Construction method of small-fragment DNA (deoxyribonucleic acid) library based on Illumina Hiseq 2500 sequencing platform

A DNA library and sequencing platform technology, applied in the field of medical molecular biology, can solve the problems of cumbersome small-fragment DNA library construction process, DNA library sample loss, and long time spent, so as to reduce the cost and shorten the library construction time , The effect of reducing the amount of reagents

Inactive Publication Date: 2015-09-30
浙江圣庭医学检验实验室有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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 precious samples; the third is that there are many kinds of reagents used for library construction. Increases the cost of the entire library construction process

Method used

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  • Construction method of small-fragment DNA (deoxyribonucleic acid) library based on Illumina Hiseq 2500 sequencing platform
  • Construction method of small-fragment DNA (deoxyribonucleic acid) library based on Illumina Hiseq 2500 sequencing platform
  • Construction method of small-fragment DNA (deoxyribonucleic acid) library based on Illumina Hiseq 2500 sequencing platform

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1: Construction of a small fragment DNA library based on the Illumina Hiseq 2500 sequencing platform.

[0045] 1. Reagents

[0046] Reagent 1 has a pH value of 7.5 and consists of deionized water, 50mM Tris-HCl, 10mM MgCl 2、 10 mM dithiothreitol (DTT), 1 mM γ-32p-ATP, 10 mM dNTPs, 10 U / ul T4 DNA Polymerase / Klenow Fragment, 10 U / ul T4 polynucleotide kinase.

[0047] Reagent 2 has a pH value of 7.6 and its components are deionized water, 25uM DNA Adapter, 200U / ul T4 DNA ligase, 10mM MgCl 2 , 50mM Tris-Hcl, 50wt% glycerol, 1mM ATP, 5mM dNTPs.

[0048] Reagent 3 has a pH value of 7.6 and its components are deionized water, 100mM Tris-HCl, 5mM MgCl 2 , 250uM dNTPs, 10U / ul Tap DNA polymerase and 12.5mM primer mixture.

[0049] 2. Experimental steps

[0050] (1) Collect maternal peripheral blood and prepare plasma

[0051] In this example, a total of 48 pregnant women were extracted with peripheral blood numbers: ST01, ST02, ST03, ST04, ST05, ST06, ST07, ST08, ST...

Embodiment 2

[0077] Library detection

[0078] Use Agilent Bioanalyzer 2100 to detect the DNA library size, and some samples are detected as figure 2 shown. The concentration of the DNA library was detected by ABI7500Q-PCR, and the specific detection results are shown in the table below.

[0079]

[0080] As can be seen from the data in the above table, the concentration of the library constructed by the reagent combination solution and library construction method of the present invention is appropriate, which is much higher than the concentration (10pM) on the HiSeq machine. The reagent combination solution and library construction method of the invention can be applied to Hiseq2500 sequencing platform.

[0081] The Illumina Hiseq 2500 sequencing platform was used to perform high-throughput sequencing of the DNA library constructed above, and the bioinformatics analysis of the sequencing data obtained the following results:

[0082] sample number Karyotype Sequencing ...

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Abstract

The invention discloses a construction method of a small-fragment DNA (deoxyribonucleic acid) library based on an Illumina Hiseq 2500 sequencing platform, which comprises the following steps: blood free DNA extraction, fragment size screening, terminal repair, 3' terminal linker addition, PCR (polymerase chain reaction) amplification, magnetic bead purification and the like. Compared with the library construction process based on a sequencing platform in the past, the method disclosed by the invention simplifies the experiment process, shortens the library construction time, optimizes the reaction system, greatly reduces the reagent consumption, lowers the library construction cost, lowers the library loss, and is suitable for constructing an artificially-fragmented small DNA library.

Description

technical field [0001] The invention relates to the field of medical molecular biology, in particular to a method for constructing a small fragment DNA library based on the Illumina Hiseq 2500 sequencing platform. 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 are 1 / 800, 1 / 6000 and 1 / 1000 newborns respectively. Currently, there is no effective treatment for such diseases, and prenatal diagnosis is being used to reduce the number of births of affected children. [0003] Among the existing detection methods for this type o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C40B50/06C12Q1/68
Inventor 王震徐飞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
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