AS-PCR Assay for 20 mtDNA SNP Typing and Haplotype Frequency / 法医学杂志

ABSTRACT

Objective To develop a multiplex allele-specific PCR (AS-PCR) assay with three-color fluo-rescence labeling for mitochondrial DNA (mtDNA) SNP typing. Methods Based on the principle of AS-PCR, the primer sets were designed for 20 SNP located on the coding region of mtDNA and divided in-to 2 groups labeled with FAM and HEX fluorescence, respectively. A primer set included two forward (reverse) allelic specific primers with different sizes and a generic reverse (forward) primer. Blood sam-ples from 200 unrelated individuals were analyzed by AS-PCR and capillary electrophoresis. Three ran-dom samples at least for each SNP site were examined and verified by direct sequencing. The haplotype frequency was investigated. Results Distinct electropherograms of 200 blood samples were obtained suc-cessfully. The typing results of direct sequencing were identical to those obtained from AS-PCR. The minimum detectable DNA concentration was 0.2 pg under the system of 10μL. The sensitivity of the DNA concentrations ranged from 0.5 to 5 pg. The 200 individuals were assigned into 15 haplotype, and the haplotype diversity was 0.906 0. Conclusion AS-PCR is a simple, rapid and efficient method for mtDNA SNP typing, and can be applied to forensic practice.