Primer combination and method for detecting human embryo Chediak-Higashi syndrome LYST gene mutation

A primer combination and syndrome technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc., can solve problems such as contamination, amplification failure, allele tripping, etc., and achieve accurate detection results. , the detection process is fast and the cost is low

Inactive Publication Date: 2019-01-01
广州达瑞生殖技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although traditional PCR is sensitive, when it is applied to PGD, there are disadvantages such as contamination, allele dropout, and amplification failure.

Method used

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  • Primer combination and method for detecting human embryo Chediak-Higashi syndrome LYST gene mutation
  • Primer combination and method for detecting human embryo Chediak-Higashi syndrome LYST gene mutation
  • Primer combination and method for detecting human embryo Chediak-Higashi syndrome LYST gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1: Pre-implantation diagnosis of LYST gene mutation in 4 embryos with Chediak-Higashi syndrome based on the Ion Torrent sequencing platform

[0087] 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), DNA Ligase (Life Corporation), XP Reagent (Beckman Corporation), Platium TM PCR SuperMix High Fidelity (Life Company), Library Amplification Primer Mix (Life Company), Nuclease-FreeWater, absolute ethanol.

[0088] Before the experiment of this method, a reagent needs to be freshly prepared: 75% ethanol.

[0089] Instruments and equipment needed for this experiment: centrifuge, magnetic stand, pipette, PCR instrument, oscillator, fluorometer Qubit 4.0.

[0090] Main experimental steps:

[0091] (1) Sample source: a couple carrying LYST mutation gene, f...

Embodiment 2

[0184] Embodiment 2: Sanger generation sequencing verification of embodiment 1

[0185] 1. Nested PCR amplification:

[0186] 1. Sample:

[0187] 1.1 8 WGA samples: No. 1~8

[0188] 1.2 2 whole blood DNA samples of parents: M (mother), F (father)

[0189] 2. There are 2 sets of 3 pairs of PCR primers:

[0190] 2.1 Primer A composition: ① G1-5Fa: GAGGTTCAGGAAGATTTTGTG, G1-5Ra:

[0191]

[0192] 2.2 Primer B composition: ②G1-3Fa: CAGCTGATAATGACCCAAGAA, G1-3Ra:

[0193] ACTGGAAATGTTTCAAAGGAA---650bp; ③G1-3F: AGGCAGGAGAATGGTGTGAAC, G1-3R: GCAATCATAGCTCACTCACCG---356bp

[0194] 3.PCR method:

[0195] 3.1 Primer A for ordinary PCR, that is, use primer ① to perform PCR amplification according to the following PCR reaction system / conditions

[0196] 3.2 Primer B for nested PCR, that is, first use primer ② to perform PCR amplification according to the following PCR reaction system / conditions, and then

[0197] Dilute the PCR product 10,000 times, and use primer ③ to carry ou...

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Abstract

The invention provides primers and a method for detecting Chediak-Higashi syndrome LYST gene mutation based on a high-throughput sequencing technology, wherein the used primer composition comprises the primers for specifically amplifying a LYST gene and specifically amplifying the closely linked SNP sites within a 2 Mb range from the upstream to the downstream of the LYST gene. According to the present invention, the method has advantages of versatility, multi-site SNP sequencing, high throughput, low cost, high sensitivity and strong specificity, can perform embryo pre-transplantation detection on all Chediak-Higashi syndrome gene mutation, and can achieve the rapid detection process and the accurate detection result.

Description

technical field [0001] The invention provides a kit for detecting gene mutations of Chediak-Higashi syndrome based on high-throughput sequencing technology. Wherein, the primer composition used includes primers for specifically amplifying the LYST gene and for specifically amplifying closely linked polymorphic sites (SNPs) within 2 Mb upstream and downstream of the LYST gene. The method of the present invention has the advantages of versatility, multi-site SNP sequencing, high throughput, low cost, high sensitivity, and strong specificity, and can detect all Chediak-Higashi syndrome gene mutations before embryo transfer, and the detection process is more efficient. Faster and more accurate test results. technical background [0002] Chediak-Higashi syndrome (Chediak-Higashi syndrome, CHS, OMIM 214500) is a rare autosomal recessive genetic disease, clinical manifestations include hypopigmentation, immune deficiency, mild bleeding tendency, progressive nervous system abnormal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156
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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