Subtyping mtDNA haplogroup H by SNaPshot minisequencing and its application in forensic individual identification

Abstract

Sequence variation of the hypervariable segments (HVS) I/II of mitochondrial DNA (mtDNA) and the haplogroup affiliation were determined in a sample of 271 Italian subjects. This analysis showed that 42% of the individuals could be ascribed to H, the most frequent haplogroup in European Caucasian populations. This fraction was then screened for specific single nucleotide polymorphisms located in the coding region to identify H subclades H1-H15. We set up two multiplex polymerase chain reactions and specific SNaPshot assays to investigate the frequency distribution of these subgroups in our population sample and to examine their usefulness in discriminating among commonly shared HVS I/II sequences. This allowed the assignment of a large portion of the mtDNAs ( approximately 70%) to specific subhaplogroups, with H1 and H5 being the most represented. About two-thirds of the individuals sharing common HVS I/II sequences were subdivided and ascribed to specific H subhaplogroups with a significant reduction of the frequencies of the most common mtDNA haplotypes. Haplogroup H subtyping could thus be extremely useful in forensic identification when many samples have to be analysed and compared, avoiding excessive time-consuming and labor-intensive sequencing analysis.

Simultaneous Amplification, Detection, and Analysis of Common Mutations in the Galactose-1-Phosphate Uridyl Transferase Gene

“…The primers were extended by adding a single fluorescently labeled ddNTP to its 3Ј-end. [22][23][24][25] Fluorescent dyes attached to each ddNTP are as follows: dR6G (green) for A, dTAMRA (yellow, which appears black on the electropherogram) for C, dR110 (blue) for G, and dROX (red) for T. The reactions were cycled with the following conditions: 25 cycles at 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 30 seconds. Samples were treated with one unit of shrimp alkaline phosphatase at 37°C for 45 minutes, followed by heat inactivation of enzyme at 80°C for 15 minutes.…”

Low volume amplification and sequencing of mitochondrial DNA on a chemically structured chip

“…The detection and analysis of mitochondrial SNPs allows the assignment of individuals to specific mitochondrial haplogroups [6,12].…”

Molecular Epidemiology of Mycoplasma pneumoniae: Genotyping Using Single Nucleotide Polymorphisms and SNaPshot Technology

“…The SNaPshot assay is currently widely used in forensic and population genetics (26,28) and has recently raised interest in microbiological research (25,27). A major advantage of this technology is that the eight SNP sites can be simultaneously analyzed, significantly reducing the cost and analysis time, as the automated fluorescent capillary electrophoresis analysis of minisequencing products requires only 30 min.…”

“…This approach is based on the single-base extension (SBE) of an unlabeled minisequencing primer that anneals one base upstream of the relevant SNP using a fluorochrome-labeled dideoxynucleotide (ddNTP). The allelic state is subsequently determined after separation of the extension products and detection of their fluorescence and size using capillary electrophoresis (25)(26)(27)(28).…”