Snowball the Cat and Other Tales of Forensic Science

1. Snowball the Cat and Other Tales of Forensic Science Donna C. Sullivan, PhD Division of Infectious Diseases University of Mississippi Medical Center

2. Polymorphism • A DNA polymorphism is a sequence difference compared to a reference standard that is present in at least 1–2% of a population. • Polymorphisms can be single bases or thousands of bases. • Polymorphisms may or may not have phenotypic effects.

3. Types of Polymorphic DNA Sequences • RFLP: restriction fragment length polymorphisms • VNTR: variable number tandem repeats (8 to >50 base pairs) • STR: short tandem repeats (1–8 base pairs) • SNP: single-nucleotide polymorphisms

4. DNA DIFFERS PERSON TO PERSON • All individuals genetically unique (except identical twins) • Differences in DNA sequence detected by • RFLP using large quantities intact DNA • PCR in conjunction with various methods • Single nucleotide polymorphisms (SNPs)

5. DNA Profiles from12 Unrelated Individuals At ONE Polymorphic Locus

6. RFLP ANALYSIS: Detect Alleles by Southern Blot Hybridization • Analysis of VNTR locus by Southern blot most commonly results in two-band pattern, comprised of a band inherited from each parent • Single band patterns possible with 2 alleles of same size • For 3 different alleles, six different genotypes possible

7. RFLP Analysis: Detect Alleles by Southern Hybridization

8. Restriction Fragment Length Polymorphisms • RFLP genotypes are inherited. • For each locus, one allele is inherited from each parent. Southern blot band patterns

9. CALCULATION OF POSSIBLE GENOTYPES • For N different alleles, total possible genotypes: (n X (n+1)) 2 • For 3 alleles, 6 genotypes • For 4 alleles, 10 genotypes • For 5 alleles, 15 genotypes

10. VNTR: Variable Number Tandem Repeats • Short sequence of DNA repeated in head-to-tail fashion • Occur at specific chromosomal locations • Interspersed throughout human genome • Number of repeated units vary between individuals

11. Multiple Alleles at VNTR loci

12. Distinguish Individuals by Combination of Alleles at VNTR Loci

13. Short Tandem Repeat Polymorphisms (STR) • STR are repeats of nucleotide sequences. • AAAAAA… - mononucleotide • ATATAT… - dinucleotide • TAGTAGTAG… - trinucleotide • TAGTTAGTTAGT… - tetranucleotide • TAGGCTAGGCTAGGC… - penta nucleotide • Different alleles contain different numbers of repeats. • TTCTTCTTCTTC - four repeat allele • TTCTTCTTCTTCTTC - five repeat allele

15. 11 repeats 5 repeats (Allelic ladder) Genotype: 6,8 Genotype: 7,9 Short Tandem Repeat Polymorphisms • Allelic ladders are standards representing all alleles observed in a population.

16. M S1 S2 V E M M S1 S2 V E M M S1 S2 V E M Locus 1 Locus 2 Locus 3 Evidence Testing by STR-PCR • Which suspect—S1 or S2—was at the crime scene? (V = victim, E = crime scene evidence, M = molecular weight standard)

19. FGA TPOX D8S1179 vWA PentaE D18S51 D2S11 THO1 D3S1358 Short Tandem Repeat Polymorphisms by Multiplex PCR

20. Child’s alleles Mother’s alleles Father’s alleles STR-PCR • STR genotypes are inherited. • One allele is inherited from each parent.

21. Single Nucleotide Polymorphisms (SNP) • Single-nucleotide differences between DNA sequences. • One SNP occurs approximately every 1,250 base pairs in human DNA. • SNPs are detected by sequencing, melt curve analysis, or other methods. • 99% have no biological effect;60,000 are within genes.

22. Top Ten Ways to Know that You are a Forensic DNA Scientist 10.You have your children’s DNA profiles framed on your desk instead of their pictures. 9.When your children hurt themselves, you are more interested in collecting their blood to generate a DNA profile than getting them cleaned up… 8.Your pockets are full of napkins with DNA sequences written on them. 7.You want to name your first four kids: Adenine, Thymine, Guanine, and Cytosine. 6.You wonder how jello would work as a separation medium…and have tried it when no one else was around…but were too afraid to publish the results.

23. Top Ten Ways to Know that You are a Forensic DNA Scientist 5.You want to start a paternity testing business for all those who appear on the Jerry Springer show. 4.You know that DNA stands for more than the National Dyslexics Association. 3.You want to do a study on the genetics of inbreeding…and have selected the Reservoir deer population for a case study.

24. Top Ten Ways to Know that You are a Forensic DNA Scientist 2.You know that “scientists” on the popular TV show CSI: Crime Scene Investigation cannot possibly get their DNA results within the timeframe of a single commercial break. 1.Your license plate reads: OJ DID IT!

25. LOCARD TRANSFER THEORY “When two objects come in contact, traces from one will be transferred from one to another, and in both directions.”

26. FORENSCI SCIENCE CONCEPTS • Identification • Classification: characteristics shared by items (Firearms, Shoes) • Groups (Same type of gun, same size shoe) • Individualization • Created by random acts (rifling on bullets) • Fingerprints (influenced by environmental, developmental factors) • Traits that are so rare, alone or in combination, that they can not be duplicated by chance

27. DNA Identification Applications • Sexual assault • Homicide and other violent crimes • Exculpate wrongly accused suspects • Identify serial crimes • Identify human remains • Sex offender tracking • Parentage testing

28. In The Beginning • Alec Jeffreys, geneticist at University of Leicester, UK • Coined the term “DNA fingerprinting” • RFLP analysis • Demonstrated that forensic samples, even dried stains several years old, contained sufficient DNA to yield conclusive results

29. Finding A Pitchfork In The Haystack: Midlands Rapes • Two British schoolgirls raped and murdered • Kitchen porter at an insane asylum confessed to one of the murders • Were both crimes committed by the same man? • Did the kitchen porter do it?

30. Finding A Pitchfork In The Haystack: Midlands Rapes • Semen samples from rape/murder scenes: • Both crimes committed by same man • Blood sample from kitchen porter: • Did not match crime scene evidence • Police felt that the murderer was a local • Tested DNA from every man between the age of 17-34 (4,582 men) in the local area • No matches found

31. Finding A Pitchfork In The Haystack: Midlands Rapes • Local baker over-heard some colleagues in a pub • Colin Pitchfork, another baker, had convinced another employee to give blood in his place • Police arrested Pitchfork (he immediately confessed) • Pitchfork’s DNA was perfect match to crime scene evidence (4,583rd man tested)

34. Evaluation Of Evidence • Establish type of biological material present in sample • Is it blood? Catsup? • Is it human blood? • “State of the DNA” • How much degradation of the DNA is there? • How much total DNA is present? • How much DNA is human?

36. Forensic Blood Typing • ABO groups • Phosphoglucomutase (PGM) • Erythrocyte acid phosphatase (EAP) • Haptoglobin • Adenylate kinase (AK)

37. Semen Can Be Identified With Several Laboratory Tests • Acid Phosphatase - a presumptive test • Anti P30 - a quantitative and qualitative test that identifies the presence of the protein P30 found only in human semen • Spermatozoa search - a microscopic analysis visually observing the presence of sperm cells

38. HLA DQ/HLA DQA1 System • HLA: human lymphocyte antigens • Determine whether patient has antibodies against a potential donor’s HLA antigens • HLA DQ  is historic name for this region • A 242 bp region with variation detected with specific probes for subregions

39. HLA DQ/HLA DQA1 System • Original test could detect 6 common DQ  alleles, 21 possible genotypes • Subsequent analysis, renaming by geneticists resulted in DQA1 test, increased number of subtypes detected (up to 28 detectable types) • Cheap, fast color reactions

40. Mixed Vs Contaminated Samples • MIXED SAMPLES • Contain DNA from more than one individual • Mixing occurs before or during commission of a crime • CONTAMINATED SAMPLES • Materials deposited during collection, preservation, handling or analysis

41. HOW MUCH SAMPLE DO YOU NEED?

42. Gender Identification • Amelogenin gene for tooth pulp found on sex chromosomes ( one on X, one on Y) • Different size PCR products from X and Y copies of the gene: • X Chromosome 106 bp PCR product • Y Chromosome 112 bp PCR product • “Right side primers” • Located to right of amelogenin gene, included amelogenin gene • X chromosome 549 bp (set 1), 977 bp (set 2) • Y Chromosome 360 bp (set1), 788 bp (set2)

44. DNA/CSI Quiz True or false: • DNA results take 8 minutes • DNA results can tell investigators what a suspect looks like • All CSIs are good looking and solve crimes wearing Armani • CSI investigators drive Hummers, especially in Florida • Compliments of Linda Ledray

45. The Combined DNA Index System (CoDIS) • A database of DNA profiles from violent felons and crime scene samples • Laws concerning who is eligible for the database vary from state to state • Database currently contains about 2,038,470 felons and 93,956 crime scene profiles (19,00 hits so far)

46. The Mystical Power of CoDIS • Extremely powerful investigative tool, linking crimes, and pulling suspects out of thin air! • Can prevent, as well as solve crimes! • NOT!!!!

47. Different Systems, Different Markers

48. CODIS Profile

49. STR Data (cont’d)

50. STR data (cont’d) “The DNA profile obtained from Item 25(S) matches the DNA profile of the suspect. The combination of genetic marker types exhibited by Item 25(S) and the suspect occurs in approximately one in one hundred quadrillion (1017) individuals…”