To detect sequence variation in 105 Croatian individuals by the use of duplex polymerase chain reaction amplification of full-length hypervariable region I and II (HVI/HVII) products and subsequent hybridization to a linear array of 27 immobilized sequence-specific oligonucleotide (SSO) probes, which targets six regions within HVI and HVII, and two additional sites, 189 and 16093. Chelex-extracted bloodstains were used for amplification of HV regions. In all cases, a single robust amplification

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... was sufficient for immobilized 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 skeletal elements recovered from a mass grave. Using a panel of immobilized SSO probes, we have developed a rapid screening approach to mitochondrial DNA (mtDNA) haplotyping before direct sequence analysis. We established a reference sequence database of mtDNA haplotypes for 105 randomly selected Croatian individuals. Fifty different mitotypes were observed (33 unique). The most frequent mitotypes occurred 18 times or approximately 17.1% [111111 189 (A) 16093 (T)] and 11 times or approximately 10.5% [131111 189 (A) 16093 (T)]; all other mitotypes occurred 5% or less. The corresponding genetic diversity value for this database was approximately 0.952. The usefulness of establishing an mtDNA reference database with immobilized SSO probe testing has been demonstrated by determining the strength of a match comparison obtained for one skeletal element and a corresponding maternal reference from 18 specimens recovered from a mass grave. The sequence variation detected by the panel of immobilized SSO probes is sufficiently diverse to be used for identification of human skeletal remains from mass graves. The immobilized SSO typing strip targets polymorphic regions within HVI and HVII and is a useful identification tool for mass grave and mass disaster analysis, as well as for criminal casework testing.