DNA Typing and the Development of Methods for Determination of Degraded and Compromised Forensic Samples

1. DNA Typing and the Development of Methods for Determination of Degraded and Compromised Forensic Samples Bruce R. McCord Department of Chemistry Florida International University mccordb@fiu.edu www.fiu.edu/~mccordb

2. The Process of DNA Typing via the PCR

3. With 23 pairs of Chromosomes you can get Specific CSF1P0 TPOX D3S1358 FGA D5S818 D8S1179 THO1 vWA D7S820 D16S531 D13S317 D18S51 D21S11 AMEL Genotype The Random Match Probability for this profile in the FBI Caucasian population is 1 in 1.56 quadrillion (1015) Melanie McCord

4. The Problem: In the US an average of 115,000 rapes and attemped sexual assaults are reported each year. Another 250,000 are not reported There are also 16,500 murders and several million robberies Rape victim being examined by forensic nurse http://www.ojp.usdoj.gov/bjs/pub/pdf/rsarp00.pdf

5. The Issues • The current estimated backlog of untested forensic DNA samples is 540,000 • The number of untested rape kits nationwide is estimated to be 180,000 to 500,000 • 3. DNA is collected from criminals and data based in a system known as CODIS

6. But How To Process All This Data? Hundreds of thousands of samples? Lab Floors like a Darkroom! Fingers like an Iraqi voter ! Silver Stained Slab Gel?

7. Capillary Electrophoresis The alternative • Injection, separation, and detection are automated. • Rapid separations are possible • Peak information is automatically stored for easy retrieval.

8. Capillary Electrophoresis System Capillaryfilled withentangledPolymer Argon Ion Laser Capillary - + 5-20 kV Buffer Buffer (Sample) Deconvoluted Result

10. Separation Mechanism Electrophoretic flow DNA-- DNA-- DNA-- PDMA Polymer Structure (POP4, POP6)

11. The DNA molecules move through the polymer under the influence of the electric field and are separated low moderate high Follow the dancing DNA

12. R g V Ogston Sieving Entanglement Reptation ~ 0e-NC  ~1/N ~ f(1/CN)

13. Right Polymer, Right Voltage means: Drop Dead Beautiful Results !

14. Large Multiplex Kits provide Efficient and Rapid Analysis of Convicted Offender Samples WTC Disaster Jane Doe 231657 But what about degraded DNA ? Such samples present a special challenge Skeletal material being preped for extraction

15. DNA Degradation • polymer hydrolyzes • (nucleic acids break apart • Pyrimidinedimers • (bases X-link) • Chemical oxidation • (bases become unreadable)

16. DNA Degradation Note loss of intensity of larger alleles Powerplex 16 9947A Positive Control 0.250 ng/ 12.5 ul Bone Sample 2003.5.6 0.250 ng/ 12.5 ul

17. CSF1P0 THO1 CSF1P0 TH01 TPOX Miniplex 1 vs Powerplex 16

18. DNA typing of STRs on microfluidic chips A genotype in under a minute on a portable system Agilent Gene Chip

19. ABI MiniSTRs

20. Recovery of DNA from degraded Samples UT Forensic Anthropology Center Implications for Mass Disasters And Questions about Recovery of Ancient DNA

21. Application of Miniplexes to Casework • Skeletal remains found on April 7, 2002 on the bank of a stream • Caucasian Female, 40-60 years of age • State crime lab would not attempt nuclear DNA due to the fact the body had been in water at some point • A forensic artist produced a sketch based on age ranges, cranial features, and biological profile

22. In February of 2003, DNA extraction and amplification with the Miniplex sets were performed Low amounts of DNA yielded a profile for 12 CODIS loci covered by the Miniplexes Profile information was given to the forensic anthropologist and the coroner’s office October, 2004: the coroner was contacted by a woman who had seen the sketch on the internet and thought it looked like her mother Buccal swabs from the suspected daughter were taken and sent to the McCord lab at FIU Application of Miniplexes

23. Likelyhood Ratio = 7,611 Identify confirmed as a 54 year old woman missing since December 2000 Application of Miniplexes

24. Result Identified as Roberta Gile, Age 54 Missing since December, 2000

25. Application of MiniSTRs in bone/bone reassociation Yugoslavia Parsons et al, Forensic Science International: Genetics 1 (2007) 175–179

26. The Problem of Degradation vs Inhibition in DNA typing 0 ng 5 ng 10 ng 15 ng Degraded DNA Sample Humic Acid Inhibited DNA Sample Ski slope effect Less predictable effects Big Mini Powerplex 16 9947A Positive Control 0.250 ng/ 12.5 ul CSF1PO TH01 D7S820 FGA TPOX RFU Bone Sample 2003.5.6 0.250 ng/ 12.5 ul

27. The Issue: • With increasing interest in the forensic community in the interpretation of compromised samples and mixtures, we need to be able to better interpret electropherograms in court • We need to determine the relative effects of DNA degradation and inhibition on peak height ratios. • We need to understand the combinatorial effects of different inhibitors • We need to understand the environmental aspects of degradation and soil inhibition • We need to explore the interpretation of low level mixtures in the presence of a major contributors

29. A B Control Control Lowest inhibitor concentration Lowest inhibitor concentration Highest inhibitor concentration Highest inhibitor concentration C Control Lowest inhibitor concentration Highest inhibitor concentration Calcium qPCR Calcium Inhibition Take off cycle • No shift in take off cycle • No change in melting curve • Efficiency of amplification affected • No difference for size or Tm Melt curve Conclusion: Taq Inhibitor

30. Inhibition of PP16 with CaHPO4 Control Male500pg CaHPO4 1mM CaHPO4 1.5 mM CaHPO4 2mM CaHPO4 2.7mM D16, TPOX D18, CSF, FGA PD, PE

31. qPCR Humic Acid Inhibition Take off cycle • Shift in take off cycle • Change in melting curve • No efficiency of amplification change • Size effects on melt curve Melt curve Conclusion: Sequence specific Inhibitor

32. Inhibition of PP16 with Humic Acid Control Male500pg Humic Acid 16ng/uL Humic Acid 24ng/uL Am D3 d18 CSF PE PD

33. Conclusions • The key to automated forensic DNA typing was multiplex PCR amplification with capillary gel electrophoresis with laser induced fluorescence • Redesign of PCR primers using MniSTRs permits recovery of badly degraded DNA • Mechanisms for understanding DNA degradation can be determined using real time PCR. • Technology transfer: multiplex capillary electrophoresis, miniSTRs, realtime PCR technologies impact peoples lives. For example:this year the incidence of reported rape has hit a 20 year low

34. Acknowledgements • Funding • National Institute of Justice • National Science Foundation • J Edgar Hoover Foundation • Federal Bureau of Investigation • TSWG • Collaborators • John Butler, NIST • Eric Buel and Jan Nicklas, Vermont Forensic Laboratory • George Duncan, BSO Crime Lab • Ira Lurie, DEA • Sonja Rawn, OSFM Forensic Lab • Kelly Mount FBI • Researchers • Yin Shen • Jiri Drabek • Maximilien Blas • Maribel Funes • Silvia Zoppis • Kerry Opel • Denise Chung • Maurice Aboud • Heather LaSalle • Tanya Madi • Robyn Thompson • Brittany Hartzell • Oscar Cabrices • Stefano Boulas • William Kennedy

35. Thank you