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Mito Typing

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Submitted By slet
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Because Mitochondrial DNA (mtDNA) is abundantly available in a cell (about 1000 copies per cell) and has the ability to be used for successful DNA testing of degraded DNA, shed hairs and tracing maternal lineage, researchers are aggressively exploring its use over nuclear DNA. However, sequencing of the mtGenome of some individuals can be difficult due to the fact that traditional sequencing methods targets only about 610 base pairs in hypervariable regions 1 & 2 which in some instances a portion of the population in particular Caucasians, share the same or very similar hypervariable regions. In previous studies by Coble et. al., single nucleotide polymorphisms (SNPs) were discovered in the control and coding regions of the mtGenome that can be used to discriminate between samples that have a common HV1/HV2 sequence. To type these SNP sequence, Vallone and his team used the multiplex SNaPshot chemistry assay where allele-specific primer binds to a complimentary SNP template in the presence of fluorescently labeled ddNTps, buffer and polymerase. The primer is extended by one base unit as the fluorescently labeled ddNTps is added to the 3’ end. Additionally, a poly T-tail may be added to the 5’ end of each primer to allow electrophoretic mobility of the extension primers. Vallone used this particular assay because it is a single tube reaction that can be cycled on readily available laboratory instruments such as the ABI 310 and 3100 and it has the ability to genotype multiple SNP’s simultaneously. The result was the development of a multiplex panel “A” that targets 11 SNP sites that could be used to differentiate between individuals that share the same or similar HV1/HV2 type which according to previous studies by Coble accounts for about 21% of the West European Caucasian population. Vallone tested the assay’s sensitivity as well as its performance with

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