Fluorescent Duplex Allele-Specific PCR and Amplicon Melting for Rapid Homogeneous mtDNA Haplogroup H Screening and Sensitive Mixture Detection Harald Niedersta ¨ tter*, Walther Parson Institute of Legal Medicine, Innsbruck Medical University, Innsbruck, Austria Abstract Background: 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. Methodology/Principal Findings: A duplex ARMS assay amplifying either the ancestral mtDNA 2706G allele (non-hg H samples) or the derived 7028C allele (hg H samples) in the presence of SYBR Green fluorescent reporter dye was developed and characterized. Product detection, allele calling, and hg inference were based on the amplicon-characteristic melting- point temperatures obtained with on-line post-PCR fluorescent dissociation curve analysis (DCA). The analytical window of the assay covered at least 5 orders of magnitude of template DNA input with a detection limit in the low picogram range of genomic DNA. A set of forensically relevant test specimens was analyzed successfully. The presence of mtDNA mixtures was detected over a broad range of input DNA amounts and mixture ratios, and the estimation of allele proportions in samples with known total mtDNA content was feasible with limitations. A qualified DNA analyst successfully analyzed ,2,200 DNA extracts within three regular working days, without using robotic lab-equipment. By performing the amplification on-line, the assay also facilitated absolute mtDNA quantification. Conclusions: Although this assay was developed just for a particular purpose, the approach is general in that it is potentially suitable in a broad variety of assay-layouts for many other applications, including the analysis of mixtures. Homogeneous ARMS-DCA is a valuable tool for large-volume studies targeting small numbers of single nucleotide polymorphisms (SNPs). Citation: Niedersta ¨tter H, Parson W (2009) Fluorescent Duplex Allele-Specific PCR and Amplicon Melting for Rapid Homogeneous mtDNA Haplogroup H Screening and Sensitive Mixture Detection. PLoS ONE 4(12): e8374. doi:10.1371/journal.pone.0008374 Editor: Manfred Kayser, Erasmus University Medical Center, Netherlands Received June 26, 2009; Accepted November 23, 2009; Published December 18, 2009 Copyright: ß 2009 Niedersta ¨tter, Parson. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: harald.niederstaetter@i-med.ac.at Introduction 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)popu- lation, 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]). At large, individuals display - within the detection limits associated to current sequencing technology - only a single mtDNA haplotype (homoplasmy), but individuals can also carry more than just one mtDNA variant, a state known as heteroplasmy (see e.g. [5,6]). With increasing numbers of analyzed samples as well as improvements in detection chemistries and instrumentation it became clear that heteroplasmy occurs (also in individuals unaffected by mitochondrial disorders) at considerably higher rates PLoS ONE | www.plosone.org 1 December 2009 | Volume 4 | Issue 12 | e8374