International Journal of Legal Medicine

, Volume 122, Issue 5, pp 385–388

‘Mitominis’: multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples

Original Article

DOI: 10.1007/s00414-008-0227-5

Cite this article as:
Eichmann, C. & Parson, W. Int J Legal Med (2008) 122: 385. doi:10.1007/s00414-008-0227-5


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.


mtDNA CRMini-ampliconsMultiplexForensic science

Supplementary material

414_2008_227_MOESM1_ESM.doc (35 kb)
Table S1Amplification and sequencing primers for mtDNA mini-amplicons covering the entire mitochondrial control region (16024-576). (DOC 35 KB)
414_2008_227_MOESM2_ESM.xls (26 kb)
Table S2Overview 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)
414_2008_227_MOESM3_ESM.xls (18 kb)
Table S3Species 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)
414_2008_227_MOESM4_ESM.pdf (17 kb)
Fig. S1Frequency 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)

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  1. 1.Institute of Legal MedicineInnsbruck Medical UniversityInnsbruckAustria