Primer combination and method for detecting human embryo alpha-thalassemia gene mutation

A thalassemia, primer combination technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve problems such as low polymorphism, PCR amplification failure, complex mechanism, etc., to achieve the prevention index The effect of sexual amplification and increased homogeneity

Inactive Publication Date: 2018-09-14
广州达瑞生殖技术有限公司
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Problems solved by technology

The mechanism of its occurrence is complicated, and it may be related to chromosomal mosaicism, haploid cells in some diploid cells, and the temperature of PCR denaturation. There is a region on the α-thalassemia HBA gene located at the short arm telomere of chromosome 16, and the homology High, low polymorphism, high GC and other reasons lead to the failure of PCR amplification, and there are few effective SNP sites to choose from, which ultimately leads to the failure of constructing haplotypes

Method used

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  • Primer combination and method for detecting human embryo alpha-thalassemia gene mutation
  • Primer combination and method for detecting human embryo alpha-thalassemia gene mutation
  • Primer combination and method for detecting human embryo alpha-thalassemia gene mutation

Examples

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Embodiment 1

[0129] Example 1: Pre-implantation detection of α-thalassemia embryos in 4 cases of embryos based on the Ion Torrent sequencing platform

[0130] This embodiment adopts the following reagents: 5×Ion Ampliseq TM HiFi Master Mix (Life Company), 2 x Ion Ampliseq TM Primer Pool (Life), FuPa Reagent (Life), Ion P1 Adapter and Ion Xpress TM Barcode X (Life Corporation), Switch Solution (Life Corporation), DNALigase (Life Corporation), XP Reagent (Beckman Corporation), Platium TM PCR SuperMix High Fidelity (Life Company), Library Amplification Primer Mix (Life Company), Nuclease-FreeWater, absolute ethanol.

[0131] Before the experiment of this method, a reagent needs to be freshly prepared: 75% ethanol.

[0132] Instruments and equipment needed for this experiment: centrifuge, magnetic stand, pipette, PCR instrument, oscillator, fluorometer Qubit 4.0.

[0133] Main experimental steps:

[0134] (1) DNA extraction and whole genome amplification of the sample to be tested ...

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Abstract

The invention provides a primer combination and method for detecting alpha-thalassemia gene mutation based on a high throughput sequencing technology. The primer combination comprises a specific amplification HBA gene and single nucleotide polymorphism (SNP) primers closely linked within the 2Mb ranges of the upstream and the downstream of the specific amplification HBA gene. The method has the advantages of universality, multi-site SNP sequencing, high throughput, low cost, high sensitivity and strong specificity, all alpha-thalassemia mutation can be detected before embryo transplantation, the detection process is rapider, and detection results are more accurate.

Description

technical field [0001] The invention provides a primer combination and method for detecting α-thalassemia gene mutations in human embryos based on high-throughput sequencing technology, and belongs to the technical field of gene detection. Background technique [0002] Thalassemia (hereinafter referred to as "thalassemia") is one of the most common human monogenic blood diseases in the world. It is listed by the World Health Organization as six common diseases that endanger human health. It is also the most common and most harmful disease in southern China genetic disease. In the high-incidence areas of α-thalassemia in southern my country, the total incidence rate is 2.46%, and the incidence rates in Guangxi, Guangdong, Jiangxi, Sichuan and Zhejiang provinces (districts) are 14.95%, 4.11%, 2.60%, 1.92% and 1.20% respectively. %. Thalassemia is divided into alpha-thalassemia and beta-thalassemia. At present, there is no effective cure for α-thalassemia, and the main focus ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6869C12N15/11
CPCC12Q1/6869C12Q1/6883C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2525/191C12Q2535/122
Inventor 邓红辉卢绍月方雅亮
Owner 广州达瑞生殖技术有限公司
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