Constructing method of maternal plasma free DNA library and typing method of parental alleles

A DNA library and construction method technology, which is applied in DNA preparation, recombinant DNA technology, DNA/RNA fragments, etc., to achieve the effect of wide application, simple method, cost and cycle reduction

Active Publication Date: 2017-09-01
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The present invention solves the problem of accurate typing and identification of paternal alleles in maternal plasma free DNA, and utilizes the advantages of new generation sequencing technology in processing mixing, degradation (fragmentation), and micro-test materials with accurate typing results and high-throughput operations Advantages, it has a very good application prospect in the field of forensic genetics such as non-invasive paternity identification, polymorphism detection, and gene frequency investigation

Method used

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  • Constructing method of maternal plasma free DNA library and typing method of parental alleles
  • Constructing method of maternal plasma free DNA library and typing method of parental alleles
  • Constructing method of maternal plasma free DNA library and typing method of parental alleles

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Experimental program
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Embodiment 1

[0044] According to the gene sequences of 22 human autosomes and Y chromosomes, specific amplification primers were designed for 720 SNP sites evenly distributed in the genome (as shown in Table 1, and the sequences are shown in SEQ ID NO.1-1440 in turn. ), with a wide range of applications, and the amplicon length does not exceed 140bp.

[0045] The distribution (A) of the 720 target SNPs detected by this primer set on each chromosome and the length distribution (B) of SNP amplicons are as follows figure 1 shown. 697 SNP sites (A-SNPs) located on 22 autosomes and 23 SNP sites (Y-SNPs) on the Y chromosome were selected and specific primers were designed. The lengths of the 720 amplicons were very concentrated, more than 80% of them were 136-139bp in length, the average was 137bp, the shortest was 124bp, and the longest was 139bp.

[0046] Table 1

[0047]

[0048]

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[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057] ...

Embodiment 2

[0074] This implementation takes a pregnant woman at 12+5 weeks of pregnancy as an example to describe the content of the present invention in detail. The free DNA based on the high-throughput sequencing platform used in the present invention The method for DNA library construction, sequencing and paternal SNP allele detection comprises the following steps:

[0075] 1. Sample collection, processing and DNA extraction provide templates for library preparation in subsequent steps

[0076] Following the principle of "informed consent", 5 mL of peripheral blood was drawn from pregnant women before puncture, stored in EDTA anticoagulant tubes, centrifuged at 1600 g for 10 min at 4 °C within 8 hours, and the supernatant was centrifuged at 16000 g for 10 min to harvest plasma; the precipitated blood cell part was 10000 g Centrifuge for 5 minutes, and discard the residual supernatant. Fetal villus tissue was collected by B-ultrasound under sterile conditions and washed thoroughly wit...

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Abstract

The invention discloses a constructing method of a maternal plasma free DNA library and a typing method of parental alleles. Firstly, a group of specific primers for amplifying SNP in maternal plasma free DNA is designed, and comprises 720 pairs of primers, and the sequences of upstream and downstream primers are shown as SEQ ID NO:1-1440. Then, peripheral blood of pregnant women of 9-20 weeks is acquired for the extraction of the plasma free DNA, and a multiple composite PCR amplification technology is applied to the construction of the free DNA library; after the library is purified and quantified, computer sequencing is performed on an Ion TorrentTM platform. The same method is used for performing library preparation and sequencing on corresponding maternal cells and fetal tissue genomic DNA. According to sequencing results, the maternal cells are selected as homozygous SNP sites, the percentage concentrations of non-parental alleles of the free DNA in maternal plasma at the corresponding sites are analyzed, and the purpose of determining a paternal allele in the free DNA is achieved.

Description

technical field [0001] The invention belongs to the technical field of biology. More specifically, it relates to a method for constructing a maternal plasma cell-free DNA library and a method for typing paternal alleles, including the design of library construction and high-throughput sequencing methods and reagents for maternal plasma cell-free DNA, as well as paternal source in cell-free DNA, etc. How to identify genes. Background technique [0002] In the past, prenatal fetal genetic diagnosis was mainly based on invasive sampling, including chorionic villus sampling (CVS) and amniocentesis. Although these diagnostic methods have high accuracy, their operations are invasive and can lead to intrauterine abnormalities. Infection, miscarriage and stillbirth and other adverse reactions of pregnancy, and the sampling time should not be earlier than 10 weeks. In 1997, the discovery of fetal cell-free DNA (cff DNA) in the plasma of pregnant women made non-invasive prenatal tes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C40B50/06C12Q1/68
CPCC12N15/1093C12Q1/6806C12Q1/6869C12Q1/6876C40B50/06C12Q2600/172
Inventor 欧雪玲杨冬桂梁灏
Owner SUN YAT SEN UNIV
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