1. Fundamentals of Forensic DNA Typing Slides prepared by John M. Butler June 2009 Chapter 14 Forensic Challenges

2. Chapter 14 – Forensic Challenges Chapter Summary DNA can be damaged or destroyed as it is exposed to environmental elements present at crime or disaster scenes. Degraded DNA often results in partial STR profiles or none at all. The use of reduced size PCR products, also known as “miniSTRs”, can in some cases enable recovery of information from degraded DNA or samples containing PCR inhibitors. Mixtures of DNA from two or more sources occur in some crime scenes and resolving the components of a mixture can be challenging in many situations. Depending on the type of mixture obtained including the ratio of contributors, the peak heights of STR alleles observed in a mixture can be used to group alleles to decipher the mixture component genotypes. Low-level DNA analysis, sometimes referred to as “low-copy number” or LCN testing, involves attempting to detect a DNA profile from only a few cells often through boosting the number of PCR cycles to enhance sensitivity. LCN testing is prone to problems, such as allelic drop-out (due to stochastic amplification effects) and allelic drop-in (due to sporadic contamination), and rigorous rules are generally applied including generating composite profiles of replicated alleles from multiple amplifications of the same DNA extract.

3. Degraded DNA sample D5S818 D13S317 D7S820 D16S539 CSF1PO Penta D Agarose yield gel results Smear of degraded DNA fragments High relative molecular mass DNA in a tight band (a) (b) Good quality DNA Degraded DNA John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 14.1

4. Full Profile (Good Quality) Partial Profile (Poor Quality) (a) (b) DNA size (bp) relative to an internal size standard (not shown) Relative fluorescence units (RFUs) John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 14.2 Comparison of Results with Good and Poor Quality DNA

5. STR repeat region miniSTR primer Conventional PCR primer (a) (b) Conventional STR test (COfiler kit) MiniSTR assay (using Butler et al. 2003 primers) 150 bp smaller John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 14.3 miniSTRs (Reduced size amplicon

6. Compromised Sample Improvements Better DNA extraction/recovery Continued use of miniSTRs –to improve success rates for recovery of information from compromised DNA evidence Replicate results for reproducibility –to improve reliability with low-template DNA testing

7. (a) Single Source D3S1358TH01D13S317D16S539D2S1338 16,169,9.38,129,917,19 (b) Mixed Source D3S1358TH01D13S317D16S539D2S1338 John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 14.4

8. Type AType BType C John M. Butler (2009) Fundamentals of Forensic DNA Typing, D.N.A. Box 14.1 Different Types of DNA Mixtures According to the German Stain Commission Classification

9. >2 alleles at a locus, except tri- allelics? Single Source DNA Sample NO Mixed DNA Sample YES Differentiate a Major/Minor Component? Determine STR profile and compute RMP YES Is the sample a mixture? TYPE B NO YES Stochastic Effects ? Possible Low Level DNA) ? YES Assume # Contributor s? TYPE C TYPE A NO A biostatistical analysis must be performed Probability of Exclusion [CPE] “RMNE” Likelihood Ratio [LR] YES NO Are # of contributors defined? A biostatistical analysis should not be performed Determine component profile(s) and compute RMP for major John M. Butler (2009) Fundamentals of Forensic DNA Typing, D.N.A. Box 14.1 Mixture Classification Flowchart

10. Identify the Presence of a Mixture Consider All Possible Genotype Combinations Estimate the Relative Ratio of the Individuals Contributing to the Mixture Identify the Number of Potential Contributors Designate Allele Peaks Compare Reference Samples Step #1 Step #2 Step #3 Step #4 Step #5 Step #6 John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 14.5 Steps in the Interpretation of Mixtures Defined by Clayton et al. 1998

11. 100 pg 50 pg 10 pg Allele dropout Severe imbalance Good heterozygote peak balance John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 14.6 Heterozygote Sample Result at a Single STR Locus at Different DNA Amounts

12. Tri-allelic pattern TPOX Variant allele D7S820 D3S1358 Stutter products 6.0%7.8% TH01 Variant allele Incomplete adenylation D8S1179 -A +A -A +A Some Example Biological Artifacts with STR Markers From Butler, J.M. (2004) Short tandem repeat analysis for human identity testing. Current Protocols in Human Genetics, John Wiley & Sons, Hoboken, NJ, Unit 14.8, (Supplement 41), pp. 14.8.1-14.8.22

13. DNA Degradation Intact sample 300 base pair PCR product can be produced Target region for PCR 300 base pair PCR product can not be produced or only in limited quantities Degraded sample Target region for PCR is fragmented

14. Degraded DNA Larger segments of DNA cannot be recovered when DNA molecules have fragmented into small pieces (caused by heat, water, or bacteria) D19 AMEL D3 D8 VWA TH01 D21 FGAD16D18 D2 “Degraded DNA” (falls apart with high temperatures) “Decay curve” of degraded DNA

15. DNA Degradation Means Less Loci Work Control (high quality DNA) Degraded 4000 rfu 600 rfu Much lower peak heights and loss of alleles With degraded DNA samples, information is simply lost at the larger sized STR loci

16. Full Profile (Good Quality) Partial Profile (Poor Quality) 4000 RFUs 600 RFUs Smaller sized DNA works With degraded DNA samples, information is simply lost at the larger sized STR loci Typing “1 ng” degraded DNA Same DNA with Different Quality Signal Strength is Lower

17. Impact of Degraded DNA Samples Comparison to a phone number (string of 13 numbers) 001-301-975-4049 If you only had “4049”…this information would be of limited value since it is not as specific (and could match other phone numbers from different area codes) DNA profiles are essentially a string of numbers – if the DNA is damaged, then the string of numbers is shorter and less informative… ------------4049----301-9-------or

18. STR repeat region miniSTR primer Conventional PCR primer (A) (B) Conventional STR test (COfiler™ kit) MiniSTR assay (using Butler et al. 2003 primers) Smaller PCR products work better with low copy number or fragmented DNA templates miniSTRs: new tool for degraded DNA 150 bp smaller

19. <15% Stutter region >70% 100% Heterozygous peak region 85% MIXTURE REGION 9% Higher than typical stutter product (>15%) 100% <15% >70% 60% 10% 25% Wrong side of allele to be typical stutter product Smaller peak area than normally seen with heterozygote partner alleles(<70%) (a) (b)

20. Mixture Basics Mixtures arise when two or more individuals contribute to the sample being tested. Mixtures can be challenging to detect and interpret without extensive experience and careful training. Differential extraction can help distinguish male and female components of many sexual assault mixtures. From J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition, p. 154 Even more challenging with poor quality data when degraded DNA is present… Y-chromosome markers can help here in some cases…

21. More on Mixtures... Some mixture interpretation strategies involve using victim (or other reference) alleles to help isolate obligate alleles coming from the unknown portion of the mixture Most mixtures encountered in casework are 2-component mixtures arising from a combination of victim and perpetrator DNA profiles major minor Ratios of the various mixture components stay fairly constant between multiple loci enabling deduction of the profiles for the major and minor components Torres et al. (2003) Forensic Sci. Int. 134:180-186 examined 1,547 cases from 1997-2000 containing 2,424 typed samples of which 163 (6.7%) contained a mixed profile with only 8 (0.3%) coming from more than two contributors 95.1% (155/163) were 2-component mixtures Ann Gross will discuss some recent collected casework summaries

22. AmelogeninD8S1179D21S11D18S51 Example Mixture Data (MIX05 Study-Profiler Plus) Single Source Sample (Victim) Evidence Mixture (Victim + Perpetrator) X,Y12,1228,31.215,16 True “Perpetrator” Profile Obligate Alleles (not present in the victim reference) Y122816 http://www.cstl.nist.gov/biotech/strbase/interlab/MIX05.htm MIX05 Case #1; Profiler Plus green loci Victim = major Perpetrator = minor

23. Sources of DNA Mixtures Two (or more) individuals contribute to the biological evidence examined in a forensic case (e.g., sexual assault with victim and perpetrator or victim, consensual sexual partner, and perp) Contamination of a single source sample from –evidence collection staff –laboratory staff handling the sample –Low-level DNA in reagents or PCR tubes or pipet tips Reference elimination samples are useful in deciphering both situations due to possibility of intimate sample profile subtraction Victim Reference and Spouse or Boyfriend Reference Examine Staff Profiles (Elimination Database), etc.

24. http://www.cstl.nist.gov/biotech/strbase/interlab/MIX05.htm MIX05 Case #1; Identifiler green loci Mixtures: Issues and Challenges The probability that a mixture will be detected improves with the use of more loci and genetic markers that have a high incidence of heterozygotes. The detectability of multiple DNA sources in a single sample relates to the ratio of DNA present from each source, the specific combinations of genotypes, and the total amount of DNA amplified. Some mixtures will not be as easily detectable as other mixtures. From J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition, p. 155 Mixture Mixture? Mixture Mixture?

25. D5S818 D13S317 D7S820 D8S1179D21S11 D18S51 Amel VWAFGAD3S1358 blue panel green panel yellow panel DNA Size (bp) Relative Fluorescence Units

26. amelogenin X-Y peak imbalance A B C B A C D D C B A 3 peaks at D8S1179 4 peaks at D21S11 4 peaks at D18S51 X Y DNA Size (bp) RFUs

27. AA BC AB BC BC AC AB AC CC AB BB AC 2 = major component 1 = major component 1 2 1 2 1 2 1 2 1 2 1 2 A BC

28. Mixture Interpretation – A Major Challenge… Not a clear cut answer because DNA result is from multiple contributors

29. STR LocusLikely “true” genotype for other component D3S135814,17 TH018,9.3 D13S3178,10 D16S53911,12 D2S133817,23 Single Source Sample Single Source vs. Mixture Samples Different possible combinations could have given rise to the particular mixture observed One or two peaks observed at each locus (tested DNA region) Locus 1Locus 2Locus 3Locus 4Locus 5 16,169,9.38,129,917,19 Mixture Sample More than two peaks observed at more than two loci (tested DNA regions) Locus 1Locus 2Locus 3Locus 4Locus 5

30. With Some Mixtures, Multiple Genotype Combinations Are Possible ABCD AC BD AB CD BC AD Peak Height Ratios (PHR) Minimum Peak Height (mPH) Proportion (p) or mixture proportion (M x ) Depends on PHR and proportion of mixture components from the various contributors

31. Common Casework Challenges D3S1358TH01D13S317D16S539D2S1338 MIXTURES DEGRADED DNA D5S818 D13S317 D7S820 D16S539CSF1PO Penta D From Butler, J.M. (2004) Short tandem repeat analysis for human identity testing. Current Protocols in Human Genetics, John Wiley & Sons, Hoboken, NJ, Unit 14.8, (Supplement 41), pp. 14.8.1-14.8.22 Loss of signal at larger size loci More than two alleles at multiple loci

32. DNA Testing Has Become Extremely Sensitive… What does it mean to obtain a DNA match between a suspect and material from a crime scene? Is the fact that a DNA profile obtained mean that this information is probative? More complicated samples (mixtures) and more items per case being submitted to labs

33. Time Line Showing the Potential for DNA Deposition/Transfer Time Crime Event Opportunity for DNA Transfer from Perpetrator Opportunity for Adventitious Transfer Adapted from Gill, P. (2002) BioTechniques 32(2): 366-385, Figure 5 Potential to “Contaminate” Discovery Investigators arrive, detect, and recover evidentiary material Laboratory analysis Analysis completed Higher sensitivity techniques are most likely to pick up previously deposited (background) DNA

34. Chapter 14 – Points for Discussion Discuss advantages and disadvantages of miniSTR assays. Name at least two ways that the presence of a mixture can be detected. What are the causes of allele drop-in and allele drop-out in the context of amplifying low amounts of DNA template?