CN-121992071-A - DNA library construction kit and application thereof

CN121992071ACN 121992071 ACN121992071 ACN 121992071ACN-121992071-A

Abstract

The invention belongs to the technical field of library construction, and particularly relates to a DNA library construction kit and application thereof. The invention provides a DNA library construction kit which comprises a reagent combination or a reagent kit for joint connection, wherein the reagent combination or the reagent kit for joint connection comprises a connection reaction liquid, the connection reaction liquid comprises a buffer solution, magnesium ions, a mercaptan reducing agent, adenine nucleoside triphosphate (ATP) and propylene glycol, the connection reaction liquid does not comprise polyethylene glycol (PEG), and the polyethylene glycol is replaced by the propylene glycol, so that the content of the PEG in a connection reaction system is reduced, and the generation of a chimeric body is reduced.

Inventors

WU JINFENG
LIU JIAHUI
ZHANG HONGZHI
FENG QIANQIAN
Liang Caijiao
GUAN YANFANG
HUANG YI

Assignees

吉因加生物医学科技(绍兴)有限公司
苏州吉因加生物医学工程有限公司
长沙吉因加生物科技有限公司

Dates

Publication Date: 20260508
Application Date: 20251226

Claims (10)

1. A DNA library construction kit comprises a reagent combination or a reagent kit for joint connection, wherein the reagent combination or the reagent kit for joint connection comprises a connection reaction liquid, the connection reaction liquid comprises a buffer solution, magnesium ions, a mercaptan reducing agent, adenine nucleoside triphosphate (ATP) and propylene glycol, and the connection reaction liquid does not comprise polyethylene glycol (PEG).
2. The kit according to claim 1, wherein, The buffer solution comprises at least one of citrate buffer solution, MES buffer solution, phosphate buffer solution, bis-Tris buffer solution and Tris hydrochloric acid buffer solution, and further comprises Tris hydrochloric acid buffer solution; Preferably, the concentration of the buffer solution in the ligation reaction solution is 0.1-1M; preferably, the concentration of the magnesium ions in the connection reaction liquid is 0.01-1M; Preferably, the thiol reducing agent comprises at least one of tris- (2-formylethyl) phosphine hydrochloride (TCEP), dithiothreitol (DTT) and glutathione reduced (GSH); Preferably, the concentration of the thiol reducing agent in the ligation reaction solution is 0.01-1M; Preferably, the concentration of ATP in the ligation reaction solution is 0.001-0.1M; Preferably, the concentration of the propylene glycol in the connecting reaction solution is 40-80% by volume percent; Preferably, the reagent combination or kit for adaptor ligation further comprises a ligase; Preferably, the Ligase comprises at least one of T4 DNA Ligase, T3 DNA Ligase, T7 DNA Ligase, pfu DNA Ligase, hiFi DNA Ligase and Quick Ligase; Preferably, the reagent combination or kit for adaptor ligation further comprises a adaptor.
3. The kit according to any one of claim 1 to 2, wherein, The DNA library construction kit also comprises a reagent combination or a kit for repairing the tail end and adding 'A'; Preferably, the reagent combination or kit for repairing the tail end and adding 'A' comprises a tail end repairing reaction liquid, wherein the tail end repairing reaction liquid comprises buffer solution, magnesium ions, potassium ions, thiol reducing agents, adenine nucleoside triphosphate (ATP), dNTP and nonionic surfactant; Preferably, the buffer solution comprises at least one of citrate buffer, MES buffer solution, phosphate buffer, bis-Tris buffer and Tris-hydrochloric acid buffer; Preferably, the concentration of the buffer solution in the end repair reaction solution is 0.1-1M; Preferably, the concentration of the magnesium ions in the end repair reaction solution is 0.01-1M; preferably, the concentration of the potassium ions in the end repair reaction solution is 0.01-1M; Preferably, the thiol reducing agent comprises at least one of tris- (2-formylethyl) phosphine hydrochloride (TCEP), dithiothreitol (DTT) and glutathione reduced (GSH); preferably, the concentration of the thiol reducing agent in the end repair reaction liquid is 0.01-1M; Preferably, the concentration of the ATP in the end repair reaction solution is 0.001-0.1M; preferably, the concentration of dNTPs in the end-repair reaction solution is 0.001-0.1M; preferably, the nonionic surfactant comprises at least one of Tween 20, triton X-100, NP-40, pluronic F-68; Preferably, the concentration of the nonionic surfactant in the end repair reaction liquid is 0.01-1% by volume percent; preferably, the reagent combination or kit for end repair and addition of "A" further comprises an end repair enzyme; Preferably, the end repair enzyme comprises a polymerase; Preferably, the end repair enzyme further comprises a polynucleotide kinase.
4. The kit according to any one of claim 1 to 3, The DNA library construction kit further comprises a reagent combination or a kit for library amplification; Preferably, the reagent combination or kit for library amplification comprises library-building primers and a DNA polymerase reaction solution; preferably, the DNA library construction kit further comprises magnetic beads; preferably, the DNA library construction kit further comprises a substance for disrupting DNA; Preferably, the DNA library construction kit further comprises a nucleic acid extraction reagent combination or kit; preferably, the DNA library construction kit may further comprise reagents for obtaining a targeting library.
5. Use of the kit of any one of claims 1-4 in any one of a 1) -a 3): a1 Constructing a DNA library; a2 Preparing a sequencing kit; a3 Sequencing.
6. A DNA library construction method comprising the step of using the DNA library construction kit of any one of claims 1 to 4.
7. The method of claim 6, wherein the step of providing the first layer comprises, The method comprises the following steps: b1 End repair of DNA fragment and addition of "A"; b2 Carrying out joint connection on the product obtained in the step b 1); b3 Purifying the product obtained in b 2); b4 Library amplification of the product obtained in b 3); The adaptor ligation employs the reagent combination or kit for adaptor ligation as described in any one of claims 1 to 4; preferably, the method for connecting the joints comprises the steps of mixing the product obtained in the step b 1), the joint connection reaction liquid and the ligase for reaction; preferably, the reaction time is 10-25min; Preferably, the method for purifying the product obtained in b 2) comprises the steps of mixing the product obtained in b 2) with magnetic beads, incubating, washing with ethanol, drying and eluting; preferably, the volume ratio of the product obtained in b 2) to the magnetic beads is 1 (1-1.4).
8. The method of claim 7, wherein the step of determining the position of the probe is performed, The library amplification employs the reagent combination or kit for library amplification as set forth in claim 4; Preferably, the library amplification method comprises mixing the product obtained in b 3), the library-building primer and the DNA polymerase reaction solution, and performing PCR reaction; Preferably, the concentration of the library primer in the PCR reaction system is 1-3 mu M; preferably, the end repair and addition "a" employs a combination of reagents or a kit for end repair and addition "a" as described in any one of claims 3 to 4; Preferably, the method for repairing the tail end and adding the 'A' comprises the steps of mixing and reacting the DNA fragment, the tail end repairing reaction liquid and the tail end repairing enzyme.
9. A DNA library obtained by the construction method of any one of claims 6-8.
10. A sequencing method for sequencing the DNA library of claim 9.

Description

DNA library construction kit and application thereof Technical Field The invention belongs to the technical field of library construction, and particularly relates to a DNA library construction kit and application thereof. Background The clinical significance of high-throughput sequencing data is mainly embodied in two aspects, one can directly serve for diagnosis of a detected person, the other is used as a part of big data, a part of the big data is accumulated for human analysis of unrecognized diseases to be accumulated, and the part of the big data plays a role in data support and also a reference role. Second generation sequencing (NGS) is the most commonly used technique in modern high-throughput sequencing research in molecular biology. Second generation sequencing DNA sequencing mainly comprises two processes of library construction and on-machine sequencing. Library construction procedures typically include (1) sonicating or enzymatically fragmenting the non-fragmented genomic DNA (e.g., using plasma free DNA or other fragmented DNA, which may be omitted), (2) end gap fill repair of the DNA fragments, (3) 5 'end phosphorylation of end repaired DNA fragments, 3' addition of single base A (adenine) to obtain cohesive ends with single base, (4) ligation of cohesive end A DNA fragments with adaptors designed according to different sequencing platforms (adaptor may carry sample tags) to obtain ligation products, (5) if targeted enrichment region sequencing (e.g., whole exome sequencing or NGS panel sequencing) is performed, PCR is performed on the ligation products by universal primers on each sequencing platform on the adaptor to obtain a sufficient amount of amplified DNA library (sample tags may be introduced in this step), optionally, (6) target region enrichment of the amplified DNA library by probe capture or targeted primer method to prepare a sequencing library. The current DNA library construction method still has the problems of low library yield and/or low template conversion rate. Disclosure of Invention The object of the first aspect of the present invention is to provide a DNA library construction kit. The object of the second aspect of the invention is to provide the use of a kit according to the first aspect of the invention. The object of the third aspect of the present invention is to provide a method for constructing a DNA library. The object of the fourth aspect of the present invention is to provide a DNA library. The fifth aspect of the present invention is directed to a sequencing method. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: In a first aspect of the present invention, there is provided a DNA library construction kit comprising a reagent set or a kit for adaptor ligation, the reagent set or the kit for adaptor ligation comprising a ligation reaction solution comprising a buffer solution, magnesium ions, a thiol reducing agent, adenine nucleoside triphosphate (ATP), and propylene glycol, wherein the ligation reaction solution does not comprise polyethylene glycol (PEG). In the invention, propylene glycol is adopted to replace polyethylene glycol, so that the content of PEG in a connection reaction system is reduced, and the generation of chimera can be reduced. In some embodiments, the buffer comprises at least one of a citrate buffer, an MES buffer, a phosphate buffer, a Bis-Tris buffer, a Tris-HCl buffer, and further a Tris-HCl buffer. In some embodiments, the concentration of the buffer in the ligation reaction is 0.1 to 1M, further 0.1 to 0.4M, and still further 0.1 to 0.2M. In some embodiments, the pH of the buffer is 7-9, further 7.4-7.8. In some embodiments, the magnesium ion is from MgCl 2. In some embodiments, the concentration of the magnesium ions in the ligation reaction solution is 0.01 to 1M, further 0.01 to 0.04M, and still further 0.01 to 0.02M. In some embodiments, the thiol reducing agent comprises at least one of tris- (2-formylethyl) phosphine hydrochloride (TCEP), dithiothreitol (DTT), glutathione reduced (GSH), and further TCEP. In some embodiments, the concentration of the thiol reducing agent in the ligation reaction is 0.01 to 1M, further 0.01 to 0.04M, and still further 0.012 to 0.014M. In some embodiments, the ATP is present in the ligation reaction at a concentration of 0.001 to 0.1M, further 0.001 to 0.01M, and further 0.001 to 0.002M. In some embodiments, the concentration of the propylene glycol in the connection reaction solution is 40% -80%, further 50% -70%, and still further 55% -65% by volume. In some embodiments, the reagent combination or kit for adaptor ligation further comprises a ligase. In some embodiments, the Ligase comprises at least one of T4 DNA Ligase, T3 DNA Ligase, T7 DNA Ligase, pfu DNA Ligase, hiFi DNA Ligase, quick Ligase, further T4 DNA Ligase. In some embodiments, the reagent combination or kit for adaptor ligation further comprises a adaptor. In some embodiments, t