阅读说明：本技术 一种利用高效地连接技术快速制备dna测序文库的方法和试剂盒 (Method and kit for rapidly preparing DNA sequencing library by utilizing efficient ligation technology ) 是由 冯延叶 刘娜 李艳玲 赖煦卉 孙大鹏 于 2019-08-29 设计创作，主要内容包括：本发明提供了一种利用高效地连接技术快速制备DNA测序文库的方法及试剂盒,其中方法包括以下步骤：DNA 3’接头连接、互补链的合成、DNA 5’接头连接以及文库扩增。试剂盒包含以下组分：工具酶、工具酶反应体系、接头体系、延伸体系、PCR反应体系以及Low-EDTA-TE。本发明实现对各种痕量DNA样本的成功建库,打破了常规的建库手段,规避了末端修复造成的DNA的损失,尽可能的反应样本的真实信息。(The invention provides a method and a kit for rapidly preparing a DNA sequencing library by utilizing an efficient ligation technology, wherein the method comprises the following steps: DNA3 'linker ligation, synthesis of complementary strand, DNA5' linker ligation, and library amplification. The kit comprises the following components: a tool enzyme, a tool enzyme reaction system, a linker system, an extension system, a PCR reaction system and Low-EDTA-TE. The method realizes the successful library construction of various trace DNA samples, breaks through the conventional library construction means, avoids the DNA loss caused by end repair, and reflects the real information of the samples as much as possible.)

1. A method for rapidly preparing a DNA sequencing library by using an efficient ligation technology is characterized by comprising the following steps:

s1, DNA3' joint connection: firstly, adding a section of polynucleotide to the 3 'end of DNA to be detected by using terminal transferase, wherein the polynucleotide is selected from PolyA, PolyC, PolyG or PolyT to obtain a DNA-polynucleotide structure, finishing the connection of the 3' end of the DNA-polynucleotide and a T7Truncated Adapter joint by using DNA repair enzyme, DNA polymerase and DNA ligase, and synthesizing a DNA-polynucleotide-T7 Truncated Adapter;

in the step S1, the DNA to be detected is a single-stranded DNA fragment or a double-stranded DNA fragment, or includes both a single-stranded DNA fragment and a double-stranded DNA fragment, and when the DNA to be detected includes a double-stranded DNA, the step S1 further includes a denaturation step, where the denaturation step denatures the double-stranded DNA into a single-stranded DNA;

s2, synthesis of complementary strand: synthesizing a complementary strand by using DNA polymerase to the DNA-poly oligonucleotide-T7 Truncated Adapter single strand added in the step S2, and synthesizing double-stranded DNA;

s3 and DNA5' joint connection: connecting a T5Truncated Adapter adaptor to the 5' end of the DNA double strand in the step S3 by using DNA ligase through blunt end or AT connection mode;

s4, library amplification: amplifying a PCR library by using a DNA polymerase reaction solution to obtain a library sample capable of being subjected to on-machine sequencing;

the T7Truncated Adapter consists of a first chain of T7Truncated Adapter and a second chain of T7Truncated Adapter, the second chain of T7Truncated Adapter is T7Truncated Adapter chain 2a, T7Truncated Adapter chain 2b, T7Truncated Adapter chain 2c or T7Truncated Adapter chain 2d, the 5' end region of the first chain of T7Truncated Adapter and the 5' end region of the second chain of T7Truncated Adapter are complementary double-stranded DNA, the 3' end of the second chain of T7Truncated Adapter is a polynucleotide selected from PolyT, PolyA, PolyG or PolyC; the 5 'end of the first chain of the T7Truncated Adapter is modified by phosphate, the 3' end of the first chain of the T7Truncated Adapter is modified by blocking, the 5 'end and the 3' end of the second chain of the T7Truncated Adapter are modified by blocking, and the blocking modified group is selected from amino, C3Space, C6Space or biotin;

the nucleotide sequence number of the first chain of the T7Truncated Adapter is shown as SEQ ID No.1, the nucleotide sequence number of the 2a chain of the T7Truncated Adapter is shown as SEQ ID No.2, the nucleotide sequence number of the 2b chain of the T7Truncated Adapter is shown as SEQ ID No.3, the nucleotide sequence number of the 2c chain of the T7Truncated Adapter is shown as SEQ ID No.4, and the nucleotide sequence number of the 2d chain of the T7Truncated Adapter is shown as SEQ ID No. 5;

the T5Truncated Adapter consists of a first strand of the T5Truncated Adapter and a second strand of the T5Truncated Adapter, wherein the 3' end region of the first strand of the Truncated Adapter and the 5' end region of the second strand of the T5Truncated Adapter are complementary into double-stranded DNA, and the 5' end region of the first strand of the T5Truncated Adapter exists in a single-stranded form; the 5 'end of the first chain of the T5Truncated Adapter is subjected to blocking modification, the 3' end of the first chain of the T5Truncated Adapter is subjected to thio modification, the 5 'end and the 3' end of the second chain of the T5Truncated Adapter are subjected to blocking modification, the blocking modification group is selected from amino, C3 Spacer, C6Spacer or biotin, and the first chain of the T5Truncated Adapter is T5Truncated Adapter chain 1a or T5Truncated Adapter chain 1 b;

the nucleotide sequence number of the T5Truncated Adapter chain 1a is shown as SEQ ID No.6, the nucleotide sequence number of the T5Truncated Adapter chain 1b is shown as SEQ ID No.7, and the nucleotide sequence number of the T5Truncated Adapter chain second is shown as SEQ ID No. 8.

2. The method for rapid preparation of DNA sequencing libraries according to claim 1, using high efficiency ligation technology, characterized by: the DNA ligase is selected from T4DNA ligase, Taq DNA ligase and E.

3. The method for rapid preparation of DNA sequencing libraries according to claim 1, using high efficiency ligation technology, characterized by: the DNA polymerase is selected from Taq polymerase, A family DNA polymerase and B family high fidelity polymerase.

4. The method for rapid preparation of DNA sequencing libraries according to claim 1, using high efficiency ligation technology, characterized by: the DNA repair enzyme is selected from Klenow, T4PNK FEN1 and Endonuclease V.

5. The method for rapid preparation of DNA sequencing libraries according to claim 3, using high efficiency ligation technology, characterized by: the linker system of the T7Truncated Adapter is a combinatorial enzyme selected from TDT, E.coli DNALigase, T4PNK, Klenow, FEN1, and Endonuclease V.

6. A kit for rapidly preparing a DNA sequencing library by using a high efficiency ligation technique according to the method of claim 1, which comprises the following components:

(1) tool enzyme: the tool enzyme comprises terminal transferase, DNA repair enzyme, DNA polymerase and DNA ligase;

(2) tool enzyme reaction system: buffer solution required by the reaction corresponding to each tool enzyme;

(3) a linker system: t7Truncated Adapter and T5Truncated Adapter;

(4) an extension system: a T7 primer;

(5) a PCR reaction system;

(6)Low-EDTA-TE；

the T7Truncated Adapter and the T5Truncated Adapter are linkers containing special modified bases;

the specific modified base includes a base for increasing the ligation efficiency at the 5 'end and preventing chain extension or a base for preventing OH extension at the 3' end of the target DNA library; the special modified basic group is phosphate group or amino, C3 Spacer, C6Spacer, biotin and the like.

7. The kit for rapidly preparing a DNA sequencing library by using the high efficiency ligation technology according to claim 6, wherein:

the T7Truncated Adapter consists of a first chain of T7Truncated Adapter and a second chain of T7Truncated Adapter, the second chain of T7Truncated Adapter is T7Truncated Adapter chain 2a, T7Truncated Adapter chain 2b, T7Truncated Adapter chain 2c or T7Truncated Adapter chain 2d, the 5' end region of the first chain of T7Truncated Adapter and the 5' end region of the second chain of T7Truncated Adapter are complementary double-stranded DNA, the 3' end of the second chain of T7Truncated Adapter is a polynucleotide selected from PolyT, PolyA, PolyG or PolyC; the 5 'end of the first chain of the T7Truncated Adapter is modified by phosphate, the 3' end of the first chain of the T7Truncated Adapter is modified by blocking, the 5 'end and the 3' end of the second chain of the T7Truncated Adapter are modified by blocking, and the blocking modified group is selected from amino, C3Space, C6Space or biotin;

the nucleotide sequence number of the first chain of the T7Truncated Adapter is shown as SEQ ID No.1, the nucleotide sequence number of the 2a chain of the T7Truncated Adapter is shown as SEQ ID No.2, the nucleotide sequence number of the 2b chain of the T7Truncated Adapter is shown as SEQ ID No.3, the nucleotide sequence number of the 2c chain of the T7Truncated Adapter is shown as SEQ ID No.4, and the nucleotide sequence number of the 2d chain of the T7Truncated Adapter is shown as SEQ ID No. 5;

the T5Truncated Adapter consists of a first strand of the T5Truncated Adapter and a second strand of the T5Truncated Adapter, wherein the 3' end region of the first strand of the Truncated Adapter and the 5' end region of the second strand of the T5Truncated Adapter are complementary into double-stranded DNA, and the 5' end region of the first strand of the T5Truncated Adapter exists in a single-stranded form; the 5 'end of the first chain of the T5Truncated Adapter is subjected to blocking modification, the 3' end of the first chain of the T5Truncated Adapter is subjected to thio modification, the 5 'end and the 3' end of the second chain of the T5Truncated Adapter are subjected to blocking modification, the blocking modification group is selected from amino, C3 Spacer, C6Spacer or biotin, and the first chain of the T5Truncated Adapter is T5Truncated Adapter chain 1a or T5Truncated Adapter chain 1 b;

the nucleotide sequence number of the T5Truncated Adapter chain 1a is shown as SEQ ID No.6, the nucleotide sequence number of the T5Truncated Adapter chain 1b is shown as SEQ ID No.7, and the nucleotide sequence number of the T5Truncated Adapter chain second is shown as SEQ ID No. 8.

8. The kit for rapidly preparing a DNA sequencing library by using the high efficiency ligation technology according to claim 7, wherein:

the DNA repair enzyme is selected from T4PNK, Klenow, FEN1 and Endonuclase V;

the DNA polymerase is selected from Taq polymerase, A family DNA polymerase and B family high-fidelity DNA polymerase;

the DNA ligase is selected from T4DNA ligase, Taq DNA ligase and E.

9. The kit for rapidly preparing a DNA sequencing library by using the high efficiency ligation technology according to claim 7, wherein: the linker system of the T7Truncated adapter is a combinatorial enzyme selected from TDT, E.coli DNALigase, T4PNK, Klenow, FEN1, and Endonuclease V.

Technical Field

The invention relates to the field of biotechnology, in particular to a technology for efficiently connecting sequencing joints at the tail ends of DNA (deoxyribonucleic acid) in sequence to realize library construction of various complex samples and trace DNA.

Background

AT present, the conventional library construction strategy is to repair the ends of fragmented DNA, then to connect sequencing adapters through AT, and to amplify the library by PCR. However, the conventional library construction requires high quality of sample DNA and large input amount, and cannot realize normal library construction of samples seriously damaged, especially samples mainly existing in single-stranded DNA. And when the input amount is too low, the conventional library building cannot be finished.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method and a kit for rapidly preparing a DNA sequencing library by utilizing an efficient connection technology, so that successful library construction of various trace DNA samples is realized, the conventional library construction means is broken, the DNA loss caused by terminal repair is avoided, and the real information of the samples is reflected as much as possible.

The invention relates to a method for rapidly preparing a DNA sequencing library by utilizing an efficient ligation technology, which is characterized by comprising the following steps of:

s1, DNA3' joint connection: firstly, adding a section of polynucleotide PolyA or PolyC or PolyG or PolyT to the 3 'end of DNA to be detected by using terminal transferase to obtain a DNA-polynucleotide structure, finishing the connection of the 3' end of the DNA-polynucleotide and a T7Truncated Adapter joint by using DNA repair enzyme, DNA polymerase and DNA ligase, and synthesizing DNA-polynucleotide-T7 Truncated Adapter;

in the step S1, the DNA to be detected is a single-stranded DNA fragment or a double-stranded DNA fragment, or includes both a single-stranded DNA fragment and a double-stranded DNA fragment, and when the DNA to be detected includes a double-stranded DNA, the step S1 further includes a denaturation step, where the denaturation step denatures the double-stranded DNA into a single-stranded DNA;

s2, synthesis of complementary strand: synthesizing a complementary strand by using DNA polymerase to the DNA-poly oligonucleotide-T7 Truncated Adapter single strand added in the step S2, and synthesizing double-stranded DNA;

s3 and DNA5' joint connection: connecting a T5Truncated Adapter adaptor to the 5' end of the DNA double strand in the step S3 by using DNA ligase through blunt end or AT connection mode;

s4, library amplification: amplifying a PCR library by using a DNA polymerase reaction solution to obtain a library sample capable of being subjected to on-machine sequencing;

the T7Truncated Adapter consists of a first chain of T7Truncated Adapter and a second chain of T7Truncated Adapter, the second chain of T7Truncated Adapter is T7Truncated Adapter chain 2a, T7Truncated Adapter chain 2b, T7Truncated Adapter chain 2c or T7Truncated Adapter chain 2d, a5' end region of the first chain of T7Truncated Adapter and a5' end region of the second chain of T7Truncated Adapter are complementary to form a double-stranded DNA, the 3' end of the second chain of T7Truncated Adapter is a polynucleotide selected from PolyT, PolyA, PolyG or PolyC; the 5 'end of the first chain of the T7Truncated Adapter is modified by phosphate, the 3' end of the first chain of the T7Truncated Adapter is modified by blocking, the 5 'end and the 3' end of the second chain of the T7Truncated Adapter are modified by blocking, and the blocking modified group is selected from amino, C3Space, C6Space or biotin;

the nucleotide sequence number of the first chain of the T7Truncated Adapter is shown as SEQ ID No.1, the nucleotide sequence number of the 2a chain of the T7Truncated Adapter is shown as SEQ ID No.2, the nucleotide sequence number of the 2b chain of the T7Truncated Adapter is shown as SEQ ID No.3, the nucleotide sequence number of the 2c chain of the T7Truncated Adapter is shown as SEQ ID No.4, and the nucleotide sequence number of the 2d chain of the T7Truncated Adapter is shown as SEQ ID No. 5;

the T5Truncated Adapter consists of a first strand of the T5Truncated Adapter and a second strand of the T5Truncated Adapter, wherein the 3' end region of the first strand of the Truncated Adapter and the 5' end region of the second strand of the T5Truncated Adapter are complementary into double-stranded DNA, and the 5' end region of the first strand of the T5Truncated Adapter exists in a single-stranded form; the 5 'end of the first chain of the T5Truncated Adapter is subjected to blocking modification, the 3' end of the first chain of the T5Truncated Adapter is subjected to thio modification, the 5 'end and the 3' end of the second chain of the T5Truncated Adapter are subjected to blocking modification, the blocking modification group is selected from amino, C3 Spacer, C6Spacer or biotin, and the first chain of the T5Truncated Adapter is a T5Truncated Adapter chain 1a or a T5Truncated Adapter chain 1 b;

the nucleotide sequence number of the T5Truncated Adapter chain 1a is shown as SEQ ID No.6, the nucleotide sequence number of the T5Truncated Adapter chain 1b is shown as SEQ ID No.7, and the nucleotide sequence number of the T5Truncated Adapter chain second is shown as SEQ ID No. 8.

Further, the DNA ligase is selected from T4DNA ligase, Taq DNA ligase, e.

Still further, the DNA polymerase is selected from Taq polymerase, A family DNA polymerase, B family high fidelity DNA polymerase.

Further, the DNA repair enzyme is selected from the group consisting of T4PNK Klenow, FEN1, and Endonuclease V.

Still further, the linker system of T7Truncated adapter is a combinatorial enzyme selected from the group consisting of TDT, e.coli DNA Ligase, T4PNK, Klenow, FEN1, and endonuclearase V.

Furthermore, in step S1, the DNA to be tested is one of genomic gDNA, FFPE DNA, cell free DNA, ChIP DNA, archaea, virus DNA, RNA-seq single-stranded cDNA and Bisiute DNA.

The invention also provides a kit for rapidly preparing the DNA sequencing library by using the method and utilizing the efficient ligation technology, which is characterized by comprising the following components:

(1) tool enzyme: the tool enzyme comprises terminal transferase, DNA repair enzyme, DNA polymerase and DNA ligase;

(2) tool enzyme reaction system: buffer solution required by the reaction corresponding to each tool enzyme;

(3) a linker system: t7Truncated Adapter linker and T5Truncated Adapter linker;

(4) an extension system: a T7 primer;

(5) a PCR reaction system;

(6)Low-EDTA-TE；

the T7Truncated Adapter and the T5Truncated Adapter are linkers containing special modified bases;

the specific modified base includes a base for increasing the ligation efficiency at the 5 'end and preventing chain extension or a base for preventing OH extension at the 3' end of the target DNA library; the special modified basic group is phosphate group or amino, C3 Spacer, C6Spacer, biotin and the like.

Further, the T7Truncated Adapter joint is designed as shown in FIG. 1;

the T7Truncated Adapter consists of a first chain of T7Truncated Adapter and a second chain of T7Truncated Adapter, the second chain of T7Truncated Adapter is T7Truncated Adapter chain 2a, T7Truncated Adapter chain 2b, T7Truncated Adapter chain 2c or T7Truncated Adapter chain 2d, a5' end region of the first chain of T7Truncated Adapter and a5' end region of the second chain of T7Truncated Adapter are complementary to form a double-stranded DNA, the 3' end of the second chain of T7Truncated Adapter is a polynucleotide selected from PolyT, PolyA, PolyG or PolyC; the 5 'end of the first chain of the T7Truncated Adapter is modified by phosphate, the 3' end of the first chain of the T7Truncated Adapter is modified by blocking, the 5 'end and the 3' end of the second chain of the T7Truncated Adapter are modified by blocking, and the blocking modified group is selected from amino, C3Space, C6Space or biotin;

the nucleotide sequence number of the first chain of the T7Truncated Adapter is shown as SEQ ID No.1, the nucleotide sequence number of the 2a chain of the T7Truncated Adapter is shown as SEQ ID No.2, the nucleotide sequence number of the 2b chain of the T7Truncated Adapter is shown as SEQ ID No.3, the nucleotide sequence number of the 2c chain of the T7Truncated Adapter is shown as SEQ ID No.4, and the nucleotide sequence number of the 2d chain of the T7Truncated Adapter is shown as SEQ ID No. 5;

the T5Truncated Adapter consists of a first strand of the T5Truncated Adapter and a second strand of the T5Truncated Adapter, wherein the 3' end region of the first strand of the Truncated Adapter and the 5' end region of the second strand of the T5Truncated Adapter are complementary into double-stranded DNA, and the 5' end region of the first strand of the T5Truncated Adapter exists in a single-stranded form; the 5 'end of the first chain of the T5Truncated Adapter is subjected to blocking modification, the 3' end of the first chain of the T5Truncated Adapter is subjected to thio modification, the 5 'end and the 3' end of the second chain of the T5Truncated Adapter are subjected to blocking modification, the blocking modification group is selected from amino, C3 Spacer, C6Spacer or biotin, and the first chain of the T5Truncated Adapter is T5Truncated Adapter chain 1a or T5Truncated Adapter chain 1 b;

the nucleotide sequence number of the T5Truncated Adapter chain 1a is shown as SEQ ID No.6, the nucleotide sequence number of the T5Truncated Adapter chain 1b is shown as SEQ ID No.7, and the nucleotide sequence number of the T5Truncated Adapter chain second is shown as SEQ ID No. 8.

Still further, the DNA repair enzyme is selected from T4PNK, Klenow, FEN1, and endonucleoclean V;

the DNA polymerase is selected from Taq polymerase, A family DNA polymerase and B family high-fidelity DNA polymerase;

the DNA ligase is selected from T4DNA ligase, Taq DNA ligase and E.

Still further, the linker system of the T7Truncated Adapter is a combinatorial enzyme selected from the group consisting of TDT, e.coli DNA Ligase, T4PNK, Klenow, FEN1, and endonuclean V.

The invention has the beneficial effects that: the invention realizes the successful bank building of various DNA samples, breaks through the conventional bank building means, avoids the loss of DNA caused by end repair and reflects the real information of the samples as much as possible. The invention has great advantages compared with the prior art especially when establishing a library aiming at a trace amount of DNA samples of 5 pg-1 mu g: 1. the invention has no selectivity to DNA samples, and the DNA to be detected can be double-stranded DNA, single-stranded DNA, complete DNA or damaged DNA fragments; 2. the invention can be used for constructing a library aiming at trace DNA samples, and the weight of the samples can be as low as 5 pg-1 mug; 3. efficient DNA library construction can be tested for simple samples or complex samples.

Drawings

FIG. 1 is a schematic diagram of a T7Truncated Adapter structure using the method of the present invention;

FIG. 2 is a schematic structural diagram of a T5Truncated Adapter using the method of the present invention;

FIG. 3 is a schematic representation of the efficiency analysis of the addition of PolyA at the end of oligonucleotide N1 using the method of the invention;

FIG. 4 is a schematic diagram of the analysis of efficiency of terminal transferase with PolyA at different incubation times in a method for rapidly preparing a DNA sequencing library by using an efficient ligation technique according to the present invention;

FIG. 5 is a schematic diagram of the analysis of 3' linker efficiency of a method for rapidly preparing a DNA sequencing library by using an efficient ligation technique according to the present invention;

FIG. 6 is a schematic diagram of the analysis of 3' linker efficiency in a method for rapidly preparing a DNA sequencing library by using an efficient ligation technique according to the present invention;

FIG. 7 is a schematic diagram of the analysis of the complementary strand synthesis of the method for rapid preparation of DNA sequencing libraries using efficient ligation;

FIG. 8 is a schematic diagram of 5' linker efficiency analysis of a method for rapid preparation of DNA sequencing libraries using efficient ligation techniques according to the present invention;

FIG. 9 is a schematic diagram of PCR amplification efficiency analysis of a method for rapidly preparing a DNA sequencing library by using a high-efficiency ligation technique according to the present invention;

FIG. 10 is a schematic diagram of library preparation of different samples of a method for rapid preparation of DNA sequencing libraries using efficient ligation techniques according to the present invention;

FIG. 11 is a library preparation diagram of trace samples of a method for rapidly preparing a DNA sequencing library by using an efficient ligation technique according to the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings in conjunction with the following detailed description. It should be understood that the description is intended to be exemplary only, and is not intended to limit the scope of the present invention. Moreover, in the following description, descriptions of well-known structures and techniques are omitted so as to not unnecessarily obscure the concepts of the present invention.