Characterization of mtDNA SNP typing and mixture ratio assessment with simultaneous real-time PCR quantification of both allelic states

Abstract

We performed a study on the forensic utility of allele-discriminatory quantitative real-time PCR (rtPCR) using Minor Groove Binder TaqMan probes, targeting the highly variable mitochondrial single nucleotide polymorphism 16519T/C. The apparent single-cycle PCR efficiency was virtually 100% for both 16519 alleles. The allele designations made by rtPCR were concordant with the results obtained in a previous study by sequencing analysis. In heteroplasmic samples, minor allele proportions down to 9% were unambiguously detected and quantified. The variation in allele proportion estimates was essentially the same within and between different rtPCR runs, and the differences between total copy number estimates found for rerun samples were comparable to those found with non-allele-discriminatory quantitative rtPCR assays.

First successful assay of Y-SNP typing by SNaPshot minisequencing on ancient DNA

“…The allele designation is then possible by separating extended products and detecting the fluorescence by capillary electrophoresis. Currently, various multiplex minisequencing-based assays have been successfully validated for the analysis of mitochondrial DNA [14][15][16][17], autosomes [18,19], the Y-chromosome [20][21][22][23][24], Duffy and ABO group system [25,26], and the melanocortin 1 receptor gene as indicator of red hair phenotype [27]. All these studies were consistent with the fact that this SNP typing methodology is robust, reliable, and extremely sensitive.…”

Population differences of two coding SNPs in pigmentation-related genes SLC24A5 and SLC45A2

“…However, such methods are only useful for the purposes of identification when the suspect has been identified or when the genotype or pattern matches one already present on the DNA database. In addition to these markers, SNPs are now useful in the forensic field because the simplicity and population specificity of SNPs allow inferences to be drawn about the population origin of interest for forensic purposes [3,4,11,20,16]. Recently, research targeted toward identifying specific genetic markers that may provide further information on the physical characteristics of the individuals from which the sample came [2] has identified markers such as AIMs.…”

Forensic mass screening using mtDNA

“…Population studies published in the literature [18,25,29] and databases [4,21,26,27,38] may be helpful to obtain information on whether a specific haplotype is rare enough to perform a screening. Mention should also be made of the opportunity to subtype mtDNA of a more frequent haplotype with regard to HV1 and HV2, using further d-loop polymorphisms [17,19,24,36] and even SNPs in the coding area [6,8,20,30]. Plus signs (+) indicate cleavage of a polymorphic restriction site in the amplicon a The existence of a non-polymorphic restriction site…”