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
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[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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