complementarity.The non-stickiness part refers to the double stranded section of cohesive terminus,cohesive termini connector.

As an example, the extension primer uses：

Primer5：5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-3’(SEQ ID NO.5).

The DNA cloning reagent includes archaeal dna polymerase, dNTPs, DNA polymerase buffer liquid, amplimer.

The sequence of the non-stickiness part of the sequence of the amplimer and any one chain of cohesive terminus,cohesive termini connector all or Partial complementarity.The non-stickiness part refers to the double stranded section of cohesive terminus,cohesive termini connector.

As an example, the amplimer uses：

Primer6：

5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’(SEQ ID NO.6)；

Primer7：

5’-CAAGCAGAAGACGGCATACGAGATtgccgaGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT- 3’(SEQ ID NO.7)。

2nd, library kit is built using DNA and library foundation is carried out to sample genomic dna

(1) dephosphorylation is carried out to the genomic DNA of sample, prevents connecting certainly between the genomic DNA of sample, this Step is optional step

Specifically, according to the form below 5 adds in configuration reaction solution：

Table 5

Reagent	Volume (ul)
		10x T4DNA ligation buffer	4
1%Tween 20	1
		FastAP(1U/μl)	1
The genomic DNA (6pg-100ng) of sample	14
		Total volume	20

Here the genome of sample can unicellular sample be cracked or using genomic DNA using cell pyrolysis liquid Extraction agent extracts many cells sample and obtains.It can also use by other people using technology known in the art via slender The sample genomic dna that the cracking of born of the same parents' sample is obtained or obtained by the extracting of many cells sample.

(2) to make sample genomic dna using well known to a person skilled in the art for example high-temperature denatured rear sudden cold schemes of technology It is denaturalized into single stranded DNA

Specifically, after sample being flicked mixing, according to the form below 6 is reacted：

Table 6

Temperature	Time
		37℃	30min
95℃	5min

Sample is immediately transferred on ice.

(3) single stranded DNA is attached by ligase and cohesive terminus,cohesive termini connector, obtains the single stranded DNA that one end carries connector Specifically, according to the form below 7 adds in configuration reaction solution：

Table 7

Flick mixing, total volume 40ul.

Then, according to the form below 8 is reacted：

Table 8

Temperature	Time	Recurring number
			37℃	30min	1
95℃	5min	1
			4℃	forever	1

Sample in -20 DEG C can be preserved or directly carry out next step reaction.

As shown in figure 3, the bonding ratio using 3% agarose gel electrophoresis detection single stranded DNA and the double-stranded adapters with stickiness. Wherein, it is cloudy right：Not plus the negative control of ligase, 10ng；1：Represent No. 1 sample, 10ng；2：Represent No. 2 samples, 50ng；3： Represent No. 3 samples, 100ng.S+A：The sample of successful connection adds connector, and product length is more than sample therefore shows what is run on glue It is relatively slow；S：Sample is not connected with connector, and molecular weight is smaller, therefore the very fast of race is shown on glue；A：Connector.From figure 3, it can be seen that In different sample initial amounts, there is preferable connection result, top connection can be fully connected substantially when sample initial amount is low. When sample size increases, have sample segment could not successful connection, but since connector has been depleted, therefore improving connector content can improve Measure the bonding ratio of sample.

DNA sample can not have to FastAP processing when being connect with the double-stranded adapters with stickiness, preferably sharp With FastAP processing.The 1%Tween 20 used in reaction can be replaced by NP40, triton x-100 of same concentration Generation；T4 DNA ligases can be substituted by other enzymes with double-strand or single-stranded connection enzymatic activity, preferably there is HiFi Taq DNA ligase, T4RNA ligases 2,Ligase, 9 ° of N^TMDNA ligase, Taq DNA ligases, T7 DNA connections Enzyme, T3 DNA ligases, electricity turn ligase, flat end/TA ligases premixed liquid, instantaneous stickiness ligase premixed liquid, One or more of Circligase ssDNA ligase, 5 ' AppDNA/RNA thermostabilization ligases.It is preferably used T4DNA ligases.

(4), magnetic capture one end carries the single stranded DNA of connector, and the visual concrete condition selection of this step, is optional step

1st, the magnetic bead for modifying Streptavidin, at least balances 30 minutes, vortex mixing in room temperature.

2nd, it takes in the EP pipes of beads to the 1.5ml of 20ul (20ul/ samples).

3rd, 1xBinding buffer (1M NaCl, the 10mM Tris-HCl (pH 8.0), 1mM of 500 μ l of Beads EDTA (pH 8.0), 0.05%Tween-20,0.5%SDS) it washs 2 times.

4th, beads is resuspended in the 1xBinding buffer of 100ul.

5th, sample (40ul) is heated into 1min at 95 DEG C, is quickly transferred on ice, place at least 2-5min.

6th, the beads mixings for completing sample with washing.

7th, 40min, and continuous mixing are being incubated at room temperature.

8th, wink from, EP pipes are placed on magnetic frame, in removal please.

9th, wash bufferI (0.1M NaCl, 10mM Tris-HCl (pH 8.0), 1mM the EDTA (pH of 200 μ l are added in 8.0), 0.05%Tween-20,0.5%SDS), beads is resuspended in vortex 8s.

10th, wink from, EP pipes are transferred on magnetic frame, remove supernatant.

The magnetic bead of wherein Streptavidin modification can be Dynabeads^TMM-280Streptavidin、 Dynabeads^TMMyOne^TMStreptavidin C1、Dynabeads^TMThe commercial products such as M-270 Streptavidin, also may be used To be similar Magnetic bead sample that the Streptavidin that is voluntarily coupled is modified.

(5), it extends into double-stranded DNA using single stranded DNA to extend single stranded DNA of the one end with connector with reagent, shape The double-stranded DNA of connector is carried into one end

Specifically, according to the form below 9 adds in configuration reaction solution：

Table 9

Reagent	Volume (ul)
		10x Klenow reaction buffer	5
10mM dNTPs	0.4
		1%Tween-20	2.5
Primer5(10μM)	1
		Water	39.1
Total volume	48

Primer5：5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-3’(SEQ ID NO.5).

Optionally, primer5 can carry out thio-modification, also can preferably carry out thio-modification, more without thio-modification Preferably whole primer carries out thio-modification, and more preferably the 10 of 3 ends base carries out thio-modification；More preferably 3 5 bases at end carry out thio-modification；A base at more preferably 3 ends carries out thio-modification.

1. beads is transferred on magnetic frame, it please in removal.

2. adding in the mixture in upper table 9, vortex makes the abundant mixings of beads.

3. 95 DEG C are transferred the sample into, 2min.

4. it is quickly transferred on ice, at least place 2-5min.

5. sample is transferred in room temperature in EP pipes.

6. adding in the Klenow fragment (10U/ μ l) of 2ul, beads is resuspended mixing.

7.25 DEG C of incubation 5min, per 60s mixings 2s, 1500rpm.

8.35 DEG C of incubation 25min, per 60s mixings 2s, 1500rpm.

Wherein Klenow fragment can also be substituted by other polymerases, such as：Vent archaeal dna polymerases, T7 Archaeal dna polymerase, Bsu archaeal dna polymerases large fragment, T4 archaeal dna polymerases, Klenow segments, DNA polymerase i (E.coli), Therminator^TMArchaeal dna polymerase, Sulfolobus DNA polymerase i V, phi29 archaeal dna polymerase, Bst2.0Archaeal dna polymerase, 2.0 archaeal dna polymerases of Bst, Bst archaeal dna polymerases, Deep VentR (exo -) DNA polymerizations Enzyme, Deep VentR^TMArchaeal dna polymerase, VentR (exo -) archaeal dna polymerase,Archaeal dna polymerase,Heat opens Dynamic Taq archaeal dna polymerases,Thermal starting Taq archaeal dna polymerases,Taq archaeal dna polymerases, thermal starting Taq archaeal dna polymerases, Taq archaeal dna polymerases large fragment, Taq archaeal dna polymerases,Thermal starting archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Flex archaeal dna polymerases,Super fidelity dna polymerase, Thermal starting surpass fidelity dna polymerase,The combination of the one or more enzyme such as super fidelity dna polymerase.

Above-mentioned reaction system is served only for example displaying rather than limits.

(6), it is cleaned after expanding, is optional step

1. sample wink from, be transferred on magnetic frame, remove supernatant.

11st, wash bufferI (0.1M NaCl, the 10mM Tris-HCl (pH 8.0), 1mM EDTA of 200 μ l is added in (pH 8.0), 0.05%Tween-20,0.5%SDS).

2. by sample blending, wink is from being transferred on magnetic frame, remove supernatant.

3. add in the wash buffer II (0.5xSSC, PH7.0) of 100 μ l.

4. sample is incubated at room temperature 3min.

5. wink from, be transferred on magnetic frame, in removal please.

6. add in wash bufferI (0.1M NaCl, 10mM Tris-HCl (pH 8.0), 1mM the EDTA (pH of 200 μ l 8.0), 0.05%Tween-20,0.5%SDS), beads is resuspended in vortex 8s.

Above-mentioned cleaning program only with example show rather than limit.

(7), the other end of double-stranded DNA of the one end with connector is connected to using double-stranded adapters, both-end is formed and carries connector Double-stranded DNA

According to the form below 10 adds in configuration reaction solution：

Table 10

1. beads is removed into supernatant, with the mix mixings in upper table 10.

2. sample is paid attention into mixing in 25 DEG C of incubation 1h, incubation.

T4 DNA ligases can be substituted by other enzymes with double-strand or single-stranded connection enzymatic activity, preferably there is HiFi Taq DNA ligases, T4 RNA ligases 2,Ligase, 9 ° of N^TMDNA ligase, Taq DNA ligases, T7 DNA Ligase, T3 DNA ligases, electricity turn ligase, flat end/TA ligases premixed liquid, instantaneous stickiness ligase premixed liquid, T4 DNA ligase, Circligase ssDNA ligase, 5 ' AppDNA/RNA thermostabilizations ligases, etc. one or more of enzymes group It closes.T4DNA ligases are preferably used.

(8), washing and sample elution after connecting

1. by sample wink from, be transferred on magnetic frame, in removal please.

2. add in wash bufferI (0.1M NaCl, 10mM Tris-HCl (pH 8.0), 1mM the EDTA (pH of 200 μ l 8.0), 0.05%Tween-20,0.5%SDS).

3. by sample blending.

4. wink from, be transferred on magnetic frame, remove supernatant.

5. add in the wash buffer II (0.5xSSC, PH7.0) of 100 μ l.

6. by sample at 45 DEG C, it is incubated 3min.

7. wink from, be transferred on magnetic frame, in removal please.

8. add in wash bufferI (0.1M NaCl, 10mM Tris-HCl (pH 8.0), 1mM the EDTA (pH of 200 μ l 8.0), 0.05%Tween-20,0.5%SDS).

9. transferring the sample into mixing, it is transferred on magnetic frame, it please in removal.

10. add in the seedless sour water of 50 μ l.

11. by sample after 10min is incubated at room temperature, 95 DEG C of heating 2min.

12. transferring the sample on magnetic frame, supernatant is transferred in the EP pipes of clean low suction.

Above-mentioned double-stranded adapters connection scheme is served only for example explanation rather than limits.

(9), double-stranded DNA of the both-end with connector is expanded using DNA cloning reagent, it is (optional obtains sequencing library Step) the mixing pcr amplification raw materials of according to the form below 11：

Table 11

Sample	Volume (ul)
		KAPA HiFi HotStart ReadyMix(2x)	25
Primer6(10μM)	1
		Primer7(10μM)	1
NF water	23
		Total volume	50

By the sample of elution or directly with the following reaction of magnetic bead progress, (each sample can do 2 or multiple parallel PCR Amplified reaction can increase the yield of sample).

Mixing sample, according to the form below 12 are expanded：

Table 12

Primer6：

Primer7：

5’-CAAGCAGAAGACGGCATACGAGATtgccgaGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT- 3’(SEQ ID NO.7)；

Wherein underscore part is index sequences, could alternatively be other index sequences, specifically refers to The index sequences that illumina sequencing instruments are applicable in.

If you do not need to carrying out library sequencing, only expanded, then carried out using following primer：

Primer8：5’-CACTCTTTCCCTACACGACGCTCTTCCGATCT-3’(SEQ ID NO.8)；

Primer9：5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’(SEQ ID NO.9)；

The overall length primer that Primer8 or Primer9 can wherein be chosen is expanded or chooses a part therein It is preferred that overall length primer is selected to be expanded as PCR primer.

Wherein KAPA HiFi HotStart ReadyMix (2x) can also be substituted by other polymerases, such as vent Archaeal dna polymerase, T7 archaeal dna polymerases, Bsu archaeal dna polymerases large fragment, T4 archaeal dna polymerases, Klenow segments, DNA polymerase i (E.coli)、Therminator^TMArchaeal dna polymerase, Sulfolobus DNA polymerase i V, phi29 archaeal dna polymerase, Bst2.0Archaeal dna polymerase, 2.0 archaeal dna polymerases of Bst, Bst archaeal dna polymerases, Deep VentR (exo -) DNA polymerizations Enzyme, Deep VentR^TMArchaeal dna polymerase, VentR (exo -) archaeal dna polymerase,Archaeal dna polymerase,Heat opens Dynamic Taq archaeal dna polymerases,Thermal starting Taq archaeal dna polymerases,Taq archaeal dna polymerases, thermal starting Taq archaeal dna polymerases, Taq archaeal dna polymerases large fragment, Taq archaeal dna polymerases,Thermal starting archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Flex archaeal dna polymerases,Super fidelity dna polymerase, Thermal starting surpass fidelity dna polymerase,Super fidelity dna polymerase, KAPA HiFi Uracil+ReadyMix (2x), The combination of the one or more enzyme such as PfuTurbo Cx thermal starting archaeal dna polymerases.

Above-mentioned reaction is served only for example displaying rather than limits.

(10), Ampure XP beads purification of samples

1. AMPure XP BeadsDNA are purified into magnetic bead in equilibrium at room temperature at least 30min.

2. in the sample for taking 40 μ lDNA purifying magnetic bead Agencourt AMPure XP Beads to 50 μ l, vortex mixing. If same sample has carried out parallel PCR reactions, multiple samples can be mixed into a pipe.Wherein DNA purifying magnetic beads Agencourt The dosage of AMPure XP Beads can more need to be adjusted, preferably 40-50ul.

3. being placed at room temperature for 5min, sample is made fully to be combined with DNA.

4. transferring the sample on magnetic frame, discarded supernatant after sample clarification.

5. with the ethyl alcohol (now with the current) of fresh 200ul 80% (vol/vol), beads is cleaned, can be turned upside down, turned Dynamic EP pipes, make washing abundant.

6. it discards supernatant.

7. with the ethyl alcohol (now with the current) of fresh 200ul 80% (vol/vol), beads is cleaned, can be turned upside down, turned Dynamic EP pipes, make washing abundant.It discards supernatant.

8. wink from primary, residual ethanol is discarded after sample clarification.

The waiting 9. room temperature is uncapped, makes ethyl alcohol fully volatilize, until small slight crack occurs in surface.(overdrying or there can not be the ethyl alcohol residual It stays).

10. suitable seedless sour water is added in, with the abundant mixing of sample.

11. in incubation at room temperature at least 2min.

12. supernatant is transferred in the EP pipes of clean NF, remaining 2ul carries out quantitative and quality inspection.

Wherein DNA purifying magnetic beads can be that AMPure XP Beads can be by waiting other to have effects that identical magnetic bead replaces In generation, in addition to that can use magnetic beads for purifying sample, can also use has effects that identical kit or scheme substitute.

The present embodiment library outbound chip testing result figure is as shown in figure 4,2100 highly sensitive chip of Agilent detects library Clip size after outbound, X-axis are clip size, and Y-axis is fluorescence intensity.The size in the library is 100bp-400bp, wherein dividing Segment of the cloth in 220bp or so reaches peak value.

Lower machine data Analysis of quality control, as shown in table 13：

Table 13

Sample ID	Sample source	Data volume (G base)	Sequencing pattern	Yield (ng)	Q20	Q30	map_rate
								Sample1	FFPE samples	1.05	PE150	356	79.59	75.91	21.18%
Sample2	FFPE samples	1.11	PE150	653	80.12	76.64	20.71%
								Sample3	FFPE samples	1.13	PE150	643	83.75	80.79	20.36%
Sample4	FFPE samples	1.06	PE150	581	93.74	87.66	62.83%
								Sample5	FFPE samples	1.23	PE150	573	93.75	87.68	62.88%
Sample6	FFPE samples	1.15	PE150	652	94.3	88.6	62.41%

If certain base mass value is 20 (Q20), then it represents that the probability of base sequencing error is 0.01.If certain base Mass value is 30 (Q30), then it represents that the probability of base sequencing error is 0.001.Map_rate：Can money order receipt to be signed and returned to the sender to genome number According to the ratio for accounting for lower machine data.The sample source of Sample1, Sample2, Sample3, Sample4, Sample5, Sample6 For the DNA of FFPE sample extractions, 6 parts are equally divided into, every part of 20ng.Sample1, Sample2, Sample3 are using conventional The scheme of illumina double-strands connection build library (specifically using kit such as：IlluminaSample Preparation Kits；Custom Amplicon orAmplicon-Cancer Panel；Targeted RNA Expression；HT Sample Prep Kits；v1/v2/LT Sample Prep Kits；Synthetic Long-Read DNA Library Prep Kits； Small RNA Sample Prep Kits；DNA Sample Prep Kit：Multiplexing Sample Prep Oligo Only Kit；v1 and v1.5Small RNA Kits；Sample Preparation Kits), and Sample4, Sample5, Sample6 using the Novel DNA in the present invention build library kit by single stranded DNA with it is glutinous Property end fitting connection mode carry out building library.Two schemes as can be known from Table 13, yield is suitable, but is adopted in the quality of data The side that library kit in a manner that single stranded DNA is connect with cohesive terminus,cohesive termini connector build library is built with the Novel DNA in the present invention Formula is substantially better than the scheme of conventional illumina double-strands connection.

In addition, parallel samples be respectively adopted traditional two chain build library scheme and the solution of the present invention structure text into library, Using identical experiment condition, the result figure of the content of library molecule is identified using the mode of relative quantification, as shown in Figure 5 It is tested by 3 repetitions, the connection mode of single stranded DNA about improves 2 times of molecular amounts than double-stranded DNA connection mode.

In conclusion the present invention effectively overcomes various shortcoming of the prior art and has high industrial utilization.

The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe The personage for knowing this technology all can carry out modifications and changes under the spirit and scope without prejudice to the present invention to above-described embodiment.Cause This, those of ordinary skill in the art is complete without departing from disclosed spirit and institute under technological thought such as Into all equivalent modifications or change, should by the present invention claim be covered.

Sequence table

<110>Shanghai Major Biological Medical Technology Co., Ltd.

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Claims

1. a kind of DNA builds library kit, include at least：Cohesive terminus,cohesive termini connector, ligase, DNA extension reagents and DNA cloning Reagent.

2. DNA according to claim 1 builds library kit, which is characterized in that the kit further includes slender cellular lysate Liquid, the unicellular lysate are used to crack unicellular sample, obtain unicellular sample genomic dna.

3. DNA according to claim 1 builds library kit, which is characterized in that the kit further includes double-stranded adapters.

4. DNA according to claim 3 builds library kit, which is characterized in that

The cohesive terminus,cohesive termini connector is used to be connected to the single stranded DNA of sample genomic dna denaturation, obtains one end and carries connector Single stranded DNA；

The ligase is used to connect cohesive terminus,cohesive termini connector and the single stranded DNA of sample genomic dna denaturation；

The DNA extensions reagent is used to extend single stranded DNA of the described one end with connector, forms one end and carries connector Double-stranded DNA；

The double-stranded adapters are used to be connected to the other end of double-stranded DNA of the described one end with connector, form both-end and carry connector Double-stranded DNA；

The DNA cloning reagent is used to expand double-stranded DNA of the both-end with connector, obtains library.

5. DNA according to claim 1 builds library kit, which is characterized in that the cohesive terminus,cohesive termini connector is with stickiness The double-stranded adapters of end, one end of the cohesive terminus,cohesive termini connector is the first cohesive terminus,cohesive termini, and first cohesive terminus,cohesive termini includes at least Random sequence.

6. DNA according to claim 5 builds library kit, which is characterized in that the other end of the cohesive terminus,cohesive termini connector is Flat end or the second cohesive terminus,cohesive termini.

7. DNA according to claim 6 builds library kit, which is characterized in that when the other end of the cohesive terminus,cohesive termini connector During for the second cohesive terminus,cohesive termini, any one of following characteristics or multinomial are further included：(1) second cohesive terminus,cohesive termini is or stochastic ordering The repetitive sequence of row or A/T/C/G oligonucleotides, preferably repetitive sequence；(2) cohesive terminus,cohesive termini may be other sequences Row, overall length or part such as the joint sequence of illumina microarray datasets to form cohesive end, prevent connector from connecting certainly；(3) institute The length for stating the second cohesive terminus,cohesive termini is 2-10nt, preferably 2-5nt.

8. DNA according to claim 5 builds library kit, which is characterized in that first cohesive terminus,cohesive termini includes stochastic ordering Row and special sequence.

9. DNA according to claim 8 builds library kit, which is characterized in that the special sequence is the sequence that sequence determines Row.

10. DNA according to claim 5 builds library kit, which is characterized in that in the cohesive terminus,cohesive termini connector, with The single-stranded length of one cohesive terminus,cohesive termini is 10-70nt, more preferably preferably 10-30nt, 10-25nt.

11. DNA according to claim 5 builds library kit, which is characterized in that in the cohesive terminus,cohesive termini connector, with The single-stranded annealing temperature of one cohesive terminus,cohesive termini is 10-100 DEG C, preferably 20-80 DEG C, more preferably 30-60 DEG C.

12. DNA according to claim 5 builds library kit, which is characterized in that further include any one of following characteristics or It is multinomial：(1) in the cohesive terminus,cohesive termini connector, 5 ' the single-stranded ends with the first cohesive terminus,cohesive termini carry out phosphorylation modification；(2) it is described In cohesive terminus,cohesive termini connector, 5 ' the single-stranded ends with the first cohesive terminus,cohesive termini carry out spacer modifications；(3) the cohesive terminus,cohesive termini connector In, 5 ' the single-stranded ends with the first cohesive terminus,cohesive termini carry out amido modified；(4) it is glutinous with first in the cohesive terminus,cohesive termini connector Property end single-stranded 5 ' end using the modification that methylates repetition A/T/C/G sequences；(5) it in the cohesive terminus,cohesive termini connector, carries 3 ' the single-stranded ends of first cohesive terminus,cohesive termini carry out biotinylation modification；(6) in the cohesive terminus,cohesive termini connector, with the first stickiness end 3 ' the single-stranded ends at end carry out spacer modifications；(7) in the cohesive terminus,cohesive termini connector, single-stranded 3 ' with the first cohesive terminus,cohesive termini End carries out amido modified.

13. DNA according to claim 5 builds library kit, which is characterized in that in the cohesive terminus,cohesive termini connector, without That single-stranded referred to as another of first cohesive terminus,cohesive termini is single-stranded.

14. DNA according to claim 13 builds library kit, which is characterized in that in the cohesive terminus,cohesive termini connector, another Single-stranded length is 10-70nt, more preferably preferably 10-30nt, 10-25nt.

15. DNA according to claim 13 builds library kit, which is characterized in that in the cohesive terminus,cohesive termini connector, another Single-stranded annealing temperature is 10-100 DEG C, preferably 20-80 DEG C, more preferably 30-60 DEG C.

16. DNA according to claim 13 builds library kit, which is characterized in that further includes any one of following characteristics It is or multinomial：(1) in the cohesive terminus,cohesive termini connector, another 5 ' single-stranded ends carry out phosphorylation modification；(2) cohesive terminus,cohesive termini connects In head, another 5 ' single-stranded ends carry out spacer modifications；(3) in the cohesive terminus,cohesive termini connector, another single-stranded 5 ' hold into Row is amido modified；(4) in the cohesive terminus,cohesive termini connector, another 3 ' single-stranded ends carry out biotinylation modification；(5) stickiness In end fitting, another 3 ' single-stranded ends carry out spacer modifications；(6) in the cohesive terminus,cohesive termini connector, another single-stranded 3 ' ends carry out amido modified.

17. DNA according to claim 1 builds library kit, which is characterized in that the ligase is selected from HiFi Taq DNA Ligase, T4 RNA ligases 2, T4 RNA ligases,Ligase, 9 ° of N^TMDNA ligase, Taq DNA ligases, T7 DNA ligases, T3 DNA ligases, electricity turn ligase, flat end/TA ligases premixed liquid, instantaneous stickiness ligase premix Any one of liquid, T4 DNA ligases, Circligase ssDNA ligase, 5 ' AppDNA/RNA thermostabilization ligases or It is a variety of.

18. DNA according to claim 1 builds library kit, which is characterized in that it is described extension reagent be selected from archaeal dna polymerase, DNTPs, DNA polymerase buffer liquid, extension primer.

19. DNA according to claim 18 builds library kit, which is characterized in that further includes any one of following characteristics It is or multinomial：(1) archaeal dna polymerase is selected from vent archaeal dna polymerases, T7 archaeal dna polymerases, Bsu archaeal dna polymerases, T4 DNA and gathers Synthase, Klenow segments, DNA polymerase i (E.coli), Therminator^TMArchaeal dna polymerase, Sulfolobus archaeal dna polymerases IV, phi29 archaeal dna polymerase, Bst2.0Archaeal dna polymerase, 2.0 archaeal dna polymerases of Bst, Bst DNA polymerizations Enzyme, Bst archaeal dna polymerases, Deep VentR (exo -) archaeal dna polymerase, Deep VentR^TMArchaeal dna polymerase, VentR (exo -) Archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Taq archaeal dna polymerases,Thermal starting Taq Archaeal dna polymerase,Taq archaeal dna polymerases, thermal starting Taq archaeal dna polymerases, Taq archaeal dna polymerases large fragment, Taq Archaeal dna polymerase,Thermal starting archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Flex DNA gather Synthase,Super fidelity dna polymerase,Thermal starting surpass fidelity dna polymerase,In super fidelity dna polymerase The combination of one or more of enzymes；(2) a concentration of 2.5mM or 10mM of the dNTPs；(3) sequence of the extension primer is with sticking Property end fitting any one chain non-stickiness part sequence it is all or part of complementary.

20. DNA according to claim 1 builds library kit, which is characterized in that the DNA cloning reagent polymerize selected from DNA Enzyme, dNTPs, DNA polymerase buffer liquid, amplimer.

21. DNA according to claim 1 builds library kit, which is characterized in that further include any one of following characteristics or It is multinomial：(1) archaeal dna polymerase is selected from vent archaeal dna polymerases, T7 archaeal dna polymerases, Bsu archaeal dna polymerases, T4 DNA polymerizations Enzyme, Klenow segments, DNA polymerase i (E.coli), Therminator^TMArchaeal dna polymerase, Sulfolobus archaeal dna polymerases IV, phi29DNA polymerase, Bst2.0Archaeal dna polymerase, 2.0 archaeal dna polymerases of Bst, Bst archaeal dna polymerases, Bst archaeal dna polymerases, Deep VentR (exo -) archaeal dna polymerase, Deep VentR^TMArchaeal dna polymerase, VentR (exo -) DNA gather Synthase,Archaeal dna polymerase,Thermal starting Taq archaeal dna polymerases,Thermal starting Taq DNA gather Synthase,Taq archaeal dna polymerases, thermal starting Taq archaeal dna polymerases, Taq archaeal dna polymerases large fragment, Taq DNA Polymerase,Thermal starting archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Flex DNA polymerize Enzyme,Super fidelity dna polymerase,Thermal starting surpass fidelity dna polymerase,One in super fidelity dna polymerase The combination of kind or several enzymes；(2) a concentration of 2.5mM or 10mM of the dNTPs；(3) sequence and stickiness of the amplimer The sequence of the non-stickiness part of any one chain of end fitting is all or part of complementary.

22. DNA according to claim 3 builds library kit, which is characterized in that the end of the double-stranded adapters is flat end Or cohesive terminus,cohesive termini.

23. DNA according to claim 22 builds library kit, which is characterized in that the length of the cohesive terminus,cohesive termini is 2- 10nt, preferably 2-5nt.

24. DNA according to claim 22 builds library kit, which is characterized in that the cohesive terminus,cohesive termini can be with one Cohesive terminus,cohesive termini, Y types end or the repetitive sequence for A or T or C or G of a T.

25. DNA according to claim 3 builds library kit, which is characterized in that the double-stranded adapters include first chain and Article 2 chain.

26. DNA according to claim 25 builds library kit, which is characterized in that in the double-stranded adapters, first single-stranded Length for 10-70nt, preferably 10-30nt, be further preferably 10-25nt.

27. DNA according to claim 25 builds library kit, which is characterized in that in the double-stranded adapters, first single-stranded Annealing temperature for 10-100 DEG C, preferably 20-80 DEG C, be further preferably 30-60 DEG C.

28. DNA according to claim 25 builds library kit, which is characterized in that further includes any one of following characteristics It is or multinomial：(1) in the double-stranded adapters, first 5 ' single-stranded end carries out phosphorylation modification；(2) in the double-stranded adapters, the One 5 ' single-stranded end carries out spacer modifications；(3) in the double-stranded adapters, first 5 ' single-stranded end carries out biotinylation Modification；(4) in the double-stranded adapters, first 3 ' single-stranded end carries out thio-modification.

29. DNA according to claim 25 builds library kit, which is characterized in that in the double-stranded adapters, Article 2 is single-stranded Length for 10-70nt, preferably 10-30nt, be further preferably 10-25nt.

30. DNA according to claim 25 builds library kit, which is characterized in that in the double-stranded adapters, the double-strand connects In head, the single-stranded annealing temperature of Article 2 is 10-100 DEG C, and preferably 20-80 DEG C, be further preferably 30-60 DEG C.

31. DNA according to claim 25 builds library kit, which is characterized in that further includes any one of following characteristics It is or multinomial：(1) in the double-stranded adapters, 3 ' single-stranded ends of Article 2 carry out spacer modifications；(2) Article 2 is single-stranded 3 ' ends carry out biotinylation modification；(4) 3 ' single-stranded ends of the Article 2 carry out phosphorylation modification.

32. the DNA as described in claim any one of 1-31 builds library kit and is preparing gene establishment library product or gene order-checking Purposes in product.

33. the DNA as described in claim any one of 1-31 builds genome dna library of the library kit for sample and establishes or be sequenced In purposes.