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Enzyme composition, buffer reagent and sequencing library construction method for cfDNA end repair

A technology of end repair and enzyme composition, applied in biochemical equipment and methods, combinatorial chemistry, chemical library, etc., can solve the problems of inapplicable cfDNA end repair, high cost, and large amount of enzymes, and achieve low cost and high dosage The effect of less, high repair efficiency

Active Publication Date: 2021-03-19
JIANGSU COWIN BIOTECH CO LTD
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The above-mentioned patents all have some shortcomings, such as the amount of enzyme used is high, the cost is high, or it is not suitable for end repair of cfDNA with low sample volume

Method used

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  • Enzyme composition, buffer reagent and sequencing library construction method for cfDNA end repair
  • Enzyme composition, buffer reagent and sequencing library construction method for cfDNA end repair
  • Enzyme composition, buffer reagent and sequencing library construction method for cfDNA end repair

Examples

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Embodiment 1

[0049] Example 1 cfDNA library construction

[0050] cfDNA library construction, including the following steps:

[0051] 1. End repair

[0052] In each 50 μl reaction system, use the end repair buffer reagent as 10×End Repair ReactionBuffer, and its components and contents are as follows:

[0053] components content Tris-Hcl (pH8.3) 1M MgCl 2

0.2M DTT 0.2M KCl 0.2M ATP 20mM dATP 20mM dTTP 2mM dCTP 2mM dGTP 2mM NP40 0.7%v / v Tween-20 0.7%v / v

[0054] In each 50 microliter reaction system, the components and contents of the end repair enzyme composition are as follows:

[0055] components content T4 Polynucleotide Kinase 2.5U Klenow exo- 1U Taq DNA Polymerase 3U glycerin 50%v / v MgCl 2

10mM Tris-Hcl 70mM

[0056] End repair includes the following steps:

[0057] 1. Take 1 ng of free DNA in a PCR tube, the volume should be ≤42 μL,...

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Abstract

The invention provides a cfDNA (circulating cell free DNA) terminal repairing enzyme composition, which comprises the following enzymes, including an enzyme I and an enzyme II, wherein the enzyme I enables cfDNA to carry out DNA double-strand repairing and 3' terminal plus A when dNTP (deoxy-ribonucleoside triphosphate) is in the presence, and is a Taq DNA polymerase and Klenow Fragment, Exo-, andthe enzyme II enables a cfDNA 5' terminal to generate phosphorylation modification and is T4 Polynucleotide Kinase (T4 PNK for short). The invention also provides a cfDNA terminal repairing buffer solution reagent. The invention also provides a method which uses the above reagent to carry out cfDNA terminal repairing and construct a cfDNA sequencing library. The invention has the following technical effects that a use amount of reagent enzymes is small, cost is low, repairing efficiency is unexpectedly high, and therefore, the invention is suitable for carrying out terminal repairing on the cfDNA with a low sample amount.

Description

technical field [0001] The invention relates to an enzyme composition for cfDNA end repair, a buffer reagent and a method for constructing a sequencing library, belonging to the field of biotechnology. Background technique [0002] Plasma free DNA (circulating cell free DNA, cfDNA) is a kind of DNA free in body fluid outside cells, and the size of cfDNA fragments is between 150-400bp. cfDNA sequencing is widely used in the diagnosis of tumor diseases and prenatal screening. In view of the low content of cfDNA in blood and the short fragments, the sequencing depth of high-throughput sequencing is limited. [0003] Therefore, it is necessary to improve the efficiency of cfDNA end repair and A addition to ensure the efficiency of library construction, high-throughput sequencing data can cover all cfDNA, and improve the authenticity and reliability of sequencing data. [0004] Chinese patent CN201480062608.5 provides an enzyme composition for DNA end repair, adenylation, and p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12C12N9/22C12Q1/6806C40B50/06
CPCC12N9/1258C12N9/22C12Q1/6806C12Y207/07008C40B50/06C12Q2525/191C12Q2521/101
Inventor 余丽萍
Owner JIANGSU COWIN BIOTECH CO LTD
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