Forensic DNA Analysis

1. Forensic DNA Analysis SFS3f. Compare short tandem repeat patterns (STR) and relate to identifying the DNA of an individual. SFS3g. Explain the use of the DNA database for DNA profiling.

2. EQ • What is DNA? • How is DNA evidence analyzed by Criminalist & how has it become useful to criminal investigations over the past quarter century?

3. What is DNA? • DNA “fingerprinting” is a common way to ID people by their unique genetic code. • DNA is a double stranded molecule located in the nucleus of a cell; Every cell in an organism contains the same DNA. • Characteristics of DNA varies btwn individuals w/in a species & btwn species.

4. DNA- deoxyribonucleic acid. Long-chain molecule made up of four bases that pair together & held together by hydrogen bonds. A=T; G=C In humans- order of bases are 99.9% the same. What is DNA?

5. DNA is contained in chromosomes Chromosomes contain DNA & associated proteins (which allow DNA to condense/wind/coil up tightly) What is DNA?

6. Genes are sequences of DNA nucleotides Genes are located on chromosomes Alleles: different types of the same gene (dominant, recessive, co-dominant, etc). What is DNA?

7. Forensic DNA analysis seeksnucleotide variation in DNA • 1. Point mutations from errors in DNA replication • – RFLP (Restriction Fragment Length Polymorphism) analysis • 2. Variable number tandem repeats (VNTRs) • – STR (Short tandem repeat) analysis

8. All the DNA nucleotides in a cell must be copied (replicated) prior to cell division. Random copying errors are made during DNA replication. DNA replication copies DNAnucleotides Every human cell contains 3.4 billion nucleotide pairs. Why is there nucleotide variation between the DNA of individuals of the same species?

9. DNA replication

10. Objectives of Forensic DNA Testing • To link an individual to a crime scene/criminal act. • To exonerate suspects. • To identify victims of mass disasters. • To determine Paternity.

11. Specimen Samples used for Forensic DNA Testing • Blood (what components?) • Semen • Saliva • Bones • Skin scrapings • Hair follicular tag

12. Main Procedures for DNA Fingerprinting: • Isolation of the DNA to separate the DNA from the cell. • Cutting w/ a restriction enzyme to make shorter base strands. • Sorting the segments by size, using electrophoresis • Analyzing the resulting print by identifying specific alleles/ genes.

13. DNA Fingerprinting Analysis: • RFLP (Restriction Fragments Lengths polymorphism)-use restriction enzymes to cut the DNA in fragments that are many diff lengths and shapes. The length vary from person to person. • Restriction Enzyme-recognize a specific sequence of bases & cut the DNA molecule at specific points. • Electrophoresis-a procedure that sorts/ separates DNA fragments by size.

14. Early 1980s: Restriction Fragment Length Polymorphism (RFLP) • Genetic variation in the distance between restriction enzyme sites • Template DNA digested by enzymes, electrophoresed, detected via Southern blotting • Power of discrimination in the range of 106-108 for a six probe analysis Sir Alec Jeffreys

15. Mechanisms for RFLPs

16. The Catch: • RFLP testing requires a relatively large amount of HMW DNA (~50ng = thousands of cells) • Not ideal for forensic evidence, in which small, degraded samples are common

17. 2. PCR To The Rescue! • Polymerase Chain Reaction = molecular Xeroxing • Three temperature phases, carried out in a Thermal Cycler, replicate/ copy or “amplify” the desired DNA fragment(s) Dr. Kary Mullis Eccentric Genius

19. PCR (cont’d) • Works with lower quantity (1-2ng), lower quality samples • Power of discrimination goes from 102-106...not good enough for databasing

20. 3. The Current Method of Choice: Autosomal Short Tandem Repeats (STR) • Non-coding, tetranucleotide sequences which vary greatly from person to person in the number of repeating units • Requires <1ng of DNA to type 13-15 STR loci • Power of discrimination ranges from 1014-1023. World population is 109 so bring on the database!

21. Applied Biosystems 310 Genetic Analyzer

22. The Process In a Nutshell Amplified DNA samples are injected into a capillary. Fluorescent tags on the DNA fragments are excited by a laser as they pass a window in the capillary, the fluorescence is recorded by a camera, and this signal is converted into a “peak” by the computer software.

23. STR data X, Y, X Y

24. STR data (cont’d)

25. 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…”

26. How are these astronomical figures derived? The product rule: combined probability of a series of independent events is determined by multiplying the probabilities of each event. STR loci are inherited independently (unlinked) Homozygous loci: p2 (same allele inherited from mother and father) Heterozygous loci: 2pq (either allele could be inherited from either parent) p(17)2 x 2p(15)q(17) x 2p(23)q(26)…. (.223)2 x 2(.083)(.25) x 2(.14)(.02) = .000013, which is equivalent to a probability of one in 76,000 using just 3 of the 13 loci!

27. STR Artifacts -A (“minus A”): Incomplete addition of nucleotide ‘A’ by DNA polymerase; results in a peak that is one base pair smaller than allele peak.

28. STR Artifacts Stutter: Slippage of DNA polymerase; results in a peak that is four base pairs (one repeat unit) smaller than allele peak.

29. STR Artifacts Pull-up: Incomplete filtration of spectral overlap in fluorescent detection system.

30. Pull-up

31. DNA Mixtures When more than one source of DNA is detected in a sample, assignment of genotypes becomes more difficult.

32. Degraded/Trace DNA Samples Larger alleles “drop-out” when template DNA is low in quantity or quality, reducing certainty of genotypes.

33. 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)

34. 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!

35. The Dark Side of CoDIS(What the FBI doesn’t want you to know.) • DNA mixtures and degraded DNA profiles have lead to spurious matches • Stringent laws explicitly permit databasing innocent people • Adding arrestees to database violates presumption of innocence • However, the prosecution rate on case to offender matches is shockingly low! (~10%)

36. PCR • Isolate the DNA from crime scene evidence to get a pure sample and cut target strands. • Mix the DNA samples, primer, DNA polymerase enzyme & nucleotides in tube. Place in PCR chamber. • Heat the dsDNA to separate to 95°C. • Cool to 50°C so that primers can attach to ssDNA. • Reheat to 72 °C so that DNA polymerase can attach to end of primer. • The DNA polymerase will now add complementary nucleotides to the ssDNA. Two new strands are made. • Repeat cycle until desired # of DNA is copied.

37. Electrophoresis • Isolate pure sample & cut the DNA into fragments. • Make agarose gel. • Add DNA with a buffer solution to wells in gel. • Place the gel in the electrophoresis plate so that a current passes through. • The DNA will migrate from one end to the next based on size. • Stain the gel with Ethidium Bromide when the electrophoresis is done. • Place under UV light to observe band. • Analyze/ compare bands for size comparison.

38. Take this scenario:A woman gets out of bed, watches TV, talks on the phone, pets her cat, and then shoots her husband. She hides the gun and runs away. Which object would be most useful to the forensic serologist in finding out who the woman was? A. the cat B. the phone C. the TV D. the TV remote E. none listed What system used by the FBI compiles DNA of known violent offenders from all over the nation and can be used to match DNA with a sample found at a crime scene? A. IAFIS B. IBIN C. SICAR D. CODIS E. DNAW REview

39. Review Review: Who’s DNA is the unknown? Suspect A Unknown Suspect B

40. In this gel, the base-pair lengths are listed to the right. Which end of the DNA fingerprint was plugged to the NEGATIVE terminal during electrophoresis? A. Top B. Bottom C. Left D. Right Review

41. In a talk-show scandal, it is revealed that one of three siblings has a different father. GASP! Using the DNA fingerprint of just the mother, daughter and two sons, which sibling has a DIFFERENT father. Daughter Son 1 Son 2 all have the same father—go figure! Review

42. Which suspect's DNA matches the felon's DNA left at the crime scene? A. suspect 1 B. suspect 2 C. suspect 3 D. none listed Review

43. Whose the daddy? This mother is trying to decide between two men who desperately want to support her and her newborn baby. Both want to be a part of the baby's life, because they love the mother so much. Who gets the honor and privilege? A. Dad 1 B. Dad 2 C. neither man D. both Review

44. Compare the sperm DNA to the two suspects and the boyfriend. Which male is most likely the assailant? A. suspect 1 B. suspect 2 C. boyfriend D. none listed REview

45. In this DNA fingerprint, the blood sample taken from a crime scene is not the victim's and so is assumed to be the perpetrator. Which individual is the best suspect? A. Bob B. Sue C. John D. Lisa E. none Review

46. There was a mix-up at the hospital. Which of these children belong to the parents? A. All of the children B. Children 2, 3 & 6 C. Children 1 & 5 D. Children 2 & 4 Review