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Method for preparing nucleic acid for sequencing and use thereof

A nucleic acid and sequencing technology, used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as slowing down the running speed, affecting the structure of the sequencing library, and affecting the ratio of effective signal output, etc. Achieve the effect of large output of sequencing data and good library structure

Active Publication Date: 2021-07-30
ANNOROAD GENE TECH BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Based on the polymerase's preference for purine, it will inevitably lead to the two enzymes on the sequencing molecule chasing each other, because one of the enzymes will be fixed at the bottom of the ZMW. When the two enzymes are close, there will be two results: 1) Two fluorescent signals are generated at the same time, and the two signals overlap and interfere with each other, which affects the output ratio of effective signals; 2) The rear enzyme molecule pushes away the front enzyme molecule fixed on the ZMW, or the front molecule restricts The speed of the enzyme molecule fixed on the ZMW slows down the running speed, increases the probability of DNA strand shedding, and the sequencing signal is thus suspended, which affects the structure of the sequencing library
[0003] Therefore, the construction method of the sequencing library for the PB platform needs to be optimized

Method used

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  • Method for preparing nucleic acid for sequencing and use thereof
  • Method for preparing nucleic acid for sequencing and use thereof
  • Method for preparing nucleic acid for sequencing and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0068] 1. Utilize the method of the embodiment of the present invention to construct a sequencing library, the method is as follows:

[0069] (1) Take 8 μg of extracted mouse lung genomic DNA, cut it into large fragments of 30-40K, perform end repair and add A treatment, and the reaction reagents include T4 DNAP, T4 PNK, HemoklenTaq and universal buffer.

[0070] (2) The genome end repair product was ligated with the lox66 sequence coupled with cohesive ends, and the reaction reagents included T4 DNA ligase and Transformer buffer.

[0071] (3) The ligation product was purified using Ampure purification magnetic beads to obtain the purified product.

[0072] (4) UDG cleavage and circularization of the ligation product, the reagents include but not limited to T4 DNA ligase, UDG enzyme and matching buffer.

[0073] (5) Digestion, the reagents include T4 exonuclease 7 and matching buffer.

[0074] (6) Site-specific recombination of the digestion product with the hetero-adapter, ...

Embodiment 2

[0097] 1. Utilize the method of the embodiment of the present invention to construct a sequencing library, the method is as follows:

[0098] (1) Take 8ug of extracted mouse lung genomic DNA, cut it into large fragments of 10-15K, and perform end repair and A treatment. The reaction reagents include T4 DNAP, T4 PNK, HemoklenTaq and universal buffer.

[0099] (2) The genome end repair product was ligated with the lox66 sequence coupled with cohesive ends, and the reaction reagents included T4 DNA ligase and Transformer buffer.

[0100] (3) The ligation product was purified using Ampure purification magnetic beads to obtain the purified product.

[0101] (4) UDG cleavage and circularization of the ligation product, the reagents include but not limited to T4 DNA ligase, UDG enzyme and matching buffer.

[0102] (5) Digestion, the reaction reagents include T4 exonuclease 7 and supporting buffer.

[0103] (6) Site-specific recombination of the digestion product with the hetero-ada...

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Abstract

The invention discloses a method for preparing a nucleic acid for sequencing and applications thereof, wherein the method for preparing a nucleic acid for sequencing includes: providing a first sequence, the first sequence comprising a target sequence and a first recombinase recognition sequence; providing The second sequence, the second sequence includes: middle segment: the middle segment forms at least one duplex region, and contains a second recombinase recognition sequence, and the first recombinase recognition sequence and the second recombinase recognition sequence The enzyme recognition sequence has at least one identical enzyme cutting sequence; two linker segments: the two linker segments are respectively located at the ends of the middle segment, and each of the linker segments contains at least one single-stranded body region, and wherein A linker segment contains at least one primer binding site; and the first sequence and the second sequence are contacted to perform a recombination reaction, so as to obtain the nucleic acid for sequencing, the nucleic acid for sequencing contains the target sequence.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to the field of gene detection. Background technique [0002] Pacbio (PB) sequencing uses phi29 for basic modification, which slows down the travel speed of sequencing enzymes, so that more accurate base discrimination can be obtained from continuous video, but this modification also introduces higher purine-pyrimidine travel speed difference. The purine preference of polymerase is a widely reported phenomenon, and the purine-pyrimidine complementarity of DNA double strands causes the purine-pyrimidine imbalance on DNA double strands. For the real-time and continuous synthesis of polymerases on the PB platform, it will inevitably lead to the polymerase on the complementary double strand with purine-pyrimidine imbalance running faster on one strand and slower on the other, and the difference depends on the difference in the ratio of purine-pyrimidine . The library structure us...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806C12N15/11C12Q1/6869
CPCC12Q1/6806C12Q1/6869C12Q2521/507C12Q2525/191C12Q2525/301C12Q2531/119
Inventor 潘伟业王亚蕾杨新芳李艳秋张介中李志民李大为玄兆伶王海良王娟
Owner ANNOROAD GENE TECH BEIJING
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