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Single tube nested type allele specific PCR method

A technology of chain reaction and polymerase, applied in the field of molecular biology, can solve the problems of amplification products, pollution, complicated procedures, etc.

Inactive Publication Date: 2005-08-17
徐定邦 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention provides a single-tube nested allele-specific polymerase chain reaction method to overcome the pollution of the amplification products of the above-mentioned various SNP detection methods, slow speed, complicated procedures, difficult automation, high cost and special equipment. and other shortcomings and limitations, to develop a method that is simple, sensitive, fast, economical and accurate

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Targeting the human beta actomyosin gene (National Center for Bioinformatics Gene Bank No. BC016045), it is assumed that the allelic specific mutation occurs at base 1281 (change from G to C) or base 1356 (Changed from C to G), the primer sequence and characteristics are shown in Table 6. A1 is the primer for the jacket enrichment template, and A2 and A3 are the allele-specific PCR primers for the inner jacket. The allele-specific primer of A2 is a positive primer, and the third base from the 3'end of the primer is indicated in bold bold for allele-specific mutations; the allele-specific primer of A3 is a reverse primer, and the base of the 3'end of the primer Allele-specific mutations are indicated in bold and bold type.

[0040]

[0041] * Melting temperature calculated by nearest base method

[0042] The outer primer "A1" does not contain introns between the positive and negative primers, the amplified product is 295 bases long, and the melting temp...

Embodiment 2

[0047] Example 2 also targets the human beta actomyosin gene (National Center for Bioinformatics Gene Bank No. BC016045), assuming that the allelic specific mutation occurs at base 1281 (change from G to C). The primers for the jacket enrichment template are the same as in Example 1. A4 is the inner set of allele-specific PCR primers. The primer is 20 bases long. The allele-specific primer is a positive primer. The second base from the 3'end of the primer is indicated in bold bold for allele-specific mutations. The introduction of two mismatched bases at position 8 (from T to A) and 16 (from G to C) from the 3'end of the position-specific primer is underlined. The primer sequence and characteristics are shown in Table 8.

[0048]

Name

Location

Length

Melting temperature

Degree

(℃)

Knot with template

Combined strength

Sequence (5’ to 3’)

A4 positive

Wild

1263-

1282

20

4...

Embodiment 3

[0052] Example 3 targets the human glyceraldehyde-3-phosphate dehydrogenase gene (Genebank No. XM_006959 of the National Center for Bioinformatics, USA). It is assumed that the allelic-specific mutation occurs at base 415 (change from G to C). The primer sequence and characteristics are shown in Table 9. G1 is the primer used for the jacket enrichment template, G2 is the inner set of allele-specific PCR primers, the allele-specific primer is the positive primer, and the 5th base from the 3'end of the primer is indicated in bold boldface for allele-specific mutations .

[0053]

[0054] * Melting temperature calculated by nearest base method

[0055] The length of the amplified product of the coat primer "G1" is 616 bases, and the melting temperature of the amplified product is 90.4°C. The length of the amplified product of the inner set of primer "G2" is 147 bases, and the melting temperature of the amplified product is 87.1°C.

[0056] The PCR template of Example 3 was ...

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PUM

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Abstract

The present invention provides single-tube nested PCR method with very short primer or primer containing several mismatched bases for base mutation detection. Very short primer of 6-14 bases or normal length primer containing several mismatched bases is allelically amplified specially to make 'wild allelic specific primer' to be deactivated in combining with mutant template and 'mutant allelic specific primer' to be deactivated in combining with wild template completely, so as to avoid detection pseudo positivity and raise detection reliability. The present invention is suitable for nucleic acid detection one single base mutation in molecular biology lab and clinical detection, especially the nucleic acid analysis of genetic diseases, mutant virus infection, drug-resistant bacterial infection and metabolic disease caused by single base mutation.

Description

Technical field [0001] The invention relates to molecular biology technology, in particular to a polymerase chain reaction method for detecting allele-specific mutations. technical background [0002] Single nucleotide polymorphism (SNP) refers to a DNA sequence polymorphism caused by a single nucleotide variation at the genomic level, which accounts for 90% of the various heritable polymorphisms in humans the above. It is estimated that there is one SNP in every 500-1000 base pairs in the human genome, and the total number of SNPs in the total of 3.2 billion base pairs is as high as several million, which is directly or indirectly related to the cause of disease and drug response. SNP will have an immeasurable impact on population genetics, pharmaceutical industry, forensic medicine, cancer and genetic diseases and even evolutionary research. [0003] There are many techniques for detecting single nucleotide polymorphisms (Kwok, P.Y.ed, Single Nucleotide Polymorphisms method and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C12Q1/25C12Q1/68
Inventor 徐定邦朱德芬谢文凯徐文慧
Owner 徐定邦
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