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ARMS-PCR method for mtDNA allelic gene typing and point mutation detecting

An allele and point mutation technology, applied in the field of molecular genetics, can solve the problems of high utility and difference in PCR amplification efficiency, etc., and achieve the effect of highly reliable results, simple operation and practicability

Inactive Publication Date: 2012-01-11
CENT SOUTH UNIV
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Problems solved by technology

[0006] The purpose of the present invention is to solve the deficiencies of the existing mtDNA genotype polymorphism analysis methods, aiming at the characteristics that mtDNA templates are more effective than nuclear genomic DNA and are not easy to form differences in PCR amplification efficiency between mismatched primers, an optimized and improved method is proposed. ARMS-PCR method for mtDNA point mutation detection or mtSNPs polymorphism genotype identification

Method used

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  • ARMS-PCR method for mtDNA allelic gene typing and point mutation detecting
  • ARMS-PCR method for mtDNA allelic gene typing and point mutation detecting
  • ARMS-PCR method for mtDNA allelic gene typing and point mutation detecting

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Embodiment 1

[0013] Example 1 Detection of mtSNP 8701A / G by the method of the present invention

[0014] Taking the individual genotyping of mtSNP 8701A / G as an example, aiming at the alternating changes of A and G nucleotides in different individuals, mismatched primers were designed in accordance with the principle of ARMS-PCR amplification, and the optimized 200 cases of DNA samples were tested for genotyping under amplification conditions, and the results were consistent with the results of sequence analysis and typing.

[0015] (1). Design ARMS-PCR amplification primers

[0016] Six pairs of ARMS-PCR amplification primers were designed in the experiment, such as figure 1 shown. The common upstream primer is ATF1 (np8466-8587). The 3' ends of the 6 downstream ARMS primers are fixedly terminated at the mtDNAnp8701 site, and 6 downstream primers that differ from each other at the 3' ends are designed for the nucleotide substitution of G and A between the two SNP alleles .

[0017] S...

Embodiment 2

[0025] Example 2: Detection of mt10398A / G

[0026] Taking the aforementioned individual genotyping of mtSNP10398A / G, which can be detected by Bgl I enzyme digestion, as an example, aiming at the alternating changes of A and G nucleotides in different individuals, an error in accordance with the principle of ARMS-PCR amplification was carried out. Design primers and use optimized amplification conditions for genotyping of DNA samples.

[0027] The inventor designed two pairs of primers, the shared downstream primer is (10398R):

[0028] 5'-GGTGTTGAGGGTTATGAGAGTA-3';

[0029] The two upstream primers are mismatched ARMS primers. The upstream ARMS primer 1 is (10398FG):

[0030] 5'-GACTACAAAAAGGATTAGAC AC AG-3';

[0031] The upstream ARMS primer 2 is (10398FA):

[0032] 5'-GACTACAAAAAGGATTAGAC AC AA-3'.

[0033] The bases at the 3' end of the upstream primer are complementary to A or G respectively, and consecutive mismatched bases are also added at the penultimate 3-4 ...

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Abstract

The invention provides an ARMS-PCR method for mtDNA allelic gene typing and point mutation detecting. When an idiosyncratic ARMS primer is in design, an upstream primer or a downstream primer is additionally provided with two continuous basic group mismatches at the 3 to 4 position counting backwards at the 3`end, the mismatching way is the exchanging of A to T or G to C, and the increasing quantity of templates mtDNA when in ARMS-PCR amplification is 2 to 10 times larger than the conventional using quantity. By improving ARMS-PCR, the invention is suitable for detection of mtDNA mutation or mtSNPs allelic gene typing. Compared with the present mtDNA pleomorphic or genovariation detecting technology, the method in the invention not only is fairly reliable in results and is simple as well as easy in operation, but also greatly quickens the analyzing process, lowers the cost and reduces the workload.

Description

technical field [0001] The invention belongs to the field of molecular genetics, and in particular relates to a mitochondrial DNA (mitochondrial DNA, mtDNA) polymorphic genotype identification and point mutation detection method. Background technique [0002] Case-control studies to understand the association between mtDNA mutations or polymorphisms and diseases often involve differential identification of the polymorphic genotypes of a large number of individuals; services such as molecular diagnosis, genetic counseling, and medication guidance for mtDNA-related diseases are also available. Both require fast, easy, and efficient methods for mutation analysis or genotyping. [0003] Restriction enzyme methods such as restriction fragment length polymorphisms (restriction fragment length polymorphisms, RFLPs) analysis is currently the main method for detecting mtDNA point mutations or mtDNA single nucleotide polymorphisms (mitochondrial DNA single nucleotide polymorphisms, mt...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 朱敏胡维新周钢黄河
Owner CENT SOUTH UNIV
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