Abstract
The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300–400 bp each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and more stable approach using shortened amplicons in the fragment range between 144 and 237 bp. Ten such amplicons were required to produce overlapping fragments that cover the entire human mtDNA control region. These were co-amplified in two multiplex polymerase chain reactions and sequenced with the individual amplification primers. The primers were carefully selected to minimize binding on homoplasic and haplogroup-specific sites that would otherwise result in loss of amplification due to mis-priming. The multiplexes have successfully been applied to ancient and forensic samples such as bones and teeth that showed a high degree of degradation.
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Acknowledgments
We would like to thank Volker Büchele, the Tiroler Landesmuseum Ferdinandeum, the Naturwissenschaftliche Sammlungen ‘‘Alpenzoo Innsbruck’’, and the‘‘Tierpark Hellabrunn’’ in Munich for kindly providing non-human samples. Anita Brandstätter, Anna König, Harald Niederstätter, and Florian Pitterl are acknowledged for their helpful assistance, comments, and discussion.
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Table S1
Amplification and sequencing primers for mtDNA mini-amplicons covering the entire mitochondrial control region (16024-576). (DOC 35 KB)
Table S2
Overview of the evaluation of primer specificity in a dataset of 1,544 mtDNA haplotypes. Samples that displayed mutations within the binding regions of one of the 20 PCR primers were amplified with the respective multiplex and sequenced. Four samples were carried through all processes as positive controls. hg, haplogroup; +, successful result; +/−, weak sequencing result, in some cases only single-strand sequence; −, no sequencing result. (XLS 27 KB)
Table S3
Species cross-reactivity of the two multiplexes on DNA of 20 animal species. −, no sequencing result; +, successful sequencing result sequencing a human sample; not found, sequence that could not be found through blast searches; Pan troglodytes and Rattus norwegicus Chr 1, species identified by blast search; MA, mini-amplicon. (XLS 18 KB)
Fig. S1
Frequency of polymorphic positions in HVS-I (upper panel) and HVS-II (lower panel). There were 5,173 sequences screened including mainly west European but also African and Asian sequence data to choose new primers. In this dataset, 521 positions showed at least one to nine mutations referring to the Cambridge Reference Sequence, 214 positions showed at least ten mutations, and 19 positions showed polymorphism in at least 10% of the sequences analyzed. Gray indicates all 19 positions that showed more than 10% mutation rate. (PDF 17 KB)
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Eichmann, C., Parson, W. ‘Mitominis’: multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples. Int J Legal Med 122, 385–388 (2008). https://doi.org/10.1007/s00414-008-0227-5
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DOI: https://doi.org/10.1007/s00414-008-0227-5