Method and kit for genotyping point mutation

A point mutation and kit technology, applied in the field of molecular biology, can solve the problems of unfavorable result judgment and high background

Active Publication Date: 2017-12-19
北京宏微特斯生物科技有限公司
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Problems solved by technology

However, because the concentration of human genomic DNA molecular template is generally high, the background of ARMS primers after selective amplification is still high, and for specific sequences, ARMS primers are not enough to form obvious amplification differences, which is not conducive to the judgment of results

Method used

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  • Method and kit for genotyping point mutation
  • Method and kit for genotyping point mutation
  • Method and kit for genotyping point mutation

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Experimental program
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Effect test

preparation example Construction

[0067] 3. Preparation of positive quality control products

[0068] Download the sequence of the ATP7B gene from NCBI, and design two pairs of primers near the four detection sites of c.2333G>T, c.2755C>G, c.2975C>T and c.3809A>G: SEQ ID NO : 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23; SEQ ID NO: 24, SEQ ID NO: 24, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23; ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27; SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31. Using a pair of outer primers alone: ​​SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 22, SEQ ID NO: 23; SEQ ID NO: 26, SEQ ID NO: 27; SEQ ID NO: 30, SEQ ID NO: 31 is used to amplify the wild-type target fragment containing the detection site; two pairs of primers are used at the same time, and the site-directed mutagenesis method based on overlap extension PCR can amplify the mutant target fragment containing the detection site. Then insert the wild-type target fr...

Embodiment 1

[0077] Example 1 Detection of positive quality control substances at four loci c.2333G>T, c.3809A>G, c.2975C>T and c.2755C>G of the ATP7B gene associated with Wilson's disease

[0078] The present invention uses Taqman probes and paired primers with asymmetric concentrations to make the Tm values ​​of the melting curves of the wild type and the mutant type different in real-time fluorescent quantitative PCR, and the mutation type can be interpreted through the Tm value, making the result easier to judge.

[0079] 1) The design of the reaction system, primers and probes is as above;

[0080] 2) The fluorescent quantitative PCR reaction system and procedures are as above;

[0081] 3) The wild-type positive quality control and the mutant positive quality control of the four mutation sites are carried out in two reaction wells respectively.

[0082] Test results such as figure 1 As shown, there are two melting curve peaks in the HEX channel, which are the peaks of the c.2333G>T ...

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Abstract

The invention provides a method and a kit for genotyping point mutation. The kit uses the fluorescence probe melting curve of fluorescent quantitative PCR to directly genotype the point mutation; and the kit includes a pair of upstream and downstream primers for carrying out asymmetric PCR amplification on every point mutation point of a sample to be detected, a Taqman probe designed for every point mutation point of the sample to be detected, and a positive quality control substance for every point mutation point of the sample to be detected. The method and the kit have the advantages of convenience in use for the point mutation genotyping, and convenience in promotion and use.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for directly detecting genotypes of point mutations by utilizing the melting curve of fluorescent probes of fluorescent quantitative PCR and a kit thereof. Background technique [0002] Wilson's disease is a disease that seriously affects the quality of human life. It is a genetic disease that causes defects in copper metabolism in the body due to mutations in the ATP7B gene. It has high genetic heterogeneity, and different mutation sites lead to the onset Age and clinical phenotypes vary. ATP7B gene detection for Wilson's disease patients and their families has practical significance for the treatment and early diagnosis of the disease. [0003] In recent years, with the development of sequencing technology and scientific research discoveries, a large number of point mutations of the ATP7B gene have been located, and the genetic detection of Wilson's disease ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q1/6883C12Q2600/156C12Q2600/166C12Q2531/113C12Q2563/107
Inventor 王华贵赵相胜刘利成其他发明人请求不公开姓名
Owner 北京宏微特斯生物科技有限公司
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