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

2. 50 cases between

3. BONE SAMPLE PREPARATION
Lab preparation
Sampling
Cleaning
Pulverization
Weighing

4. - 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)

5. 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

6. Cleaning 10 ml bleach invert 20 sec, discard 10 ml dH2O1x
10 ml bleach
invert 20 sec, discard 3x
10 ml dH2O 2x
10 ml absolute ethanol

7. 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

8. 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 !!

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

10. 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

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

12. 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® YM ml NMWL
(nominal molecular weight limit)
Centricon® ml, NMWL
Amicon® Ultra ml, NMWL NMWL

13. PCR & post-PCR
Quantification
PCR
STR analysis

14. 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).

15. 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

16. PCR - II
MiniElute PCR purification kit

17. 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

18. 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.

19. Thank you!