Snp-sensitive "on/off" Switch For Detection Of Snp Loci In Mitochondrial Dna Coding Region

ObjectiveTo establish a reformed allele-specific PCR, which combined phosphorothioate-modified primers with high fidelity DNA polymerase. To apply this technique in detecting mt-SNPs for population genetics and investigate the allelic frequencies and haplotypes of four mt-SNP loci in Chinese Jiangsu Han population. Methods1 We used the mtDNA 10400 locus to design unmodified and 3′phosphorothioate-modified allele-specific primers for PCR, which was performed using polymerases with and without 3′exonuclease activities. The effects on primer-extension were evaluated by 2% agarose gel electrophoresis.2 For mtDNA-SNP loci C12705T, G8701A, G8584A, C10400T, two allele-specific reverse primers with 4 bases different in size and a common forward primer were designed for every SNP in detecting. Human mtDNA samples were obtained from 60 unrelated individuals in Chinese Jiangsu Han population by Chelex-100 method. DNA was amplified with PCR using high fidelity Pfu DNA Polymerase. The genotyping of SNPs was determined by the two allele-specific fragments different in size after polyacrylamide gel and silver staining. Typing results were confirmed by direct sequencing.Results1 With the use of high fidelity Pfu DNA polymerase having 3′to 5′exonuclease activities and 3′phosphorothioate-modified allele-specific primers, we improved the specificity of AS-PCR greatly and reduced false positives remarkably in our study.2 The different mtDNA-SNP loci comprised a single band with different size respectively. Typing results were proved to be robust and reliable in 60 unrelated individuals, which were completely consistent with those by direct sequencing. The allelic frequencies of C12705T, G8701A, G8584A, C10400T were 0.37/0.63, 0.47/0.53, 0.83/0.17 and 0.53/0.47 respectively. A total of 6 different haplotypes was identified and the genetic diversity reached 0.7438.ConclusionWe successfully designed and established the SNP-sensitive"on/off"switch consisting high fidelity DNA polymerase and 3′phosphorothioate-modified allele-specific primers, which was proved to be accurate and superior when applying in detecting mt-SNPs for population genetics. The"on/off"switch is a simple, rapid, economic and efficient method, which will be very powerful in association studies by single nucleotide polymorphism identification and mtDNA forensic and population genetics.