CRAS-PCR detection method of single base mutation of gene

A single base variation and detection method technology, applied in the fields of life sciences and biology, can solve the problems of lack of reliability in genotype detection, inability to completely suppress non-specific amplification, and false positive detection results

Inactive Publication Date: 2014-11-26
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In summary, the thermodynamic characteristics of heat-resistant DNA polymerases are one of the root causes of non-specific amplification of AS-PCR products. Under the pr

Method used

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  • CRAS-PCR detection method of single base mutation of gene
  • CRAS-PCR detection method of single base mutation of gene
  • CRAS-PCR detection method of single base mutation of gene

Examples

Experimental program
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Effect test

Embodiment example 1

[0050] Implementation case one: TPMT *2 Genotype detection

[0051] 1. Design and synthesis of allele-specific scorpion primers

[0052] according to TPMT *2 (dbSNP ID: rs1800462; G238C) genotype nucleic acid sequence characteristics, respectively designed allele-specific scorpion primers matching its wild-type and mutant sequences, wherein, SEQ No. 1 and SEQ No. 2 and TPMT*2 (G238C) The reverse sequence is complementary, both are PCR amplification upstream primers, respectively used for specific amplification TPMT *2 (G238C) wild-type and mutant nucleic acid sequences. The 5'-end and 3'-end oligonucleotide sequences of the PCR blocker HEG in SEQ No.1 and SEQ No.2 are probe sequence region and primer sequence region respectively, and the effect of this PCR blocker is A common downstream primer (ie: SEQ No. 3) that prevents wild-type and mutant allele-specific scorpion primers from extending along the probe sequence. Among the above allele-specific scorpion primer sequen...

Embodiment example 2

[0068] Implementation case two: TPMT *3B and TPMT *Detection of 3C genotype

[0069] 1. Design and synthesis of single-stranded allele-specific scorpion primers and their common primers

[0070] according to TPMT *3B(dbSNP ID: rs1800460; G460A) and TPMT *3C (dbSNP ID: 1142345; A719G) genotype nucleic acid sequence characteristics, designed wild-type and mutant-matched allele-specific scorpion primers and their common primers. TPMT *3B The allele-specific scorpion primers matched by the wild type and the mutant type are Seq No. 4 and Seq No. 5, both of which are upstream primers, respectively labeled with HEX and FAM fluorescent reporter groups; the common primer is the downstream primer Seq No. 6. TPMT *The allele-specific scorpion primers matching the wild type and mutant type of 3C are Seq No. 8 and Seq No. 9, both of which are downstream primers, respectively labeled with HEX and FAM fluorescent reporter groups; the common primer is the upstream primer Seq No. 7. ...

Embodiment example 3

[0090] Implementation case three: BRAF Gene mutation detection at the 600th codon

[0091] 1. Design and synthesis of double-stranded allele-specific scorpion primers and their common primers

[0092] according to BRAF All mutations in the second base of the 600th codon (V600; GTG) were designed to target the wild type (V600; GTG) and V600E (GTG>GAG), V600A (GTG>GCG), V600G (GTG> GGG) and other three mutant double-stranded allele-specific scorpion primer probe-primer monomers. The probe-primer monomer targeting wild type (V600; GTG) is SEQ No. 11, the probe-primer monomer targeting mutant V600E (GTG>GAG) is SEQ No. 12, targeting mutant V600A The probe-primer monomer for (GTG>GCG) is SEQ No. 13, and the probe-primer monomer for targeting mutant V600G (GTG>GGG) is SEQ No. 14. The 5'-ends of SEQ No. 11~14 are respectively labeled with CY5, FAM, HEX and Texas Red fluorescent reporter groups, and between the probe sequence region and the primer sequence region is the PCR bl...

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Abstract

The invention provides a CRAS-PCR detection method of single base mutation of a gene. The detection method uses single-brand or double-brand allele specific scorpion primers capable of specifically identifying the wild and mutant nucleic acid sequences of the single base mutation, and the common reference of the allele specific scorpion primers. The different genotypes of the single base mutation can be qualitatively and quantitatively detected in a single PCR reaction tube by analyzing the type and intensity of fluorescence signals generated by all competitive allele specific scorpion primers in a PCR amplification process, the detection results are accurate and reliable, have good linear range and sensitivity, and do not comprise false positive results caused by non-specific amplification and false negative results can be completely avoided.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and is a detection method for gene single base variation (ie: genotype), which can accurately detect gene single nucleotide polymorphism and single base mutation and other genotypes . Background technique [0002] Single nucleotide polymorphisms (single nucleotide polymorphisms, SNP) refer to the sequence polymorphisms caused by the transformation, transversion, deletion and insertion of a specific base in the genomic DNA sequence, and any The population frequency of the allele is not less than 1%. SNPs are widely distributed in the human genome, and some SNPs can directly affect protein function and genetic stability. The analysis of genomic SNPs is an important clue for the study of individual, pedigree and ethnic characteristics, disease risk prediction and prevention, and individualized treatment of various diseases. It is estimated that about 10 5 A SNP molecular marker was ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2531/113C12Q2525/301C12Q2535/125
Inventor 黄庆府伟灵张阳杨昭黄君富夏涵
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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