Population variation of human mitochondrial DNA hypervariable regions I and II in 105 croatian individuals demonstrated by immobilized sequence-specific oligonucleotide probe analysis

Aim. To detect sequence variation in 105 Croatian individuals by the use of duplex polymerase chain reaction amplification of full-length hypervariabl e region I and II (HVI/HVII) products and subsequent hybridization to a lin ear array of 27 immobilized sequence-specific oligonucleotide (SSO) probes, which targets six regions within HVI and HVII, and two additional sites, 1 89 and 16093. Methods. Chelex-extracted bloodstains were used for amplification of HV reg ions. In all cases, a single robust amplification was sufficient for immobi lized SSO probe typing and subsequent direct sequence analysis for both HVI and HVII. This method, suitable for a range of forensic samples (including shaft portions of single hairs), was also applied to the analysis of 18 sk eletal elements recovered from a mass grave. Using a panel of immobilized S SO probes, we have developed a rapid screening approach to mitochondrial DN A (mtDNA) haplotyping before direct sequence analysis. Results. We established a reference sequence database of mtDNA haplotypes f or 105 randomly selected Croatian individuals. Fifty different mitotypes we re observed (33 unique). The most frequent mitotypes occurred 18 times or s imilar to 17.1% [111111 189 (A) 16093 (T)] and 11 times or similar to 10.5% [131111 189 (A) 16093 (T)]; all other mitotypes occurred 5% or less. The c orresponding genetic diversity value for this database was similar to0.952. The usefulness of establishing an mtDNA reference database with immobilize d SSO probe testing has been demonstrated by determining the strength of a match comparison obtained for one skeletal element and a corresponding mate rnal reference from 18 specimens recovered from a mass grave. Conclusion. The sequence variation detected by the panel of immobilized SSO probes is sufficiently diverse to be used for identification of human skel etal remains from mass graves. The immobilized SSO typing strip targets pol ymorphic regions within HVI and HVII and is a useful identification tool fo r mass grave and mass disaster analysis, as well as for criminal casework t esting.