DNA concentration was measured with GeneQuant pro spectrophotometer (Amersham Biosciences, Cambridge, England) at λ = 260 nm from 10 μl of total DNA volume. Purity of DNA was

assessed using the ratio of OD260 nm/280 nm. Samples were amplified using ABI 9700 thermocycler (Applied Biosystems, Foster City, CA, USA). STR genotyping was performed using PowerPlex16™ System PCR Amplification Kit (nDNA; Promega Corporation, Madison, WI, USA) version 3.1. Y-STR genotyping was conducted using AmpFlSTR® Yfiler™ Amplification Kit (Applied Biosystems, Foster City, CA, USA). Polymerase chain reaction (PCR) was carried out according to the manufacturer’s instructions. Fluorescence detection of genotypes was performed with ABI Prism® 3100-Avant Genetic Analyzer and by using Data Collection v.2.0, and Gene Mapper ID v.3.2 analysis software (Applied Biosystems, Foster City, USA). For mtDNA analysis we amplified click here HV1 (Primer L15996: 5′-CTC CAC CAT TAG

CAC CCA AAG C-3′; Primer H16401: 5′-TGA TTT CAC GGA GGA TGG TG-3′) and HV2 (Primer L29: 5′-GGT CTA TCA CCC TAT TAA CCA C-3′; Primer H389: Sirolimus 5′-CTG GTT AGG CTG GTG TTA GG-3′) regions. Considering that nucleotide positions within the mtDNA are numbered from 1 to 16,569 using the L-strand from control region, HV1 region spans positions 16,024 to 16,365 (342pb) and HV2 covers positions 73 to 340 (268pb). PCR products of mtDNA were purified from residual primers with Exonuclease I (EXO I; Amersham Biosciences – E70073Z; GE HealthCare©) and Shrimp Alkaline Phosphatase (SAP; Amersham Biosciences – E7009; GE HealthCare©), and sequenced directly by cycle sequencing. Hyper variable segments were sequenced with BigDye Terminator Cycle Sequencing kit from Applied Biosystems on ABI PRISM™ 3100-Avant DNA sequencer. ABI PRISM™ 3100-Avant Genetic Analyzer

was used for separation and detection of the fluorescence-labelled 4��8C chain termination products. The sequences of mtDNA were manually checked using CHROMAS18 and aligned with CLUSTAL-X.19 DNA profiles obtained from the teeth of the deceased were compared to the DNA profiles of reference samples obtained from close relatives. Relatives’ genomic DNA was extracted from leucocytes by a standard method.20 DNA analyses of relatives were performed as described above. This project was approved by the Research Ethics Committee of the Pontifical Catholic University of Rio Grande do Sul (Tel. +55 51 33203345; protocol #1107/05) and the consent or assent to take part in this study was obtained from Forensic Laboratory of Instituto-Geral de Perícias of Rio Grande do Sul, Brazil. Allele identification was carried out with Gene Mapper ID software version 3.2 using ABI Prism 3100 from Applied Biosystems. Comparisons between the results obtained from the different protocols were examined using ANOVA or ANOVA followed by Student Newman’s Keul Post Hoc and Pearson’s correlation (SPSS software package for Windows version 13.0; SPSS, Inc.). A P value of <0.