Abstract: Objective To develop a multiplex allele-specific PCR （AS-PCR） assay with three-color fluorescence 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 into 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 samples from 200 unrelated individuals were analyzed by AS-PCR and capillary electrophoresis. Three random 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 successfully. 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 10 μL. 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.

Key words: forensic genetics, polymorphism, single nucleotide, alleles, polymerase chain reaction, haplotype