Method and system for determining haplotype of fetal duchenne muscular dystrophy

A technology for muscular dystrophy and haplotypes, applied in the fields of genomics, biochemical equipment and methods, and microbial determination/inspection, which can solve the problem of sample size, difficulty in sampling, and number of detection sites that cannot detect maternal mutations Less problems, to achieve the effect of avoiding bleeding, strong practicability, and simple samples

Active Publication Date: 2022-05-24
GUANGZHOU JINGKE DX CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have disadvantages such as the small number of detection sites, the inability to detect maternal mutations, or the need for a large sample size, which is difficult to sample.

Method used

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  • Method and system for determining haplotype of fetal duchenne muscular dystrophy
  • Method and system for determining haplotype of fetal duchenne muscular dystrophy
  • Method and system for determining haplotype of fetal duchenne muscular dystrophy

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Experimental program
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Effect test

Embodiment 1

[0172] 1. Preparation of Duchenne Muscular Dystrophy Gene Hybridization Capture Chip

[0173] Determine the region where the common Duchenne muscular dystrophy is located, the preferred region is 30897180 to 33561648 on chromosome X is the region of Duchenne muscular dystrophy.

[0174] To remove duplicated fragments, the preferred method is to remove the regions with more than 200 repeated fragments, that is, if a certain fragment can be compared with more than 200 regions on the chromosome, then these fragments are removed.

[0175] To obtain a chip for DMD hybridization capture, the preferred method is a liquid-phase capture chip.

[0176] Amplification and enrichment of long fragments can be performed at the same time using the PCR method, which can be used as a supplementary method to assist the chip to capture the target region.

[0177] 2. Construction of the three-generation library

[0178] a. Sample preparation: Obtain fetal paternal and maternal blood samples, and...

Embodiment 2

[0253] Non-invasive DMD detection was performed on 1 patient. In this case, both the father and mother were carriers of the c.583C>T mutation, and the fetus was homozygous for the c.583C>T mutation.

[0254] 1. Collection and processing of samples from father and mother

[0255] Streck blood collection tubes were used to collect 5 mL of peripheral blood from the father and mother according to the standard peripheral blood collection operation. After the collection was completed, the peripheral blood of the father and mother was plasma separated in time according to the standard two-step centrifugation method.

[0256] 1.1 Plasma DNA extraction

[0257] The TIANamp Micro DNA Kit was used to extract cell-free DNA from the peripheral blood plasma of the mother. The specific operation steps are as follows:

[0258] 1.1.1 Take 600μL of maternal peripheral blood plasma into a 2mL centrifuge tube, add 20μL of Proteinase K solution, mix well, and centrifuge briefly.

[0259] 1.1.2 ...

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Abstract

The present invention relates to a method for determining the haplotype of the fetal Duchenne muscular dystrophy gene, comprising the following steps: constructing a third-generation sequencing library based on blood samples, the blood samples being taken from the blood samples of the fetal father and mother; performing the third-generation sequencing library Perform third-generation sequencing to obtain third-generation sequencing results; determine the haplotype of the Duchenne muscular dystrophy gene of the father and mother according to the third-generation sequencing results; construct a second-generation sequencing library based on the detection samples, which are taken from peripheral blood samples of pregnant women; The second-generation sequencing library is subjected to second-generation sequencing to obtain the results of the second-generation sequencing; according to the results of the second-generation sequencing and the haplotypes of the Duchenne muscular dystrophy gene of the father and mother, the haplotype of the Duchenne muscular dystrophy gene of the fetus is determined.

Description

technical field [0001] The present invention relates to a method and system for determining the haplotype of fetal Duchenne muscular dystrophy gene. Background technique [0002] At present, the detection methods for fetal Duchenne muscular dystrophy (DMD) gene are divided into two types: invasive and non-invasive. In view of the invasive method has a certain risk of miscarriage, the non-invasive method is more and more recognized and popularized. [0003] At present, there are several methods to detect fetal Duchenne muscular dystrophy gene based on cell-free DNA in maternal peripheral blood: 1. Detect paternal or new mutations by droplet digital PCR. 2. Detect paternal or de novo mutations by means of target region capture high-throughput sequencing. 3. To construct haplotypes by sequencing the parents, grandparents and maternal grandparents or progenitors, and use maternal peripheral sequencing to evaluate the dose imbalance to determine the mutations inherited from the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12Q1/6869G16B30/10G16B20/20
CPCG16B20/00G16B30/00C12Q1/6869C12Q1/6883C12Q2600/156C12Q1/6827C12Q2600/172G16B20/20C12Q2535/122C12Q2537/165
Inventor 张春生郭宇来李胜杜伯乐蒋馥蔓曾晓静夏伟成王阳朱文涛
Owner GUANGZHOU JINGKE DX CO LTD
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