Method and system for determining haplotype of fetal beta thalassemia

A technology for thalassemia and haplotypes, which is applied in the fields of genomics, biochemical equipment and methods, and microbial determination/inspection. It can solve the problem of small number of detection sites, difficult sampling, and inability to detect maternal mutations. and other problems, to achieve the effect of simple sample, avoid bleeding, and strong practicability

Active Publication Date: 2022-05-24
GUANGZHOU JINGKE DX CO LTD
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  • Claims
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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 beta thalassemia
  • Method and system for determining haplotype of fetal beta thalassemia
  • Method and system for determining haplotype of fetal beta thalassemia

Examples

Experimental program
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Effect test

Embodiment 1

[0172] 1. Preparation of microarray for beta-thalassemia gene hybridization capture

[0173] Determine the region where the common beta thalassemia is located, the preferred region is the region of 5043000 to 5453000 beta thalassemia on chromosome 11.

[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 thalassaemia 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 extract whole-gen...

Embodiment 2

[0251] One patient was tested for non-invasive thalassaemia. In this case, both the father and mother were carriers of CD41-42 mutation, and the fetus was homozygous for CD41-CD42 mutation.

[0252] 1. Collection and processing of samples from father and mother

[0253] 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.

[0254] 1.1 Plasma DNA extraction

[0255] 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:

[0256] 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.

[0257] 1.1.2 Add 600 μ...

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Abstract

The invention relates to a method for determining the haplotype of the fetal beta thalassemia 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 on the third-generation sequencing library Third-generation sequencing, obtain the third-generation sequencing results; determine the haplotype of the beta thalassemia 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 the peripheral blood samples of pregnant women; The sequencing library is subjected to second-generation sequencing to obtain the second-generation sequencing results; the fetal beta-thalassemia gene haplotypes are determined according to the second-generation sequencing results and the beta-thalassemia gene haplotypes of the father and mother.

Description

technical field [0001] The present invention relates to a method and system for determining fetal beta thalassemia gene haplotype. Background technique [0002] At present, the detection methods of fetal beta thalassemia 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 beta thalassemia 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 parents. However, these methods have disadvant...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12Q1/6869G16B20/40G16B20/50G16B20/30
CPCG16B20/00G16B30/00C12Q1/6869C12Q1/6883G16B20/20G16B25/20
Inventor 蒋馥蔓杜伯乐李胜曾晓静张春生郭宇来王阳朱文涛
Owner GUANGZHOU JINGKE DX CO LTD
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