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Sequencing library construction kit and using method and application thereof

A technology for sequencing libraries and kits, applied in the field of gene detection, can solve the problems of limited sites, contamination of PCR products, cumbersome operation process, etc., and achieve the effect of improving utilization efficiency

Active Publication Date: 2019-05-24
CARRIER GENE TECH SUZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] MLPA can simultaneously detect tens or hundreds of sites in a sample, but the operation process is cumbersome, and the sites are still limited, which is easy to cause contamination of PCR products

Method used

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  • Sequencing library construction kit and using method and application thereof
  • Sequencing library construction kit and using method and application thereof
  • Sequencing library construction kit and using method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0043] The invention provides a kit for constructing a sequencing library, specifically comprising a restriction endonuclease, a restriction endonuclease buffer, an end repair reagent, a DNA ligase reagent set, a ligase buffer, and a sequencing linker (01 -96), PCR primer set, PCR reaction system.

[0044] The restriction endonuclease is one or a combination of AseI, CviQI, NdeI, AciI, AluI, BfaI, HaeIII, HhaI, MseI, AsiSI, CviAII, PacI, PvuI, PvuI-HF, HpyCH4IV, HpySE526I, It should be understood that different restriction endonucleases can be selected according to the objects of the sequencing library, including but not limited to the restriction endonucleases listed above.

[0045] The end repair reagent group includes T4PNK (T4 Polynucleotide Kinase, T4 polynucleotide kinase), T4 DNA Polymerase (T4 DNA polymerase), Klenow DNA Polymerase (Klenow DNA polymerase), dNTPs, repair buffer.

[0046]The ligase in the DNA ligase reagent group can be DNA ligase, T4 DNA ligase, taq DN...

Embodiment 2

[0051] attached figure 1 A schematic diagram of a sequencing library construction method is shown, which is described in detail as follows:

[0052] Principle step 1-1: After the different chromosomes of the sample genome are cut by the restriction endonuclease reagent group, fragments of different sizes are formed, and these fragments all have 5'-phosphate groups and 3'-hydroxyl groups that can be reconnected ;

[0053] Principle steps 1-2: In the presence of the DNA ligase reagent group, different DNA fragments are randomly connected and become long fragments again;

[0054] Principle steps 1-3: Use random interruption methods such as ultrasonic interruption to randomly interrupt the re-formed long fragments again to obtain fragments from different regions;

[0055] Principle steps 1-4: After ligation with sequencing adapters, a complete library DNA fragment that can be amplified is formed;

[0056] Principle steps 1-5: During sequencing, there will be at least two differ...

Embodiment 3

[0092] attached image 3 A schematic diagram of a sequencing library construction method is shown, which is described in detail as follows:

[0093]Principle step 2-1: The genomic DNA of the sample is first interrupted by ultrasound to form fragments of different sizes that need to be repaired at the ends and are not easy to connect;

[0094] Principle step 2-2: Add a restriction enzyme reagent group, the DNA fragment containing the endonuclease recognition site will be cut to form a port that can be connected with a 5'-phosphate group and a 3'-hydroxyl group;

[0095] Principle step 2-3: In the presence of DNA ligase reagent group, different DNA fragments with ports that can be connected are randomly connected to form a new hybrid fragment; no endonuclease recognition site, no restriction Fragments cut by endonucleases will not be ligated;

[0096] Principle steps 1-4: After end repair and ligation with sequencing adapters, complete library DNA fragments that can be amplifi...

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Abstract

The invention relates to the field of gene detection, in particular to a sequencing library construction kit and a using method and application thereof. The kit comprises restriction endonuclease, a buffer solution for a restriction endonuclease reaction, DNA ligase, a ligase buffer solution, a tail end repair reagent, a sequencing connector, a PCR primer and a PCR amplification system, wherein the tail end repair reagent comprises T4 polynucleotide kinase, T4 DNA polymerase, Klenow DNA polymerase, dNTPs and a repair buffer solution. The sequence of a genome obtained from the kit is largely derived from different positions (intervals of 500 bp or above) on an original genome, so that the utilization efficiency of original data can be improved by 150% or above when the kit is used for analyzing the copy number variation of the genome.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a sequencing library construction kit and its use method and application. Background technique [0002] Copy number variation (CNV) refers to an increase or decrease in the copy number of a genome segment, involving a genome sequence ranging in size from 1 kb to multiple Mb, mainly due to genome recombination. CNV is a genetic polymorphism phenomenon that widely exists in animal genomes, and its mutation frequency is much higher than that of SNP. Some CNVs do not cause phenotypic changes, while others can affect the expression and function of genes, and eventually lead to the occurrence of human diseases. Therefore, the study of CNVs has great scientific and clinical significance. [0003] At present, the techniques for detecting CNV mainly include: high-resolution karyotype analysis technique, immunofluorescence in situ hybridization technique (Fluorescent In Situ Hybridization, F...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C40B40/06C12Q1/6806
CPCC12N15/10C12P19/34C12Q1/68C12Q1/6806C40B40/06C40B50/06
Inventor 罗俊峰汪进平郭长全李仁强欧阳虎陈云弟
Owner CARRIER GENE TECH SUZHOU CO LTD
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