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Construction method and application of small fragment DNA library based on ion ProtonTM sequencing platform

A DNA library and sequencing platform technology, applied in the field of gene sequencing and high-throughput sequencing, can solve the problems of waste, increased cost, and cumbersome construction of small-fragment DNA libraries.

Active Publication Date: 2015-12-02
BOAO BIOLOGICAL CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] The above-mentioned library construction method based on the illumina sequencing platform has its own limitations: first, the above-mentioned small fragment DNA library construction process is cumbersome, the operation process is complicated, and it takes a long time to build the library; second, in the above-mentioned library construction process, each step They need to be prepared independently of each other and all need to be purified, which causes the inevitable loss and waste of DNA library samples, and is not suitable for the detection of samples with a small amount of DNA or other rare samples; third, there are many kinds of reagents used to construct libraries , which increases the cost of the entire library construction process

Method used

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  • Construction method and application of small fragment DNA library based on ion ProtonTM sequencing platform
  • Construction method and application of small fragment DNA library based on ion ProtonTM sequencing platform
  • Construction method and application of small fragment DNA library based on ion ProtonTM sequencing platform

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Embodiment 1

[0102] Embodiment 1: Based on IonProton TM Method for constructing small fragment DNA library of sequencing platform

[0103] 1. Reagents

[0104]This embodiment uses the following reagents: EndRepairEnzyme (LifeTechnology), DNALigase (LifeTechnology), NickRepairPolymerase (LifeTechnology), Adapter (LifeTechnology), BarcodeX (LifeTechnology), lowTEbuffer (10mMTris-HCl, 0.1mMEDTA, pH 8.0);

[0105] The pH value of reagent I is 7.5, the solvent is water, and the solute is: 10mM Tris-HCl, 100mM KCl, 2mM dithiothreitol, 10mMATP, 10mMdNTPs mixture, 6U / μl EndRepair enzyme;

[0106] The pH value of reagent II is 7.6, the solvent is water, and the solute is: 50mM Tris-HCl, 10mMMgCl 2 , 10mM dithiothreitol, 1mMATP, 2mMdNTPs mixture, 400U / μl DNA ligase and 10U / μl NickRepair enzyme;

[0107] The pH value of reagent III is 7.6, the solvent is water, and the solute is: 66mMTris-SO 4 (pH8.9), 19.8mM (NH 4 ) 2 SO 4 , 2.4mMMgSO 4 , 220μM dNTPs mixture, 22U / ml recombinant TaqDNApolymer...

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Abstract

The invention belongs to the field of gene sequencing, and discloses an establishment method for a small fragmental DNA (Deoxyribose Nucleic Acid) library based on an Ion ProtonTM sequencing platform, and a relevant solvent composition solution. The establishment method disclosed by the invention comprises the steps of DNA sample extraction, tail end repairing, fragment selection, joint connection, PCR (Polymerase Chain Reaction) amplification, library detection, high-throughput sequencing and the like, wherein a reagent I, a reagent II and a reagent III are respectively adopted in the steps of tail end repairing, joint connection and PCR amplification. The establishment method disclosed by the invention is precise and simple in step, simple to operate, small in reagent variety and low in cost, the loss and waste of the library are reduced, and the working efficiency is improved; in addition, the establishment method can be applied to noninvasive antenatal diagnosis on pregnant woman peripheral blood fetus chromosome aneuploid based on the Ion ProtonTM sequencing platform, the diagnosis result is high in accuracy rate and is safe and economic, and the birth rate of chromosome aneuploid fetuses can be effectively controlled.

Description

technical field [0001] The present invention relates to the field of gene sequencing, especially the field of high-throughput sequencing, in particular to an IonProton-based TM The construction method of the small fragment DNA library of the sequencing platform, and the application of this method in the non-invasive detection of fetal chromosomal aneuploidy in the peripheral blood of pregnant women. Background technique [0002] Chromosomal aneuploidy refers to the increase or decrease in the number of one or several chromosomes in a cell compared to the normal 46 chromosomes in humans, which is closely related to significant morbidity and mortality in infants and young children. The incidence of chromosomal abnormalities in newborns is 1 / 160, of which trisomy 21 (Down syndrome), trisomy 18 (Edward syndrome) and trisomy 13 (Patau syndrome) are three The most important autosomal aneuploidy diseases, the incidence rates in newborns are 1 / 800, 1 / 6000 and 1 / 1000[1]. Klinefelte...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C40B40/06C12Q1/68
Inventor 糜庆丰罗东红陈治李君宝饶兴蔷陈样宜
Owner BOAO BIOLOGICAL CO LTD
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