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DMD gene exon copy number variation detection method and application thereof

A gene copy number and detection method technology, applied in the field of molecular biology, can solve the problems of cumbersome detection methods and achieve superior performance, shorten detection time, and easy standardization

Active Publication Date: 2021-11-16
胜亚生物科技(厦门)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection method is cumbersome. The 79 exons need two tubes to hybridize with the target sequence DNA, ligate, PCR amplify, and then perform two-hole capillary analysis. It takes two days to get the result.

Method used

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  • DMD gene exon copy number variation detection method and application thereof
  • DMD gene exon copy number variation detection method and application thereof
  • DMD gene exon copy number variation detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Primer Design

[0055] According to NCBI database, 79 exons of DMD gene, SRY, ACTB and HBB genes were searched, and specific primers were designed. Among them, the SRY gene was used as the interpretation of male and female samples, and two internal reference genes (ACTB and HBB) were used to monitor the PCR efficiency.

[0056] Preparation of DMD reaction solution and DMD primer mixture

[0057] Table 2 DMD reaction solution component distribution table

[0058] components final concentration per reaction 10X Taq Buffer (Thermo) 1.5X 25mM MgCl 2

0.5mM dATP / dTTP / dGTP / dCTP 0.5mM each betaine 2.0M

[0059] Table 3 DMD Primer Mix 1 Component Allocation Table

[0060]

[0061]

[0062] The 5' ends of SEQ ID NO.1 and SEQ ID NO.161 in Table 3 were modified with FAM fluorescent groups.

[0063] Table 4 DMD primer mixture 2 component distribution table

[0064] Primer name Final concentration (μM) P...

Embodiment 2

[0076] Embodiment 2 PCR amplification and result analysis

[0077] 1. Sample processing: Nucleic acid extractor (MagCore) and nucleic acid extraction kit (MagCore Genomic DNA WholeBlood Kit) extract human genomic DNA for subsequent PCR reaction, DNA concentration is 2.5ng / uL~60ng / uL, OD 260 nm / OD 280 The ratio of nm is between 1.6-2.0.

[0078] 2. Preparation of amplification reagents:

[0079] (1) Take out the DMD reaction solution, DMD primer mixture 1 and DMD primer mixture 2 from the kit, thaw at room temperature, mix them upside down, and centrifuge briefly with a microcentrifuge to make all the liquid settle to the bottom of the tube.

[0080] (2) Amplification reagent preparation: prepare the amplification reagent as shown in Table 5

[0081] Table 5 Amplification Reagent Preparation Table

[0082] Amplification reagent 1 Amplification reagent 2 per reaction volume DMD reaction solution DMD reaction solution 14.5μL DMD Primer Mix 1 DMD ...

Embodiment 3

[0101] Embodiment 3 Reagent Performance Verification

[0102] 1. Assess the diagnostic accuracy of the reagent of the present invention

[0103] Detect 9 accuracy reference products, as shown in the following table, respectively detect high, medium and low concentrations, the sample concentrations are 60ng / uL, 25ng / uL, 15ng / uL respectively, each concentration is tested in triplicate, three batches of reagents are detected, DMD gene Exon copy number meets the requirements.

[0104] Table 6 Accuracy reference product DMD gene exon mutation type

[0105]

[0106] 2. Conformity rate of specific reference products

[0107] Detect 4 missing reference products, as shown in the following table, detect high, medium and low concentrations respectively, the sample concentrations are 60ng / uL, 25ng / uL, 15ng / uL, respectively, each concentration is tested in triplicate, three batches of reagents are tested, and the test results are specific The sexual compliance rate is 100%.

[0108]...

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Abstract

The invention discloses a DMD gene exon copy number variation detection method and application thereof and belongs to the technical field of molecular biology. 79 exons of the DMD gene are amplified in a PCR reaction solution, meanwhile, one or more than two groups of reference genes are used for monitoring the PCR reaction efficiency, PCR products are analyzed through capillary electrophoresis, calculation is carried out according to DMD female controls with the DMD genes being 2 copy numbers, relative quantitative detection is carried out on the copy numbers of the 79 exons of the DMD gene, the DMD genes with the copy numbers of 0, 1, 2 and 3 are calculated, normal exon copy number, deletion mutation and repeated mutation of the DMD gene are distinguished, and small deletion and mutation can be prompted. A result can be obtained within 4 hours from a DNA sample, the condition of the exon copy number of the DMD gene can be detected only through one-time PCR experiment, the detection technology is simple in process, and standardization is easy to achieve.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a detection method and an application thereof for exon copy number variation of a DMD gene. Background technique [0002] Progressive muscular dystrophy is a group of heterogeneous gene defect diseases with progressive weakness and atrophy of skeletal muscle as the main clinical manifestation. May be accompanied by central nervous system, heart, bone, respiratory and gastrointestinal tract involvement. The onset time, progression speed, extent of involvement, and severity of different types vary greatly. Duchenne muscular dystrophy (DMD) is the most common progressive muscular dystrophy, with an incidence rate of 1 / 3500 newborn male babies; Becker muscular dystrophy (BMD), newborn male babies The incidence rate is 1 / 30000. [0003] The causative gene of DMD / BMD is the DMD gene, which is located at Xp21.2, and the encoded protein is called dystrophin (Dyst...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/6883
CPCC12Q1/6851C12Q1/6883C12Q2600/156C12Q2531/113C12Q2545/101C12Q2537/16C12Q2565/125Y02A50/30
Inventor 郭亦亦邱一帆钟丽霞李淑如
Owner 胜亚生物科技(厦门)有限公司
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