Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for preparing nucleic acid for sequencing and application of nucleic acid

A sequencing and nucleic acid technology, applied in genetic testing and biological fields, can solve problems affecting the structure of the sequencing library, slow down the running speed, and the construction method of the sequencing library needs to be optimized, so as to achieve the effect of good library structure and large output of sequencing data

Active Publication Date: 2020-06-23
ANNOROAD GENE TECH BEIJING
View PDF3 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for preparing nucleic acid for sequencing and application of nucleic acid
  • Method for preparing nucleic acid for sequencing and application of nucleic acid
  • Method for preparing nucleic acid for sequencing and application of nucleic acid

Examples

Experimental program
Comparison scheme
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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention discloses a method for preparing a nucleic acid for sequencing and an application of the nucleic acid. The method for preparing the nucleic acid for sequencing comprises the following steps: a first sequence is provided and the first sequence comprises a target sequence and a first recombinase recognition sequence; a second sequence is provided and the second sequence comprises an intermediate segment, the intermediate segment forms at least one double-stranded region and also contains a second recombinase recognition sequence, and besides, the first recombinase recognition sequence and the second recombinase recognition sequence have at least one identical cleavage sequence; two linker segments are provided and the two linker segments are respectively located at endparts of the intermediate segment, besides, each of the linker segments contains at least one single-stranded region, and besides, one linker segment contains at least one primer binding site; and thefirst sequence contacts with the second sequence to perform a recombination reaction to obtain the nucleic acid for sequencing and the nucleic acid for sequencing contains a 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com