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Nucleic acid library construction method, nucleic acid library obtained through the method, and application thereof

A technology of nucleic acid library and construction method, which is applied in the direction of nucleotide library, library, chemical library, etc., and can solve problems such as expensive, difficult to popularize, and no large fragment library construction method

Pending Publication Date: 2020-07-07
MGI TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the third-generation sequencing technology was born, it is difficult to popularize it quickly due to the disadvantages of low sequencing accuracy and high price.
At present, the cPAS-based high-throughput sequencing platform does not have a corresponding large-fragment library construction method

Method used

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  • Nucleic acid library construction method, nucleic acid library obtained through the method, and application thereof
  • Nucleic acid library construction method, nucleic acid library obtained through the method, and application thereof
  • Nucleic acid library construction method, nucleic acid library obtained through the method, and application thereof

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Experimental program
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Effect test

Embodiment

[0091] 1. Custom Circularization Adapter

[0092] 1) Linker sequence A (Primer A): 5'-AGATGTGTATAAGAGACAG-3' (SEQ ID NO: 1), the first A base at the 5' end is phosphorylated, and the second T base at the 5' end is biotin (Biotin) modification; linker sequence B (Primer B): 5'-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 2), phosphorylation modification of the first C base at the 5' end.

[0093] Dissolve Primer A and Primer B to 100 μM.

[0094] 2) Prepare the PCR primer mixture (see Table 1):

[0095] Table 1

[0096]

[0097] The reaction system can be scaled up according to actual needs.

[0098] 3) Vortex to mix thoroughly, and centrifuge briefly to return the solution to the bottom of the tube. Put it in a PCR machine, and carry out the reaction program in Table 2 (heated lid: 105°C):

[0099] Table 2

[0100]

[0101] 4) After the reaction, name it Adapter Mix (Adapter Mix) and store at -20°C.

[0102] 2. Adapter Mix embedding

[0103] 1) In a sterile PCR tube, add t...

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Abstract

The invention discloses a nucleic acid library construction method, the nucleic acid library obtained through the method, and application thereof. The method comprises the steps: breaking the nucleicacid into fragments through transposase, meanwhile, connecting transposon sequences onto the two ends of the broken fragments, wherein the transposon sequences carry biotin labels; filling a gap between the transposon sequence and the broken segment through strand displacement reaction to obtain a complete double-stranded nucleic acid segment; cyclizing the double-stranded nucleic acid fragment obtained in the previous step into a cyclic nucleic acid fragment; combining streptomycin affinity magnetic beads with the cyclic nucleic acid fragments with biotin labels; breaking cyclic nucleic acidfragments combined on streptomycin affinity magnetic beads by using a breaking enzyme, and carrying out magnetic separation to obtain fragments combined on the streptomycin affinity magnetic beads. The method aims at a large-fragment library, meets the requirement of low initial quantity, can thoroughly get rid of expensive equipment required by physical interruption, can construct a single-chaincyclic library according to a preferred scheme, and enables the library to be used for next-generation sequencing.

Description

technical field [0001] The invention relates to the technical field of sequencing, in particular to a method for constructing a nucleic acid library, the obtained nucleic acid library and uses thereof. Background technique [0002] The birth of next-generation high-throughput sequencing technology has greatly reduced the cost and time required for gene sequencing, bringing revolutionary changes to the field of genomics. With the development of high-throughput sequencing, this technology has gradually become popular. However, limited by the sequencing principle and technical bottlenecks, the sequencing read length of the current second-generation high-throughput sequencer is mostly a few hundred bases, which requires bioinformaticians to restore these fragmented information into long fragments of the organism itself Chromosomal information, however, has a short read length and cannot span repetitive regions or some complex sequences, so it brings many problems to subsequent ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C40B40/06C12Q1/6869
CPCC40B50/06C40B40/06C12Q1/6869C12Q2535/122
Inventor 耿春雨张开杨心石李艳傅书锦陈芳蒋慧
Owner MGI TECH CO LTD
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