Research Article: Fluorescent Duplex Allele-Specific PCR and Amplicon Melting for Rapid Homogeneous mtDNA Haplogroup H Screening and Sensitive Mixture Detection

Date Published: December 18, 2009

Publisher: Public Library of Science

Author(s): Harald Niederstätter, Walther Parson, Manfred Kayser.

Abstract: For large scale studies aiming at a better understanding of mitochondrial DNA (mtDNA), sequence variation in particular mt haplogroups (hgs) and population structure, reliable low-cost high-throughput genotyping assays are needed. Furthermore, methods facilitating sensitive mixture detection and relative quantification of allele proportions are indispensable for the study of heteroplasmy, mitochondrial sequence evolution, and mitochondrial disorders. Here the properties of a homogeneous competitive duplex allele specific PCR (ARMS) assay were scrutinized in the light of these requirements.

Partial Text: The human mitochondrial (mt) genome consists of a small circular chromosome comprising approximately 16,568 base pairs (bp; revised Cambridge reference sequence, rCRS, [1]). Besides the non-coding control region, the mt genome contains the genes for 22 tRNAs and two rRNAs required for intra-organellar translation of the 13 polypeptide encoding mtDNA genes. A diploid human cell (e.g. a nucleated blood cell) usually contains two copies of a particular nuclear marker or gene but hundreds to thousands of mt genomes [2], [3]. Therefore, genotyping of the highly polymorphic control region (or parts thereof) has become a standard tool in forensic genetics, when analyzing biological material containing insufficient amounts of amplifiable genomic DNA (e.g. shed hairs, stains that suffered heavy environmental stress, aged biological material) for the analysis of the highly informative nuclear DNA short tandem repeat markers. Due to its maternal mode of inheritance and the apparent lack of recombination, mtDNA forms stable lineages and haplogroups. Consequently, mtDNA testing does not provide definitive identification of individuals because all members of a maternal lineage are expected to match each other as long as no mutations occur. On the other hand, this makes mtDNA typing a superb tool for forensic human identification when no close relatives are available for comparison. As the weight of a mtDNA match between evidence- and reference-sample depends on the frequency of the found haplotype in the particular (sub)population, a large mtDNA database fulfilling high quality standards is needed for the calculation of the probability of a match by chance, being an alternative explanation for the found haplotype conformity (e.g. [4]).



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