AS-PCR assay for 20 mtDNA SNP typing and haplotype frequency.
- Author:
Yan-Chai NIE
;
Chen ZHANG
;
Ya-Nan LIU
;
Jiang-Ping HUANG
;
Hai-Tao JIAO
;
Dan WU
;
Huai-Gu ZHOU
- Publication Type:Journal Article
- MeSH:
Alleles;
DNA;
DNA Primers;
DNA, Mitochondrial/analysis*;
Electrophoresis, Capillary;
Haplotypes;
Humans;
Mitochondria;
Polymerase Chain Reaction/methods*;
Polymorphism, Single Nucleotide;
Sequence Analysis, DNA
- From:
Journal of Forensic Medicine
2014;30(2):96-109
- CountryChina
- Language:Chinese
-
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 microL. 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.
