1. Chapter 5 Biochemical Forensic Analysis: DNA Prof. J. T. Spencer Adjunct Prof. T. L. Meeks

2. Learning Goals and Objectives The use of DNA evidence has become the "gold standard" of forensic investigations. In order to understand the wealth of information that can be gained from forensic DNA studies…

3. Learning Goals and Objectives The chemical structure of DNA and how it holds genetic information The transcription and translation processes of DNA What parts of DNA are involved in forensic examinations What are variable number tandem repeats (VNTR) and short tandem repeats (STR) What a mutation is How the Restriction Fragment Length Polymorphism (RFLP) method works

4. Learning Goals and Objectives How the polymerase chain reaction (PCR)/STR method of DNA typing works How frequency of occurrences of STRs in populations is determined and used What is CODIS How mitochondrial DNA can be used in forensic investigations How DNA typing is being used in plants and other living organisms

5. The Cell The smallest unit of life The nucleus is the “brain” of the cell –contains all the genetic info the cell needs to exist & to reproduce In most types of cells, genetic information is organized into structures called chromosomes

6. Chromosomes In most types of cells, genetic information is organized into structures called chromosomes –usually X shaped Y chromosome in males –23 pairs in humans one from mother & one from father

7. Genes Each chromosome contains hundreds to thousands information blocks called genes Each gene is the blueprint for a specific type of protein in the body –only identical twins will have all the genes identical

8. Chromosomes Each chromosome is a single polymeric molecule called DNA –if fully extended the molecule would be about 1.7 meters long –unwrapping all the DNA in all your cells cover the distance from earth to moon 6,000 times

9. Structure of DNA

10. Nucleotides DNA is a polymer built from monomers called nucleotides Each nucleotide is consists of –deoxyribose pentose sugar –phosphoric acid –a nitrogenous base

11. The DNA Backbone The monomers are linked together by phosphodiester bridges (bonds) –links the 3’ carbon in the ribose of one nucleotide to the 5’ carbon in the ribose of the adjacent nucleotide

12. The DNA Double Helix DNA is normally a double stranded macromolecule Two polynucleotide chains are held together by H- bonding –A always pairs with T –C always pairs with G

15. 5’ T-T-G-A-C-T-A-T-C-C-A-G-A-T-C 3’ 3’ A-A-C-T-G-A-T-A-G-G-T-C-T-A-G 5’ In a double helix the strands go in opposite directions

16. Functions of DNA Two Functions –To transmit information from one generation of cells to the next –To provide the information for the synthesis of components (proteins) necessary for cellular function

18. Cell Types Where can DNA be found? Cell Blood Sweat Hair Roots Saliva Various Tissue Semen SAME

19. Nuclear DNA Where are the types of DNA found in a cell? Mitochondrial DNA Cell

20. Sources of Biological Evidence Blood Semen Saliva Urine Hair Teeth Bone Tissue

21. Types of objects where DNA may be found  Blood Stains  Semen Stains  Chewing Gum  Stamps & Envelopes  Penile Swabs  Plant Material  Sweaty Clothing  Bone  Hair  Fingernail Scraping  Saliva  Animal Material

22. Where DNA Evidence is Found

23. Blood Hair Roots Saliva Sweat Tissue Chemical DNA Isolation of DNA

24. Semen stain Chemical Remove Epithelial DNA Differential Isolation of DNA Different Chemical Sperm DNA Semen stain Epithelial DNA Sperm DNA

25. DNA Solution Amplification (making copies)

26. T G C A T T A G C A T A G T A G A A T C A T C T Heat Step one of a single cycleDENATURE

27. T A C T A T T C T T A T C A A T A G G Step two of a single cycleANNEAL

28. Step three of a single cycle T A A A T A G G T A C T T A T C T A C T T C A A A G G G G T T T T A GEXTEND

29. 1 Cycle 2 Cycles 3 Cycles 4 Cycles 5 Cycles 28 Cycles Amplification DNA PCR (Polymerase Chain Reaction)

30. Analysis of amplified DNA Amplified DNA Profile

31. Brief History of Forensic DNA Typing 1980 - Ray White describes first polymorphic RFLP marker 1985 - Alec Jeffreys discovers multilocus VNTR probes 1985 - first paper on PCR 1988 - FBI starts DNA casework 1991 - first STR paper 1995 - FSS starts UK DNA database 1996 – First mtDNA case 1998 - FBI launches CoDIS database

32. DNA Use in Forensic Cases Most are rape cases or murders Looking for match between evidence and suspect Must compare victim’s DNA profile Mixtures must be resolved DNA is often degraded Inhibitors to PCR are often present Challenges

33. Human Identity Testing Forensic cases -- matching suspect with evidence Paternity testing -- identifying father Historical investigations-Czar Nicholas, Jesse James Missing persons investigations Mass disasters -- putting pieces back together Military DNA “dog tag” Convicted felon DNA databases

34. Sample Obtained from Crime Scene or Paternity Investigation Biology DNA Extraction DNA Extraction DNA Quantitation DNA Quantitation PCR Amplification of Multiple STR markers PCR Amplification of Multiple STR markers Technology Separation and Detection of PCR Products (STR Alleles) Sample Genotype Determination Genetics Comparison of Sample Genotype to Other Sample Results If match occurs, comparison of DNA profile to population databases Generation of Case Report with Probability of Random Match Steps in DNA Sample Processing

35. Blood Hair Roots Saliva Sweat Tissue Chemical DNA Extraction of DNA

37. RFLP Analysis Enzymes break DNA into restriction fragments Measurements taken of fragments that vary in length across people (length polymorphism) because they contain VNTRs can produce extremely low random match probabilities requires relatively large fresh samples (>50 ng DNA) slow and expensive

38. Which Suspect, A or B, cannot be excluded from the class of potential perpetrators of this assault?

39. DNA Fingerprinting The basic structure of everyone’s DNA is the same –the difference between people is the ordering of the base pairs Every person can be distinguished by the sequence of their base pairs –millions of base pairs make this impractical –a shorter method uses repeating patterns that are present in DNA

40. VNTR’s DNA strands contain information which directs an organism’s development –exons Also contain stretches which appear to provide no relevant genetic information –introns –repeated sequences of base pairs Variable Number Tandem Repeats (VNTRs) can contain anywhere from 20 to 200 base pairs

41. VNTRs All humans have some VNTRs VNTRs come from the genetic information donated by parents –can have VNTRs from mother, father or a combination

42. D1 = biological daughter of both parents D2 = child of mother & former husband S1 = couple’s biological son S2 = adopted son

43. VNTR Analysis Usually an individual will inherit a different variant of the repeated sequence from each parent

44. VNTR Analysis PCR primers bracket the locus PCR reaction forms a nucleotide chain from the template

45. VNTR Analysis The length of the amplified DNA & its position after electrophoresis will depend on the number or repeated bases in the sequence

46. Analysis used 3 different VNTR loci for each suspect giving 6 bands

47. Although some individuals have several bands in common, the overall pattern is distinctive for each

48. Suspects A & C can be eliminated B remains a suspect

49. Short Tandem Repeats (STRs) the repeat region is variable between samples while the flanking regions where PCR primers bind are constant 7 repeats 8 repeats AATG Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another

50. Short Tandem Repeat AGAT 6 4 DNA Profile =4,6 TCTA 7 5 DNA Profile =5,7 TCTA STR

51. An Example Forensic STR Multiplex Kit D3FGAvWA 5-FAM (blue) D13 D5 D7 NED (yellow) AD8D21D18 JOE (green) GS500-internal lane standard ROX (red) AmpFlSTR ® Profiler Plus™ Kit available from PE Biosystems (Foster City, CA) 9 STRs amplified along with sex-typing marker amelogenin in a single PCR reaction 100 bp 400 bp300 bp200 bp Size Separation Color Separation

52. Short Tandem Repeats (STRs) the repeat region is variable between samples while the flanking regions where PCR primers bind are constant AATG 7 repeats 8 repeats AATG Primer positions define PCR product size Fluorescent dye label

53. 13 CODIS Core STR Loci with Chromosomal Positions CSF1PO D5S818 D21S11 TH01 TPOX D13S317 D7S820 D16S539D18S51 D8S1179 D3S1358 FGA VWA AMEL

54. DNA Quantitation using Slot Blot AMEL D3 TH01 TPOX Penta D Penta E FGA D21 D18 CSF D16 D7 D13 D5 VWA D8 PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis Blood Stain Overview of Steps Involved in DNA Typing Genotyping by Comparison to Allelic Ladder

56. ABI Prism 310 Genetic Analyzer capillary Syringe with polymer solution Autosampler tray Outlet buffer Injection electrode Inlet buffer

57. Chemistry Involved Injection –electrokinetic injection process –importance of sample preparation (formamide) Separation –capillary –POP-4 polymer –buffer Detection –fluorescent dyes with excitation and emission traits –virtual filters (hardware/software issues)

58. Separation Issues Run temperature -- 60 o C helps reduce secondary structure on DNA and improves precision Electrophoresis buffer -- urea in running buffer helps keep DNA strands denatured Capillary wall coating -- dynamic coating with polymer Polymer solution -- POP-4

59. DNA Separation Mechanism + - DNA - Size based separation due to interaction of DNA molecules with entangled polymer strands Polymers are not cross-linked (as in slab gels) “Gel” is not attached to the capillary wall Pumpable -- can be replaced after each run Polymer length and concentration determine the separation characteristics

60. ABI 310 Filter Set F 520540560580 600 620640 WAVELENGTH (nm) 100 80 60 40 20 0 5-FAM JOENEDROX Laser excitation (488, 514.5 nm) Laser excitation (488, 514.5 nm) Normalized Fluorescent Intensity Fluorescent Emission Spectra for ABI Dyes

61. Sample Detection CCD Panel Color Separation Ar+ LASER (488 nm) Fluorescence ABI Prism spectrograph Capillary or Gel Lane Size Separation Labeled DNA fragments (PCR products) Detection region Principles of Sample Separation and Detection

62. Mitochondrial DNA  What is mtDNA Typing?  Database and statistical issues

63. A Mitochondrial Exclusion

64. A Mitochondrial Inclusion

66. Mitochondrial Inconclusive?

67. The Future of Forensic DNA CoDIS SNP’s & Chips

68. FBI’s CoDIS DNA Database Combined DNA Index System Used for linking serial crimes and unsolved cases with repeat offenders Launched October 1998 Links all 50 states Requires >4 RFLP markers and/or 13 core STR markers Current backlog of >600,000 samples

69. On August 25, 1979, an 8-year old girl was brutally raped and murdered in San Pablo, CA. Semen was collected from the body and placed in an evidence room, where it sat for 22 years. Through this program, a DNA profile was made and submitted to the state and federal databases. This resulted in a “cold hit” identifying Joseph Cordova Jr. as the suspect. Cordova was a habitual child molester who at the time of the DNA analysis was incarcerated in a Colorado prison. Cordova was subsequently charged with molesting, raping and murdering the 8-year old girl. On November 8, 2000, a 12 year old girl, was kidnapped off of the street in Rancho Cordova, CA, and driven to Feather River in Sutter County where she was sexually assaulted and then killed. Nine months later, Justin Weinberger was stopped for a traffic violation in New Mexico. A check by police revealed that Weinberger was wanted on a federal warrant for child pornography. He was detained and voluntarily provided a DNA sample. Analysis of that DNA sample resulted in a match with evidence identifying Weinberger as the suspect in this case. Weinberger was subsequently extradited to California where he was tried and convicted of the murder of the 12-year old girl. Cold Hits and Solved Cases

71. STR Analysis by Hybridization on Microchips