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Method for constructing blood free DNA library

A DNA library and construction method technology, applied in DNA preparation, recombinant DNA technology, libraries, etc., can solve problems such as large amplification deviation, high cost, and adverse effects of sequencing analysis, and achieve low cost, simple steps, and reduced GC amplification. effect of increasing preference

Inactive Publication Date: 2015-07-22
浙江圣庭医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above is based on Ion Proton TM Disadvantages of the library construction method of the platform: free DNA needs to be extracted; Beckman’s Agencourt AMPure XP magnetic beads are used for the magnetic beads, which is costly; the amplification deviation between different samples is large, which will adversely affect the sequencing analysis

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  • Method for constructing blood free DNA library
  • Method for constructing blood free DNA library
  • Method for constructing blood free DNA library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Based on Ion Proton TM Construction of blood cell-free DNA library for sequencing platform

[0051] 1. Reagents

[0052] This embodiment uses the following reagents: End Repair enzyme (Bioo Scientific), T 4 DNA Ligase (New England Biolabs), Bst WarmStart DNA Polymerase (New England Biolabs), Hot Start High-Fidelity DNA Polymerase (New England Biolabs), Adapter P1 (Bioo Scientific) and Tag Adapter (Bioo Scientific).

[0053] The pH value of the end repair reagent is 7.5, the solvent is water, and the solute is: 10mM Tris-HCl, 50mM NaCl, 10mM MgCl 2 , 3mM dithiothreitol, 10mM ATP, 10mM dNTPs, end repair enzyme 10U / μl End Repair enzyme.

[0054] The ligase used in the ligation reaction was T 4 DNA Ligase (400U / μl), polymerase Bst WarmStart DNA Polymerase (8U / μl), ligation reaction buffer pH value is 7.6, solvent is water, solute: 50mM Tris-HCl (PH8.0), 10mM KCl, 10mM Dithiothreitol, 1mM ATP, 2nM dNTPs mixture, 10mM (NH 4 ) 2 SO 4 , 2mM MgSO 4 .

[0055...

Embodiment 2

[0084] Example 2: Application of the constructed blood cell-free DNA library in prenatal detection.

[0085] (1) Library detection.

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Abstract

The invention discloses a method for constructing a blood free DNA library. The method comprises steps as follows: end repairing, purification with magnetic beads, joint connection, screening and purification with the magnetic beads, library mixing, library amplification, purification with the magnetic beads and the like. Compared with previous library constructing methods base on the Ion Proton<TM> platform, the method has the advantages that free DNA is not required to be extracted, meanwhile, the Biocanal Magbeads magnetic beads are adopted, the steps are simple, the cost is low, the library loss is reduced, GC amplification preferences are reduced, the library constructing success rate is increased, and the accuracy of subsequent detection results can be improved.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for constructing a blood free DNA library. Background technique [0002] my country is a country with a high incidence of birth defects. Among the more than 20 million newborns born each year, children born with congenital ignorance and disabilities account for 4%-6% of the total number of births each year, and the total number is as high as 1.2 million, accounting for 500% of the world's annual births. More than one in five children with birth defects. Our government pays about 10 billion yuan each year for medical treatment and social relief for children with Down syndrome. Most of the surviving children with birth defects are permanently disabled or mentally handicapped, which cannot be cured. This has caused serious harm to the society. The economic burden, the psychological burden and mental pain caused to the family cannot be measured by money. [0003] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C40B50/06C40B40/06
Inventor 徐飞王震蔡乐靖钱飞箭周桂兰张林华卢灵潇刘智敏陈帼婧屠勇军陈贤丰
Owner 浙江圣庭医学检验实验室有限公司