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Linkers, primer sets, kits and library building methods for cfDNA library building

A ligation product and ligation technology, applied in the field of molecular genetics, can solve problems such as self-ligation of joints, and achieve the effects of improving transformation efficiency, simple operation, and stable sequencing results.

Active Publication Date: 2020-08-11
JIANGSU COWIN BIOTECH CO LTD
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  • Abstract
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AI Technical Summary

Problems solved by technology

Although the purification steps are reduced and the loss of DNA is reduced, the problem of self-ligation of adapters is still difficult to solve

Method used

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  • Linkers, primer sets, kits and library building methods for cfDNA library building
  • Linkers, primer sets, kits and library building methods for cfDNA library building
  • Linkers, primer sets, kits and library building methods for cfDNA library building

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Embodiment 1c

[0027] Construction method of embodiment 1cfDNA library

[0028] (1) DNA end repair reaction

[0029] ① Add the following reagents to a 200μl PCR tube (place it on ice and proceed to the next step immediately):

[0030]

[0031] At this time, the total volume of the solution in the tube was 65 μl.

[0032] This end repair reaction involves adding an "A" to the 3' end.

[0033] According to the instruction manual of the QIAamp Circulating Nucletic Acid Kit (50) kit provided by QIAGEN, the free cfDNA in the plasma was extracted using the kit;

[0034] ② Gently blow and mix the above solution with a pipette tip, and centrifuge briefly to collect all components to the bottom of the tube.

[0035] ③Place the above PCR tube in the PCR instrument, and the reaction procedure is as follows:

[0036] 37℃ 10min

[0037] 72°C 10min

[0038] Hold on 4℃

[0039] (2) Adapter connection

[0040] ①Add the following reagents directly to the above reaction solution that has completed DN...

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Abstract

The invention discloses a linker, a primer group, a kit, and a library construction method used for cfDNA library construction, wherein the linker includes a top linker and a bottom linker respectively being represented as the SEQ ID No:1 and the SEQ ID No:2; the primer group includes sequences represented as the SEQ ID No:4-6; the kit includes the linker and the primer group. The library construction method of cfDNA includes steps of: (1) repairing the terminals of the cfDNA and adding an A basic group to the 3'-terminal of the cfDNA; (2) linking the terminal of the cfDNA with the upper linker to obtain a linked product; (3) performing first-round amplification to the linked product and performing magnetic bead purification to the product; (4) performing library construction amplificationto the first-round amplified product after the magnetic bead purification, and performing magnetic bead purification to the product to prepare a high-throughput sequencing library. The linker and thecfDNA library construction method are suitable for Illumina sequencing platform and can increase conversion efficiency of the library.

Description

technical field [0001] The invention relates to the technical field of molecular genetics, in particular to a linker, a primer set, a kit and a method for building a cfDNA library. Background technique [0002] Plasma cell-free DNA (cfDNA) is a kind of extracellular DNA in a cell-free state that exists in human blood. A certain amount of cfDNA can also be detected in the culture medium. Since the discovery of cfDNA by Mandel and Metais in 1947, with the rapid development of molecular detection technology, more than 300 kinds of plasma free DNA have been detected, and the same gene mutations as tumor cell DNA have been observed, making cfDNA a marker It has potential value in the diagnosis, prognosis and detection of clinical diseases. In recent years, cfDNA detection has been used in the diagnosis and prognosis evaluation of various acute or chronic diseases. [0003] Nowadays, people usually use next-generation sequencing (NGS) technology to perform high-throughput analy...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C12N15/11
CPCC12N15/11C40B50/06
Inventor 王春香张云
Owner JIANGSU COWIN BIOTECH CO LTD
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