A DNA library construction kit, library construction method and application

Abstract

The application discloses a DNA library construction kit, a library construction method and application. The kit comprises a P7 adapter and a P5 adapter, the P7 adapter is composed of a first strand and a second strand in reverse complement, the first strand has a modified group at the 5' end, the modified group comprises a phosphate group, a hydroxyl group or adenosine, the 3' end of the first strand is modified by sealing, the sealing modification comprises C6 amino modification, C12 amino modification, dideoxy modification or spacer modification, and the 3' end of the second strand is modified by sealing, the sealing modification comprises dideoxy modification. The P7 adapter and the P5 adapter are designed and specially modified, and are combined for use, so that a brand-new connection mode is realized, the connection efficiency and the sensitivity of the kit are significantly improved, and the library construction efficiency is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and relates to a DNA library construction kit, a library construction method and application. BACKGROUND

[0002] At present, a library construction kit (for example, a library construction kit provided by a manufacturer such as illumina, Roche, NEB, etc.) is usually used to construct a library, and the main process of constructing the library includes the following steps: 1. breaking a large fragment of DNA into a DNA fragment with a length of 200-500 bp; 2. performing end repair on the fragmented DNA to form a blunt end at both ends; 3. using the terminal transferase activity of a polymerase to add an A base at the 3' end of the DNA fragment; 4. hybridizing a double-stranded Y-shaped adapter with a 3' A end to the dsDNA after the hybridization and then connecting; and 5. performing PCR enrichment on the product connected with the adapter to obtain a library that can be sequenced.

[0003] CN112708939A discloses a kit and method for constructing a DNA library, wherein the kit includes a mixed enzyme solution, a mixed buffer solution, a ligase and a ligation buffer solution, the mixed enzyme solution contains endonuclease, T4 DNA polymerase, Klenow fragment, Taq DNA polymerase and T4 polynucleotide kinase, the mixed buffer solution and the ligation buffer solution contain DTT and PEG 8000, the mixed enzyme solution and the mixed buffer solution are used to form a first reaction system, the ligase and the ligation buffer solution are used to form a second reaction system, the sequencing adapter is a Y-shaped adapter, the Y-shaped adapter is formed by annealing a P5 end adapter sequence and a P7 end adapter sequence, the connection between the last two bases at the 3' end of the P5 end adapter sequence is modified by sulfur, and the first base at the 5' end of the P7 end adapter sequence is modified by phosphorylation.

[0004] However, the common adapter connection method on the market is TA connection, and four ends of double-stranded DNA need to be connected with the adapter at the same time and respectively, and if any end is not successfully connected, the template chain cannot be amplified into a library that can be sequenced; according to the evaluation of the connection efficiency of each end as 90%, the connection efficiency of the library template can only reach 60-70% at most.

[0005] In summary, it is of great significance to provide an efficient library construction kit for the field of gene sequencing. SUMMARY

[0006] In view of the deficiencies of the prior art and actual needs, the present application provides a DNA library construction kit, a library construction method and application, the special adapter reagent of the present application is specially designed, the P7 adapter and the P5 adapter are specially modified, are combined and used, and a new adapter connection mode is adopted, so that the connection efficiency and the library construction efficiency can be effectively improved.

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0008] In the first aspect, the present application provides a DNA library construction kit, the kit comprising a P7 adapter and a P5 adapter, the P7 adapter consisting of a first strand and a second strand in reverse complement, the first strand having a modification group at the 5' end, the modification group comprising a phosphate group, a hydroxyl group or an adenylyl modification group; the 3' end of the second strand is modified by blocking, the blocking modification comprising a dideoxy modification, the P5 adapter consisting of a single strand, the 5' end of the P5 adapter being modified with a hydroxyl group, the 3' end being modified with a hydroxyl group, and at least one thio modification being contained between the two bases at the 3' end.

[0009] In the present application, the P7 adapter (P7 Truncated Adapter) and the P5 adapter (P5 Truncated Adapter) are specially modified and combined for use, which can realize a new connection mode, thereby significantly improving the connection efficiency and further improving the library construction efficiency.

[0010] Preferably, the nucleic acid sequence of the first strand of the P7 adapter comprises the sequence shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 or SEQ ID NO. 7.

[0011] Preferably, the dT bases of the second strand of the P7 adapter are all replaced by dU bases.

[0012] Preferably, the sequence of the second strand of the P7 adapter consists of ribonucleic acid.

[0013] Preferably, the nucleic acid sequence of the second strand of the P7 adapter comprises the sequence shown in SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO. 11.

[0014] Preferably, the nucleic acid sequence of the P5 adapter comprises the sequence shown in SEQ ID NO. 10.

[0015] SEQ ID NO. 1 to SEQ ID NO. 11 are shown in Table 1, wherein 5p represents a 5' end containing a phosphate group, ddC represents 2', 3'-dideoxycytidine (ddC), -NH2 represents an amino group, -C6 spacer represents a six-carbon spacer, 5-OH represents a 5' end hydroxyl group, 5App represents a 5' end adenylation modification, ddT represents 2', 3'-dideoxythymidine nucleotide, * represents a thio modification, and r represents a ribonucleic acid.

[0016] Table 1

[0017]

[0018]

[0019] Preferably, the P7 linker consists of two reverse complementary chains, a structural schematic diagram of which is shown in Figure 2A The first strand has a phosphate group modification at the 5' end and a blocking modification at the 3' end, and the blocking modification includes a C6 amino modification, a C12 amino modification, a dideoxy modification, or a spacer modification; the second strand is not completely reverse complementary to the first strand, has a dideoxy modification at the 3' end, and the middle base T is replaced with base U.

[0020] Preferably, the P7 linker consists of two reverse complementary chains, a structural schematic diagram of which is shown in Figure 2B The first strand has a hydroxyl group modification at the 5' end and a blocking modification at the 3' end, and the blocking modification includes a C6 amino modification, a C12 amino modification, a dideoxy modification, or a spacer modification; the second strand is not completely reverse complementary to the first strand, has a hydroxyl group modification at the 3' end, and the middle base T is replaced with base U.

[0021] Preferably, the P7 linker consists of two reverse complementary chains, a structural schematic diagram of which is shown in Figure 2C The first strand has a phosphate group modification at the 5' end and a blocking modification at the 3' end, and the blocking modification includes a C6 amino modification, a C12 amino modification, a dideoxy modification, or a spacer modification; the second strand is not completely reverse complementary to the first strand, and the sequence consists of ribonucleic acid, which has a dideoxy modification at the 3' end;

[0022] Preferably, the P7 linker consists of two reverse complementary chains, a structural schematic diagram of which is shown in Figure 2DAs shown, the 5' end of the first strand contains an adenylylation modification group, and the 3' end is modified with a blocking modification, which includes a C6 amino modification, a C12 amino modification, a dideoxy modification, or a spacer modification; optionally, the second strand is an RNA strand composed of ribonucleotide bases; optionally, the second strand is a DNA strand, and is modified with a blocking modification at the 3' end, all of the dT bases in the middle are replaced with dU bases, the 5' end is modified with a hydroxyl group, and the 3' end is modified with a dideoxy modification.

[0023] Preferably, the first strand of the P7 adaptor is as shown in SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3, and the second strand is as shown in SEQ ID NO. 8; or the first strand is as shown in SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3, and the second strand is as shown in SEQ ID NO. 9; or the first strand is as shown in SEQ ID NO. 4, and the second strand is as shown in SEQ ID NO. 11; or the first strand is as shown in SEQ ID NO. 5 or SEQ ID NO. 6 or SEQ ID NO. 7, and the second strand is as shown in SEQ ID NO. 8 or SEQ ID NO. 9.

[0024] Preferably, the kit further comprises an enzyme composition and / or a buffer.

[0025] Preferably, the enzyme composition comprises any one or a combination of at least two of a DNA fragmentation enzyme, an end repair enzyme, a PCR amplification enzyme, or a ligase.

[0026] Preferably, the buffer comprises an end repair buffer and / or a ligation buffer.

[0027] In the present application, the end repair reagent is not particularly limited and can be commercially available, such as End prep Buffer (ABclonal cat. RM20207) and End prep Enzymes (ABclonal, cat. RM20208), and the DNA fragmentation enzyme can be DNA Frag Reaction Buffer (ABclonal cat. RM20671) and DNA Frag Enzyme Mix (ABclonal, cat. RM20672).

[0028] Preferably, the ligase comprises T4 DNA ligase, a T4 DNA ligase mutant, or T7 DNA ligase.

[0029] In a second aspect, the present application provides a DNA library construction method, which comprises:

[0030] The DNA library construction kit is used for library construction.

[0031] In the present application, a DNA library construction method is designed based on the constructed DNA library construction kit, which adopts a brand-new adapter ligation mode, i.e. Figures 1A-1F As shown in the figure, the adapter is connected in two steps, which significantly improves the ligation efficiency and further improves the library construction efficiency.

[0032] Preferably, the DNA library construction method comprises the following steps:

[0033] (1) treating the genomic DNA to obtain fragmented genomic DNA, and performing DNA end repair on the fragmented genomic DNA, purifying the product to obtain fragmented genomic DNA with a specific end structure;

[0034] (2) using a DNA ligase to ligate a P7 adapter to the 3' end of the fragmented genomic DNA with a specific end structure;

[0035] using an enzyme to ligate a P5 adapter to the 5' end of the fragmented genomic DNA with a specific end structure;

[0036] (3) purifying the ligation product, and performing PCR amplification on the ligation product to obtain the DNA library.

[0037] Preferably, the purification comprises purification using magnetic beads.

[0038] Preferably, the enzyme used for ligation of the P5 adapter comprises any one or a combination of at least two of RNase H enzyme, UDG enzyme, Endo VII enzyme, taq polymerase, A family high-fidelity polymerase, B family polymerase, taq DNA ligase or E. coli DNA ligase.

[0039] Preferably, the DNA library construction method comprises the following steps:

[0040] (1) treating the genomic DNA to obtain fragmented genomic DNA, and performing DNA end repair on the fragmented genomic DNA, purifying the product using magnetic beads to obtain fragmented genomic DNA with a specific end structure;

[0041] (2) using a DNA ligase to ligate a P7 adapter to the 3' end of the fragmented genomic DNA with a specific end structure;

[0042] using an enzyme to ligate a P5 adapter to the 5' end of the fragmented genomic DNA with a specific end structure;

[0043] (3) The ligation product is purified by using magnetic beads, and the ligation product is subjected to PCR amplification, the amplification product is purified by using magnetic beads, and the DNA library is obtained.

[0044] In a third aspect, the present application provides an application of the DNA library construction kit as described in the first aspect in constructing a DNA library.

[0045] Compared with the prior art, the present application has the following beneficial effects:

[0046] (1) In the present application, the P7 adapter (P7 Truncated Adapter) and the P5 adapter (P5 Truncated Adapter) are designed and specially modified, and are used in combination, so that a brand-new ligation mode is realized, thereby significantly improving the ligation efficiency and the sensitivity of the kit, and further improving the library construction efficiency.

[0047] (2) In the present application, a DNA library construction method is designed based on the constructed DNA library construction kit, the method adopts a brand-new adapter ligation mode, the adapter is connected in two steps, the ligation efficiency is significantly improved, and the library construction efficiency is further improved. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1A It is a diagram of the adapter ligation mode of TA ligation step-by-step connection, the P7 adapter is first connected at the 3' end of DNA, then the P7 second strand is digested by RNase H or UDG enzyme and Endo VIII enzyme, then the P5 adapter is annealed by annealing, and the P5 adapter is connected at the 5' end of DNA;

[0049] Figure 1B It is a diagram of the adapter ligation mode of step-by-step connection based on blunt ends, the P7 adapter is first connected at the 3' end of DNA, then the P7 second strand is digested by RNase H or UDG enzyme and Endo VIII enzyme, then the P5 adapter is annealed by annealing, and the P5 adapter is connected at the 5' end of DNA;

[0050] Figure 1C It is a diagram of the adapter ligation mode of step-by-step connection based on DNA 5' hydroxyl blunt ends, the P7 adapter is first connected at the 3' end of DNA, then the P7 second strand is digested by RNase H or UDG enzyme and Endo VIII enzyme, and the 5' hydroxyl end is digested by using an exonuclease to expose a phosphate end, then the P5 adapter is annealed by annealing, and the P5 adapter is connected at the 5' end of DNA;

[0051] Figure 1DFigure for TA ligation based stepwise ligation of adapters, P7 adapter is first ligated to the 5' end of the DNA, then the second strand of P7 is digested by RNase H or UDG enzyme and Endo VIII enzyme, then P5 adapter is annealed to the DNA and ligated to the 3' end of the DNA;

[0052] Figure 1E Figure for TA ligation based stepwise ligation of adapters, P7 adapter is first ligated to the 5' end of the DNA, then the second strand of P7 is digested by RNase H or UDG enzyme and Endo VIII enzyme, then P5 adapter is annealed to the DNA and ligated to the 3' end of the DNA;

[0053] Figure 1F Figure for TA ligation based stepwise ligation of adapters, P7 adapter is first ligated to the 5' end of the DNA, then the second strand of P7 is digested by RNase H or UDG enzyme and Endo VIII enzyme, then P5 adapter is annealed to the DNA and ligated to the 3' end of the DNA;

[0054] Figure 2A Figure for P7 Truncated Adapter structure, P7 adapter is double stranded, the 5' end of the first strand is modified with phosphate, the 3' end of the first strand is modified with amino, the 3' end of the second strand is modified with dT, and the dT in the second strand is replaced with dU;

[0055] Figure 2B Figure for P7 Truncated Adapter structure, P7 adapter is double stranded, the 5' end of the first strand is modified with phosphate, the 3' end of the first strand is modified with amino, the 3' end of the second strand is modified with dT, and the dT in the second strand is replaced with dU;

[0056] Figure 2C Figure for P7 Truncated Adapter structure, P7 adapter is double stranded, the 5' end of the first strand is modified with phosphate, the 3' end of the first strand is modified with amino, the 3' end of the second strand is modified with dT, and the dT in the second strand is replaced with dU;

[0057] Figure 2D Figure for P7 Truncated Adapter structure, P7 adapter is double stranded, the 5' end of the first strand is modified with phosphate, the 3' end of the first strand is modified with amino, the 3' end of the second strand is modified with dT, and the dT in the second strand is replaced with dU;

[0058] Figure 3A Agilent 2100 peak shape identification result figure of 300bp PCR fragment;

[0059] Figure 3B Agilent 2100 peak shape identification result figure of 300bp PCR fragment connected P7 adapter product;

[0060] Figure 4A Library preparation result figure of 1ng fragmented (trace DNA) DNA by the library construction method of the application;

[0061] Figure 4B Library preparation result figure of 100ng fragmented DNA by the library construction method of the application;

[0062] Figure 5 Library preparation result figure of 1ng intact gDNA by the library construction method of the application;

[0063] Figure 6 Library preparation result figure of 100ng intact gDNA by the library construction method of the application;

[0064] Figure 7 Connection efficiency evaluation result figure of traditional TA-ligation based one-step method connecting P7 adapter and P5 adapter, in which the upper, middle and lower three peak shape figures are the experimental repeats of three adapter connection tests. DETAILED DESCRIPTION

[0065] In order to further illustrate the technical means adopted by the application and its effects, the application will be further described below in conjunction with the embodiments and drawings. It can be understood that the specific embodiments described herein are only used to explain the application, and not to limit the application.

[0066] Unless specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art, or according to the product instructions are used. Unless the manufacturer of the reagent or instrument is specified, it is a conventional product that can be commercially available through a regular channel.

[0067] Example 1

[0068] This example analyzes the P7 adapter connection efficiency of the application.

[0069] (1) Preparation of end repair system

[0070] In this example, human blood DNA (mixed whole blood of healthy people) is used as a template, and a primer is used for amplification and enrichment to obtain a 300bp length PCR fragment. The reaction system is prepared according to Table 2 below, mixed by blowing and centrifuged to the bottom of the tube;

[0071] Table 2

[0072]

[0073]

[0074] (2) Put the reaction system into the PCR instrument for end repair, and the reaction conditions are shown in Table 3;

[0075] Table 3

[0076] Temperature Time 20℃ 30 min 12℃ Hold

[0077] (3) Purification is performed using 2.2x proportion of cleanup magnetic beads;

[0078] (4) The reaction system is configured according to Table 4, mixed by blowing, and centrifuged to the bottom of the tube;

[0079] Table 4

[0080]

[0081] (5) Put the reaction system into the PCR instrument for P7 Truncated Adapter connection;

[0082] Purification is performed using 2.2x proportion of cleanup magnetic beads, and then the connection product is subjected to Qsep 100 peak identification, and the structure is shown in Figure 3A and 3B Figure 3A is an Agilent 2100 peak identification result diagram of a 300bp PCR fragment, Figure 3B is an Agilent 2100 peak identification result diagram of a 300bp PCR fragment connected with a P7 adapter, and it can be known from Figure 3B and 3A that the PCR fragment has been completely connected with the P7 adapter, and the connection efficiency is more than 90%.

[0083] Example 2

[0084] In this example, human blood DNA is used as a sample for library construction.

[0085] (1) The human blood gDNA is subjected to covaris breaking, broken to about 250bp fragments, and then the reaction system is configured according to Table 5, mixed by blowing, and centrifuged to the bottom of the tube;

[0086] Table 5

[0087] Reaction Reagent Amount Fragmented DNA 100 ng End prep buffer (ABclonal cat. RM20207) 13 μL End prep Enzyme (ABclonal, cat. RM20208) 3 μL Nuclease-free water Add up to 60 μL

[0088] ​(2) Put the reaction system into the PCR instrument for end repair, and the reaction conditions are shown in Table 3;

[0089] (3) Purify using 2.2x proportion of cleanup magnetic beads;

[0090] (4) Prepare the reaction system according to Table 6, mix by blowing, and centrifuge to the bottom of the tube;

[0091] Table 6

[0092]

[0093] (5) Put the reaction system into the PCR instrument for P7 Truncated Adapter ligation, and the reaction conditions are shown in Table 7;

[0094] Table 7

[0095] Temperature Time 25℃ 15 min 65℃ 15 min 12℃ Hold

[0096] (6) Prepare the P5 Truncated Adapter ligation system according to Table 8, mix by blowing, and centrifuge to the bottom of the tube;

[0097] Table 8

[0098]

[0099] (7) Put the reaction system into the PCR instrument for P5 adapter ligation, and the reaction system is shown in Table 9;

[0100] Table 9

[0101] Temperature Time 40℃ 15 min 12℃ Hold

[0102] (8) Purify using 2.2x proportion of cleanup magnetic beads;

[0103] (9) Prepare the PCR amplification system according to Table 10, mix by blowing, and centrifuge to the bottom of the tube;

[0104] Table 10

[0105]

[0106] (10) Put the reaction system into the PCR instrument for PCR reaction, and the reaction conditions are shown in Table 11;

[0107] Table 11

[0108]

[0109] (11) Purify using 1.0x proportion of cleanup magnetic beads to obtain a library that can be sequenced;

[0110] (12) The library is quantified using Qubit, as shown in Table 12;

[0111] Table 12

[0112] DNA input PCR cycle number Library concentration (ng / μL, 30 μL water elution) 1 ng 13c 35.8 100 ng 6c 72.0

[0113] (13) Dilute the library to 2 ng / μL and perform peak shape identification using Qsep 100, such as... Figure 4A and Figure 4B As shown, Figure 4A Peak shape diagram of a library prepared from a 1 ng DNA sample. Figure 4B The peak shape of the library prepared for a 100ng DNA sample is appropriate in size and meets the Illumina sequencer's standards.

[0114] Example 3

[0115] In this embodiment, human blood DNA (mixed whole blood from healthy individuals) was used as the sample for library construction.

[0116] (1) Human blood DNA was digested with enzymes and the reaction system was prepared according to Table 13. The mixture was mixed by pipetting and centrifugation until the bottom of the tube was reached.

[0117] Table 13

[0118]

[0119] (2) The reaction system was placed in a PCR instrument for end repair, and the reaction conditions are shown in Table 14:

[0120] Table 14

[0121] Temperature Time 32℃ 10 min 4℃ Hold

[0122] (3) After the reaction is complete, immediately purify using Cleanup magnetic beads at a ratio of 2.2×.

[0123] (4) Prepare the reaction system according to Table 15, mix it by blowing and centrifuging it to the bottom of the tube;

[0124] Table 15

[0125]

[0126] (5) Place the reaction system in the PCR instrument to connect the P7 Truncated Adapter. The reaction conditions are shown in Table 7.

[0127] (6) Configure the P5 Adapter connector connection system according to Table 16, mix thoroughly by blowing, and centrifuge briefly to the bottom of the tube;

[0128] Table 16

[0129]

[0130]

[0131] (7) The reaction system was placed in a PCR instrument for P5 adapter ligation, and the reaction system was as shown in Table 9:

[0132] (8) Purification was performed using 2.2x proportion of cleanup magnetic beads;

[0133] (9) The PCR amplification system was configured according to Table 17, mixed and homogenized by blowing, and centrifuged to the bottom of the tube;

[0134] Table 17

[0135] Reaction Reagent Amount Product from the previous step 20 μL 2x PCR Master Mix (ABclonal, RM20204) 25 μL Universal PCR Primer (ABclonal, RK21632) 2.5 μL PCR Index (ABclonal, RK21632) 2.5 μL Total volume 50 μL

[0136] (10) The reaction system was placed in a PCR instrument for PCR reaction, and the reaction conditions were as shown in Table 11;

[0137] (11) Purification was performed using 1.2x proportion of cleanup magnetic beads to obtain a library that could be sequenced;

[0138] (12) The library was quantified by Qubit, as shown in Table 18 below;

[0139] Table 18

[0140] DNA input PCR cycle number Library concentration (ng / μL, 30 μL water elution) 1 ng 13c 68.6 100 ng 6c 93.4

[0141] (13) The library was diluted to 2 ng / μL, and Qsep 100 was used for peak shape identification, as shown in Figure 5 and Figure 6 , Figure 5 the peak shape graph of the library prepared for a 1 ng DNA sample, Figure 6 the peak shape graph of the library prepared for a 100 ng DNA sample; the library size was appropriate, and met the standard for on-machine sequencing of an Illumina sequencer.

[0142] Comparative Example

[0143] The comparative example uses human blood DNA (mixed whole blood of healthy people) as a sample and uses a traditional library construction method. The traditional library construction method is a TA-based method for connecting dsDNA adapters at both ends. First, end repair is performed to modify the 5' end of the DNA with a phosphate group and the 3' end with a dA tail. Then, under the catalysis of a ligase, a Y-shaped adapter is connected to both ends of the DNA. This connection product contains three types of connection products: DNA fragments, fragments with an adapter connected, and products with two adapters connected. The connection efficiency of the adapters on the market is generally about 60%, as shown in Figure 7 The present application further optimizes the connection mode of the adapters. By using a step-by-step connection method, i.e., connecting the P7 adapter and the P5 adapter to both ends of the DNA in a step-by-step manner, the connection efficiency of each step is ensured to be above 90%, thereby making the connection efficiency of the adapters above 80%, which greatly improves the connection efficiency of the adapters compared to the traditional 60% connection efficiency.

[0144] In summary, the present application designs and specially modifies the P7 adapter (P7 Truncated Adapter) and the P5 adapter (P5 Truncated Adapter) and uses them in combination to achieve a new connection mode, thereby significantly improving the connection efficiency and the sensitivity of the kit, and further improving the library construction efficiency. Based on the constructed DNA library construction kit, a DNA library construction method is designed. The method uses a new adapter connection mode to connect the adapters in two steps, significantly improves the connection efficiency, and further improves the library construction efficiency.

[0145] The applicant declares that the present application is illustrated by the above-mentioned embodiments to explain the detailed method of the present application, but the present application is not limited to the above-mentioned detailed method, i.e., it does not mean that the present application must rely on the above-mentioned detailed method to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present application. SEQUENCE LISTING <110> Shanghai Yingji Biological Technology Co., Ltd. <120> A DNA library construction kit, library construction method and application <130> 03‑May‑2022 <160> 11 <170> PatentIn version 3.3 <210> 1 <211> 31 <212> DNA <213> Artificial sequence <400> 1 agatcggaag agcacacgtc tgaactccag t 31 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <400> 2 agatcggaag agcacacgtc tgaactccag tc 32 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <400> 3 agatcggaag agcacacgtc tgaactccag tc 32 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <400> 4 agatcggaag agcacacgtc tgaactccag tc 32 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <400> 5 agatcggaag agcacacgtc tgaactccag t 31 <210> 6 <211> 32 <212> DNA <213> Artificial Sequence <400> 6 agatcggaag agcacacgtc tgaactccag tc 32 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <400> 7 agatcggaag agcacacgtc tgaactccag tc 32 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 agacgugugc ucutccgauc 20 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <400> 9 agacg tgtgc tcttccgatc 40 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <400> 10 actctttccc tacacgacgc tcttccgatc t 31 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <400> 11 agacgugugc ucutccgauc t 21

Claims

1. A DNA library construction kit, characterized in that, The kit includes a P7 connector and a P5 connector; The P7 connector is composed of a first chain and a second chain that are oppositely complementary. The first chain has a modifying group at its 5' end, which is adenosine. The first chain is blocked at its 3' end, which is a C6 amino modification. The 3' end of the second chain is closed by a dideoxy modification. The P5 connector is composed of a single chain, with a hydroxyl group modified at the 5' end and a hydroxyl group modified at the 3' end, and at least one thio modification is included between the two bases at the 3' end. The nucleic acid sequence of the first strand of the P7 adapter is shown in SEQ ID NO. 6; The nucleic acid sequence of the second strand of the P7 adapter is shown in SEQ ID NO. 8; The nucleic acid sequence of the P5 adapter is shown in SEQ ID NO.

10.

2. The DNA library construction kit according to claim 1, characterized in that, The kit also includes an enzyme composition and / or a buffer solution.

3. The DNA library construction kit according to claim 2, characterized in that, The enzyme composition includes any one or a combination of at least two of DNA breaking enzymes, terminal repair enzymes, PCR amplification enzymes, or ligases. The buffer solution includes end repair buffer and / or ligation buffer.

4. The DNA library construction kit according to claim 3, characterized in that, The ligase includes T4 DNA ligase, a T4 DNA ligase mutant, or T7 DNA ligase.

5. A method for constructing a DNA library, characterized in that, The method includes: The DNA library was constructed using the DNA library construction kit as described in any one of claims 1-4.

6. The DNA library construction method according to claim 5, characterized in that, The method includes the following steps: (1) Process the genomic DNA to obtain fragmented genomic DNA, and perform DNA end repair on the fragmented genomic DNA to purify the product and obtain fragmented genomic DNA with specific end structures; (2) Use DNA ligase to ligate the P7 adapter to the 3' end of fragmented genomic DNA with a specific end structure; The P5 adapter is used to ligate the 5' end of fragmented genomic DNA with a specific end structure using an enzyme; (3) Purify the ligation product and perform PCR amplification on the ligation product to obtain the DNA library.

7. The DNA library construction method according to claim 6, characterized in that, The purification process includes purification using magnetic beads.

8. The DNA library construction method according to claim 6, characterized in that, The enzymes used for ligation of the P5 ligase include any one or a combination of at least two of the following: RNase H, UDG, Endo VIII, Taq polymerase, A-family high-fidelity polymerase, B-family polymerase, Taq DNA ligase, or E. coli DNA ligase.

9. The DNA library construction method according to any one of claims 5-8, characterized in that, The method includes the following steps: (1) Process the genomic DNA to obtain fragmented genomic DNA, repair the DNA ends of the fragmented genomic DNA, purify the product using magnetic beads, and obtain fragmented genomic DNA with specific end structures; (2) Use DNA ligase to ligate the P7 adapter to the 3' end of fragmented genomic DNA with a specific end structure; The P5 adapter is used to ligate the 5' end of fragmented genomic DNA with a specific end structure using an enzyme; (3) Purify the ligation product using magnetic beads, and perform PCR amplification on the ligation product. Purify the amplification product using magnetic beads to obtain the DNA library.

10. The use of a DNA library construction kit as described in any one of claims 1-4 in the construction of a DNA library.

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