ABSTRACT
Three SNaPshot multiplex assays were developed to test 23 coding region single nucleo-tide polymorphisms (SNPs) and one control region SNP outside hypervariable regions (HVR) Ⅰ and Ⅱ, which was aimed at increasing the discrimination power of the mitochondrial DNA (mtDNA) typing in forensic casework, and confirming haplogroup assignments of mtDNA profiles in both hu-man population studies and medical research. The selected SNPs targeted the East Asian phylogeny. These multiplex assays were validated by comparing with the sequencing analysis of samples chosen randomly. The mtDNA variations of 100 unrelated individuals from the Wuhan population in China were examined and classified into 3 i haplotypes, and the haplotype diversity was estimated to be 0.952. The multiplex SNaPshot method is rapid and robust, and suitable for large-scale screening studies of mtDNA variability.
ABSTRACT
Several technologically sophisticated high-throughput techniques have been recently developed for the study of human single nucleotide polymorphisms and the diagnosis of point mutations in human diseases. However, there is also a need for simple and inexpensive techniques suitable for clinical services and small research laboratories. Minisequencing meets the latter requirements. It is simple, non-radioactive and can be easily multiplexed by adding oligonucleotide tails of increasing size to the sequencing oligonucleotide primers. To optimize the minisequencing protocol, we designed a test multiplex system capable of typing simultaneously 12 different human autosomal single nucleotide polymorphisms. We discovered that the quality of minisequencing primers and the careful selection of the tail sequences were especially critical for success. This optimized protocol permits rapid genotyping at low cost and can serve as a blueprint for the creation of multiplex minisequencing systems suitable to virtually any typing application in population studies and medical genetics.