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Single tube nested type allele specific PCR method

A chain reaction and polymerase technology, applied in the field of molecular biology, can solve the problems of amplification products, high cost, pollution, etc., and achieve the effects of low false positives, simple method and high reliability

Inactive Publication Date: 2008-10-29
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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  • Single tube nested type allele specific PCR method
  • Single tube nested type allele specific PCR method
  • Single tube nested type allele specific PCR method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 takes the human beta-actomyosin gene (National Center for Bioinformatics GenBank No. BC016045) as the target, assuming that the allele-specific mutation occurs at base 1281 (changed from G to C) or base 1356 (changed from C to G), the primer sequence and properties are shown in Table 6. A1 is the primer for the outer jacket enrichment template, and A2 and A3 are the inner jacket allele-specific PCR primers. 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 letters for the allele-specific mutation; the allele-specific primer of A3 is a reverse primer, and the base at the 3' end of the primer is Allele-specific mutations are indicated in bold boldface.

[0044] Table 6. Example 1 Primer order and characteristics

[0045]

name

Location

long

Spend

melting temperature

degree(℃)

knot with the template

combined st...

Embodiment 2

[0054] Example 2 also targeted the human beta-actomyosin gene (National Center for Bioinformatics GenBank No. BC016045), assuming that the allele-specific mutation occurred at base 1281 (changed from G to C). The primers for coat 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 8th (changed from T to A) and 16th (changed from G to C) counting from the 3' end of the specific primer introduces two mismatched bases, which are underlined. The primer sequence and characteristics are shown in Table 8.

[0055] Table 8. Example 2 primer sequence and characteristics

[0056]

name

Location

length

melting temperature

Spend

(℃)

knot with the template

combined strength

Sequential (5' to 3')

A4 positive

wild

...

Embodiment 3

[0060] Example 3 targets the human glyceraldehyde-3-phosphate dehydrogenase gene (National Center for Biological Information GenBank No. XM_006959). An allele-specific mutation was assumed to occur at base 415 (change from G to C). Primer sequences and properties are shown in Table 9. G1 is the primer for the outer jacket enrichment template, G2 is the inner jacket allele-specific PCR primer, the allele-specific primer is the positive primer, and the fifth base from the 3' end of the primer is indicated in bold bold for the allele-specific mutation .

[0061] Table 9. Example 3 primer sequence and properties

[0062]

name

Location

long

Spend

melting temperature

degree(℃)

knot with the template

combined strength

Sequential (5' to 3')

G1 Positive

343-

366

24

81.4

536

GCT GGC GCT GAG TAC

GTC GTG GAG

...

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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 variation. technical background [0002] Single nucleotide polymorphism (single nucleotide polymorphism, SNP) refers to the DNA sequence polymorphism caused by a single nucleotide variation at the genome level, which accounts for 90% of the various polymorphisms that can be inherited by humans 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 a total of 3.2 billion base pairs is as high as several million, which are directly or indirectly related to the cause of disease and drug response. SNP will have an immeasurable impact on the research of population genetics, pharmaceutical industry, forensic science, cancer and genetic diseases, and even evolution. [0003] There are many technologies for detecting single nucleotide polymorphisms (Kwo...

Claims

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Application Information

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Patent Type & Authority Patents(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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