CHALLENGING ASPECTS IN BONE TISSUE FORENSIC TESTING Dr. Cristian Bogdan Iancu Genetics Laboratory, NILM Bucharest

50 cases between 2008-2012

BONE SAMPLE PREPARATION Lab preparation Sampling Cleaning Pulverization Weighing

- Removing soft tissue - Lab preparation - Removing soft tissue - Mechanical removal of flesh+ Bacterial maceration (cold/warm) “Cooking”(boiling water, microwave) Chemical maceration (5%KOH, HClO, H2O2) Enzymatic maceration (pepsin, trypsin, laundry detergent) Intervertebrate maceration (dermestid beetles)

Sampling - cutting - Dry the sample Wire brush – removal of dirt Cut the sample Sand down about 2 mm of bone surface Cut into smaller pieces

Cleaning 10 ml bleach invert 20 sec, discard 10 ml dH2O 1x 10 ml bleach invert 20 sec, discard 3x 10 ml dH2O 2x 10 ml absolute ethanol

Pulverization Weighing - cryogenic mill - Best way of grinding (at low temperature) Easy cleaning Constant supply of liquid nitrogen NEEDED Expensive Weighing Retsch Mixer Mill MM 3xx

Recommendations Pulverized bone handling Record the mass make aliquots for extractions 1 + 2 store at +4⁰C for extraction within a week store at -20⁰C for extraction within months or cover with ethanol and store in a cold place Clear all surfaces, equipment, tools with 5-10% bleach solution, autoclaving and UV not effective !!

Post-extraction clean-up DNA EXTRACTION Decalcification DNA extraction Post-extraction clean-up

Decalcification general rule 1 g powder = 40 ml EDTA pH=8.0 aprox. 3 days at 4⁰C, in a rotor-shaker centrifugation 3-5 min at 4000g discard supernatant

DNA extraction PrepFiler® BTA Forensic DNA (Applied Biosystems) magnetic particles coated with polymer 4 aliquots x 50-60 mg o/n digestion standard protocol for hard tissue 50 μl elution Quick Gene Tissue Kit (Fuji Film) polymer porous membrane 4 aliquots x 50-60 mg o/n digestion special protocol for hard tissue 50 μl elution

Post-extraction clean-up Inhibitors Humic and fulvic acids, tannins – yellow to brown staining of the extracted DNA (detectable by fluorescence) Collagen - major component of organic material - coextracted only if digested bone powder is used for extraction. Ethanol precipitation with LPA (linear polyacrylamide) Microcon® YM100 - 0.5 ml 100.000 NMWL (nominal molecular weight limit) Centricon® 30 - 2.5 ml, 30.000 NMWL Amicon® Ultra 4 - 4 ml, NMWL 10-50.000 NMWL

PCR & post-PCR Quantification PCR STR analysis

Quantification DNA amount evaluated - spectrofluorimetric method using PicoGreen® dye (Molecular Probes – Life Technologies) and NanoDrop™ 3300 (Thermo Scientific) Specific human DNA + presence of PCR inhibitors evaluation - Quantifiler® Human DNA Quantification Kit (Applied Biosystems – Life Technologies, USA).

PCR - I Samples are amplified in duplicates - AmpFISTR® NGM™ PCR Amplification Kit (30 cycles)– Veriti Thermal Cycler - AmpFlSTR® MiniFiler™ PCR Amplification Kit (30 cycles) GeneAmp® 9700 Thermal Cycler - AmpFISTR® Identifiler®Plus PCR Amplification Kit (29 cycles)– Veriti Thermal Cycler - AmpFISTR® Yfiler® PCR Amplification Kit (30 cycles) GeneAmp® 9700 Thermal Cycler

PCR - II MiniElute PCR purification kit

STR analyis 3130xl Genetic Analyzer – POP7 polymer partial profiles, unbalanced peaks and allelic drop-out Consensus DNA profile from replicates Beware of contamination! 3130xl Genetic Analyzer – POP7 polymer 3100 Avant Genetic Analyzer – POP4 polymer STR profiles are analyzed using GeneMapper® ID Software v 3.2

Conclusions Bone tissue investigation requires a complex preparation step compaired to other samples The preparation step is essential for further genetic investigation; STR analysis have to take into account the risk of contamination or amplification artefacts.

Thank you!