[A simple and rapid modified--new method for SNP typing by fragment length discrepant allele specific PCR].
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.


CONCLUSIONS
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.
Huang
Dai-xin
DX
Faculty of Forensic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. huangdaixin@hotmail.com
Yang
Qing-en
QE
Zhao
Gui-sen
GS
D000483:Alleles; D020137:Base Pair Mismatch / Q000235:genetics; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods
D017931:DNA Primers; D004247:DNA