A simple and rapid modified--new method for SNP typing by fragment length discrepant allele specific PCR.
- Author:
Dai-xin HUANG
1
;
Qing-en YANG
;
Gui-sen ZHAO
Author Information
1. Faculty of Forensic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. huangdaixin@hotmail.com
- Publication Type:Journal Article
- MeSH:
Alleles;
Base Pair Mismatch/genetics*;
DNA/genetics*;
DNA Primers;
Electrophoresis, Polyacrylamide Gel;
Genotype;
Humans;
Polymerase Chain Reaction;
Polymorphism, Restriction Fragment Length;
Polymorphism, Single Nucleotide;
Sensitivity and Specificity;
Sequence Analysis, DNA/methods*
- From:
Journal of Forensic Medicine
2005;21(1):11-14
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To establish a new method for single nucleotide polymorphism (SNP) typing based on allele specific PCR: fragment length discrepant allele specific PCR (FLDAS-PCR), and study the influence on specific extension by introducing a mismatch at the third or fourth 3'-terminal base of allele specific primers.
METHODS:For SNP loci rs759117 and rs760887, two allele specific forward primers, with different length and a mismatch introduced at the third or fourth 3'-terminal base, and a public reverse primer were designed for SNP typing. The genotyping of SNP was determined by the two allele specific fragments different in size after polyacrylamide gel and silver staining.
RESULTS:The different homozygote genotypes comprised a single band with different size respectively, and the heterozygote genotypes comprised two bands. Typing results were completely consistent with those by direct sequencing. Non-specific primer extension was decreased remarkably after introducing a mismatch at the third or fourth 3'-terminal base of allele specific primers, and the stringency of PCR reaction was cut down.
CONCLUSION:FLDAS-PCR is a simple, rapid and efficient new method for SNP typing. During FLDAS-PCR, specific primers with a mismatch at the third or fourth 3'-terminal base have more power to identify two alleles.
