Test kit of preimplantation embryonic genetic diagnosis and prenatal diagnosis for DMD

A preimplantation embryo and prenatal diagnosis technology, applied in the field of preimplantation embryo genetic diagnosis and prenatal diagnosis kits, can solve the problem of not considering the genomic region amplification preference, increasing the misreading of downstream analysis, and difficult to detect SNPs. It can improve the efficiency and stability, lower the sequencing depth, and improve the detection specificity and accuracy.

Active Publication Date: 2020-05-12
PEKING UNIV THIRD HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although prior art CN110541025A (published on December 06, 2019) also discloses a DMD gene defect detection method, the 1246 sets of primers used
Due to the potential mutual interference between the primers in the PCR process, the detection accuracy is often reduced
Secondly, since the patent document provides a way to build a library based on PCR amplification, the amplified product is not suitable for the detection of CNV in embryo samples
In additio

Method used

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  • Test kit of preimplantation embryonic genetic diagnosis and prenatal diagnosis for DMD
  • Test kit of preimplantation embryonic genetic diagnosis and prenatal diagnosis for DMD
  • Test kit of preimplantation embryonic genetic diagnosis and prenatal diagnosis for DMD

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1: Design and preparation of the probe set of the present invention

[0073] 1. Screening of DMD gene-linked SNP loci

[0074] Based on the low-depth sequencing data of MALBAC amplification products of healthy embryo cells that have been sequenced and genetically abnormal embryos, 199 DMD genes in the upstream and downstream 2M regions were screened out according to the following steps. Linked SNP loci. The specific screening process and criteria are:

[0075] 1) For each existing sequencing sample, after normalizing the read coverage of the coverage site according to the sample sequencing depth, select the site whose coverage exceeds the ideal threshold (that is, the number of coverage is at least 3 times);

[0076] 2) Select sites that appear in more than 80% of all samples (in this case, a total of 24 embryos were selected from MALBAC amplification and pre-sequencing data), and merge adjacent sites, and the resulting region is determined to be a small nu...

Embodiment 2

[0086] two. Embodiment 2: Composition, preparation and use of the kit of the present invention

[0087] The detection kit for preimplantation genetic diagnosis and prenatal diagnosis of DMD gene described in this embodiment is a kit for molecular genetic detection by simultaneously detecting the mutation of DMD gene and its linked SNP site.

[0088] The kit contains the following components: the probe set obtained in Example 1, the DMD gene capture probe set (refer to CN 106834507 A), buffer HY, buffer BL, library enrichment binding buffer, washing buffer Solution WB1, washing buffer WB3, buffer NE, PCR reaction solution, product purification eluate. The specific composition is shown in Table 1. Table 1

[0089]

[0090] The using method of described test kit is:

[0091] Sample library preparation:

[0092] a) Ultrasonic fragmentation: The initial amount (the product of cellular genomic DNA amplified by MALBAC) is 3 μg, diluted to 30 ng / μL with 1× low TEBuffer. Covar...

Embodiment 3

[0127] Embodiment 3: verification of the use effect of the kit of the present invention

[0128]The present invention will be described more comprehensively through specific embodiments below. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute an improper limitation of the present invention.

[0129] The child in family 1 suffered from Duchenne muscular dystrophy (DMD, Exon31-43 deletion), X-linked recessive inheritance. The woman is a carrier (DMD, Exon31-43 heterozygous deletion), and the man is normal. See the specific genetic map figure 1

[0130] figure 2 It is the electropherogram of exon deletion of DMD gene in an example family. The order of exon loading is: MALBAC1, MALBAC2, MALBAC3, mother (carrier), father, progenitor (DMD Exon31-43 deletion), and the results show that the progenitor carries the homozygous deletion mutation of DMD gene exon31-43.

[0131] 1. Take human emb...

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Abstract

The invention discloses a test kit of preimplantation embryonic genetics diagnosis and prenatal diagnosis for DMD, which comprises a DMD gene linked SNP site capture probe group, the DMD gene linked SNP site refers to 199 or more SNPs sites which are within 2M of the upstream and downstream of the DMD gene and have high mutation frequency in people. The probe group in the test kit disclosed by theinvention can be used for rapidly and accurately capturing the DMD gene and the DMD gene linked SNP at the same time, and the detection specificity and accuracy of the DMD preimplantation embryonic genetic diagnosis can be improved.

Description

technical field [0001] The invention belongs to the field of gene detection and molecular genetics, in particular to a kit for preimplantation embryo genetic diagnosis and prenatal diagnosis of DMD. Background technique [0002] Duchenne muscular dystrophy (DMD), or Duchenne progressive muscular dystrophy, is the most common fatal X-linked recessive neuromuscular genetic disease. The incidence of DMD is 1 / 3500 of live-born male babies, about 65% of which are related to genetics. The clinical manifestations are mainly progressive skeletal muscle atrophy and weakness in the proximal extremities, and gradual loss of mobility. 90% of the children have pseudohypertrophy of the muscles, especially the gastrocnemius muscle; most of the children are accompanied by myocardial damage, and some patients are also accompanied by Mental retardation. The onset of the disease starts at the age of 3 to 5, and the patient loses the ability to walk around the age of 12. Most of them die from...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6869
CPCC12Q1/6869C12Q1/6883C12Q2600/156C12Q2535/122C12Q2565/519
Inventor 乔杰闫丽盈陈伟朱晓辉
Owner PEKING UNIV THIRD HOSPITAL
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