[Multiplexed mutagenically separated PCR assay for rapid detection of SNP loci in mitochondrial DNA coding region].
OBJECTIVE
To develop a multiplexed mutagenically separated PCR (MS-PCR) for single nucleotide polymorphism (SNP) loci typing in mitochondrial DNA coding regions and to study the applications in investigating the allele frequencies and haplotypes of four SNP loci in mitochondrial DNA coding regions in Chinese Chengdu Han population.


METHODS
Four SNP loci C12705T, A8701G, G8584A and C10400T, two allele specific forward primer with 4 bases different in size and a common reverse primer were designed for SNP typing. The primers simultaneously were amplified in a single tube. The genotyping of SNPs was determined by the two allele specific fragments different in size after polyacrylamide gel and silver staining.


RESULTS
The different SNP loci comprised a single band with different size respectively. Typing results were completely consistent with those by direct sequencing. The allelic frequencies of C12705T, A8701G, G8584A and C10400T were 0.3813/0.6187, 0.4813/0.5187, 0.8250/0.1750 and 0.4938/0.5062 respectively. A total of 6 different haplotypes was identified and the genetic diversity reached 0.7137.


CONCLUSIONS
Multiplexed MS-PCR is a simple, rapid, accurate and efficient method for SNP typing, which will be very powerful for SNPs in the database establishing of mitochondrial DNA coding regions, the testing of forensic and population genetics research.
Ran
Peng
P
College of Basic Medicine and Forensic Medicine, Sichuan University, Chengdu, Sichuan, 610041 PR China.
Li
Ying-bi
YB
Yan
Jing
J
Zhang
Bei-lei
BL
Zhang
Ji
J
Shan
Hua-xin
HX
Ye
Zhi-peng
ZP
Hou
Yi-ping
YP
Zhang
Lin
L
Liao
Miao
M
Wu
Jin
J
D001483:Base Sequence; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics*; D005787:Gene Frequency; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D017422:Sequence Analysis, DNA
D004272:DNA, Mitochondrial