AS-PCR primer design method, gene polymorphism detection method and kit

Abstract

The invention relates to an AS-PCR primer design method, a gene polymorphism detection method and a kit. The method and kit are used for detection of a target sequence which probably includes an allelic variation area so as to determine the presence of an allele variant. The AS-PCR primer design method comprises the following steps: (i) designing an AS-PCR primer for a target sequence including an allelic variation area; (ii) modifying the AS-PCR primer designed in step (i) to provide the last base at the 3' end of the AS-PCR primer with a group capable of shielding direct extension of the AS-PCR primer. Compared with traditional AS-PCR with single site detection capability, the gene polymorphism primer design method, the detection method and the kit of the invention can realize fuzzy recognition of mutation sites, can integrate a probe with the primer as one, and thus save the primer synthesis cost and design time; the design is convenient and understandable, and is easy to grasp.

Description

technical field [0001] The invention belongs to the field of nucleic acid diagnostic reagents, in particular to an allele-specific polymerase chain reaction (AS-PCR) primer design method. The present invention also relates to an AS-PCR gene polymorphism detection method and kit, which are used to detect target sequences that may (that is, suspect) contain allelic variation regions to determine allelic variation regions. body exists. Background technique [0002] Allele-specific oligonucleotide analysis (allele-specific oligonucleotide, ASO) is a hybridization-based mutation detection technique, commonly used to detect known mutations. Design an oligonucleotide fragment of, for example, 15-20 bp, which contains the site of a known mutation, and use it as a probe. When hybridizing with sample DNA immobilized on the membrane, the difference of one base will cause Tm The value drops by 5-7.5°C, so by strictly controlling the hybridization conditions, it is possible to identify...

AI Technical Summary

Problems solved by technology

[0009] First, because the AS primers are designed for the mutant type to be tested, the traditional AS-PCR technology can only detect known polymorphic sites, but cannot detect unknown mutation types

[0010] Second, the traditional AS-PCR technology in the detection of amplification results, one is the use of electrophoresis analysis method, this method is complex and time-consuming, high cost, and the technology is difficult to popularize; the other is the use of real-time fluorescent quantitative PCR, which Additional synthesis of fluorescent probes labeled with fluorophores and fluorescent quenchers is also required

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