Chapter 13 DNA Fingerprinting and Forensic Analysis

1. Chapter 13DNA Fingerprinting and Forensic Analysis

2. Forensic science is the application of science to law • previous technologies used • photography, video cameras • fingerprinting • new technologies • DNA fingerprints

3. What is a DNA fingerprint? • Every cell of an individual carries a copy of the DNA • a cell collected from a person’s skin or hair folicle contains the same DNA as from that persons heart tissue or white blood cells • Order of base pairs in the DNA of every individual is different except identical twins

4. 1986 • Alec Jefferysfirst developed a method to test for individuality from chromosomal material after the Human Genome Project mapped human chromosomes. • http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml

5. How do we distinguish one person’s DNA from another? • We do not need to sequence the entire 3 billion base pairs of a person’s DNA to distinguish it from another person’s DNA • Intron regions of DNA (junk DNA) contain sequences that are 20-100 bp in length that are repeated at different locations (loci) along the chromosome. CGGCTACGGCTACGGCTA (repeated 3 times at this location; at another location, it may be repeated 9 times) • These sequences are called Short Tandem Repeats (STRs) or VNTRs

6. STRs • Each person has some STRs that were inherited from mother and some from father • No person has STRs that are identical to those of either parent • The number of repeats at each loci on chromosome is highly variable in the population, ranging from 4 to 40. • The length of the DNA after cutting the chromosome with a restriction enzyme, and its position after electrophoresis will depend on the exact number of repeats at the locus

7. The uniqueness of an individual’s STRs provides the scientific marker of identity known as a DNA fingerprint. • In the United States the FBI has standardized a set of 13 STR assays (13 different locations on the chromosomes) for DNA typing, and has organized the CODIS database for forensic identification in criminal cases. • The United States maintains the largest DNA database in the world: The Combined DNA Index System, with over 60 million records as of 2007.

8. Preparation of a DNA fingerprintStep 1 • Specimen collection • blood, semen, etc • easy to contaminate a DNA sample with DNA from other sources (bacteria, DNA of person collecting sample) • DNA is not stable for very long-it degrades • sunlight • heat • moisture

9. DNA fingerprinting is a comparative process: • DNA from crime scene is compared with DNA of a suspect • So minimum of two samples must be prepared Step 2 • DNA extraction • standardized methods have been developed • need to separate DNA from other cell material and debris from crime scene.

10. Step 3PCR using primers targeting STRs at different loci • PCR amplify STRs using target sites on chromosome • Polymerase chain reaction; making millions of copies • RFLP ; Restriction fragment length polymorphisms • Established by enzymes that cut DNA at certain loci

11. 1 strand of DNA Heat to denature double- stranded DNA Step 3 PCR amplification of DNA Design primers that anneal to STR locus Amplify all the regions of the chromosome where the STRs exist.

12. PCR allows you to make millions of copies of the STR region from a single copy of DNA you recovered from crime scene.

13. Since the # of times sequence is repeated is different for each person, fragment size will be different.

14. Restriction Fragment Length Polymorphism Person A Forensic sample STR STR G-G-C-C-X-X-X-G-G-C-C-X-X.. G-G-G-C-C-X-X-G-G-C-C-X-X….. PCR amplify STR region C-C-X-X-G-G C-C-X-X-X-G-G well well Gel electrophoresis

15. If you do this for 13 different repeat sequences at 13different loci on the chromosome, each person produces a different band pattern when the fragments are separated by gel electrophoresis

16. Different STRs atother loci STR1 STR2 STR3

17. Other approaches to DNA fingerprinting • Dot Blotting • Genes that display high sequence variability can be substituted for STRs • An example of a gene with this property is the human leukocyte antigen (HLA). • The gene encoding this protein has lots of sequence variability across the human population. • Since this gene is not present in other life forms, it reduces the interference that could otherwise be contributed by bacteria, fungi, dog, or cat DNA picked up in the sample at crime scene.

18. Dot blotting (continued) • Because every gene amplified by PCR has the same length, we don’t need to use electrophoresis to sort and separate the fragments. • Instead, we use “blot strip” or dot blot which contains a different DNA probe that is sensitive to the sequence variability.

19. biotin C-G-T-A G-C-A-T……. Dot Blot probe Single strand of HLA gene amplified DNA from sample

20. biotin C-G-T-C Probe 3 biotin C-G-T-A G-C-A-T……. Binding of probe to complementary DNA Binding takes place No binding takes place

21. biotin C-G-T-A G-C-A-T……. Wash away unreacted probeand add biotin-reactive enzyme Colorless substrate Strepavidin (colorless enzyme) Colored product (spot lights up)

22. biotin biotin biotin C-G-T-A C-G-T-T C-G-T-C Probe 2 Probe 3 Dot Blot Probe 1 • A visual signal is produced when the different probes anneal (bind) to the complementary sequence in the DNA sample Crime scene PCR amplified DNA on each spot

23. What do we end up with? Suspect DNA Scene DNA • Blot strips show a pattern of spots that either light up or remain dark • Compare pattern produced from crime scene DNA to pattern produced from suspect DNA

24. DNA fingerprinting in practice • Rape cases often sample a victims vagina for sperm in order to get a fingerprint of the rapist • Victims vagina cells that are mixed in with rapist’s sperm cells make it difficult to get a fingerprint of the rapist’s DNA • Scientists studying sperm cells discovered that they resist lysis in certain solutions that induce lysis of vagina cells. • They used this knowledge to separate DNA from sperm and vagina cells

25. Transfer cells on swab to lysis buffer 1 Lysis buffer causes vagina cells to lyse and release DNA into solution Centrifuge tube to sediment sperm cells, then decant supernatant to remove vagina DNA Add lysis buffer 2 Lysis buffer causes remaining sperm cells to lyse and release DNA into solution

26. Always need suspect DNA • When suspect’s DNA doesn’t match sperm DNA from victim, investigation hits a barrier. • Need to find more suspects • Computer searchable DNA databases are now authorized by all 50 states • in some cases courts have agreed that collecting blood sample from someone without “probable cause” violates state and federal laws prohibiting unreasonable search and seizure.

27. Need lucky break or a lead that gets you to the perpetrator or the crime.

28. DNA profile database • CODIS Combined DNA Index System • run by FBI • contains profiles of convicted offenders • contains unidentified DNA taken from crime scenes • visit CODIS website to see how it works • www.fbi.gov/hq/lab/codis/index1.htm • CODIS allows identifying possible suspects when no prior suspect exists

29. Invasion of privacy • Some groups are worried that DNA samples will get in hands of insurance companies or potential employers • use to identify genetic defects that might cost them $$ • Why is this concern invalid? • What do you need to identify a genetic defect? • What does the STR analysis yield in the way of data that can provide information on genetic disorders? • Some groups are demanding that DNA samples be destroyed after investigation is complete.

30. DNA fingerprint can be used to refute erroneous evidence that would otherwise convict an innocent suspect • DNA evidence can expose a faulty eye-witness • Forest Hills Rapist • 3 separate victims identified assailant as Black to the police • Suspect on trial for crimes was White • DNA of white suspect was tested and matched DNA from sperm left at scene of each crime

31. Meeting Legal Standards • Court uses 5 different standards to determine whether evidence should be allowed in court • New technique must meet one or several of the standards before evidence using new technique can be introduced.

32. 5 Standards • Relevancy test • Frye standard-general acceptance test • Coppolino standard-allows new or controversial science to be used if adequate foundation can be laid • Marx standard-court must be able to understand and evaluate scientific evidence • Daubert standard requires special pretrial hearings for scientific evidence. Scientific procedure must be described in a peer-reviewed journal

33. Simpson/Goldman Murder • Pretrial hearings announced that blood collected at crime scene matched that of O.J.s • Defense argued that contamination could have occurred during sample collection and between collection of different samples • Technician admitted mislabeling samples • Possibility that evidence might be tainted was obvious to both the court and the jury • DNA evidence was not allowed as evidence • When rules of evidence are not followed, DNA samples lose their value in court.

34. Chain of custody • Requires that collection of evidence must be systematically recorded and access to evidence must be controlled • Special challenges for DNA samples • crime scene may have DNA from people other than perpetrators of crime • DNA collected from victims in a morgue can become contaminated by DNA of other bodies previously on autopsy table • during early days all procedures for processing DNA was not standardized, people running assays were not experienced and made mistakes

35. Common Problems • Band shifting

36. Maintaining High Standards • American Society of Crime Laboratories Directors • National Forensic Science Technology Center • College of American Pathologists All provide accreditation to forensic laboratories Proficiency testing of technicians “Blind” tests

37. Educating the Jury • Comparison of STR data is a statistically-based method • Jurors may not understand significance of a 1 in 50 billion chance of a random match • Attorneys must compare chance of random match of DNA data with chance that people will die by being hit by lightening over their lifetime to make them appreciate these numbers • Jurors must understand what DNA evidence offers in the way of putting suspect at a crime scene

38. Paternity testing • Verifying parents of a child to determine responsibility for child support • 250,000 cases per year in U.S. • Using amniocentisis, it is even possible to verify a child’s parents before birth • collect fetal cells from amniotic fluid • cultivate cells in tissue culture medium to produce more cells for DNA extraction and fingerprinting.

40. Tracing geneology through mother’s mitochondrial DNA • Inside each cell of the body is an organelle called the mitochondria • The mitochondria has chromosomes that were only inherited from the mother (MtDNA) • comes from the cytoplasm of the egg. • The DNA of the mother’s mtDNA is the same as her mother’s MtDNA, and so on, back through the maternal bloodline.

41. MtDNA is used to reunite families separated by corrupt governments • Junta in Argentina arrested pregnant women and took their newborn infants and gave them to supporters of the regime without consent of mother. • AAAS helped reunite 51 children with their natural mothers after the Junta regime collapsed. • MtDNA can be used to identify a corpse that has been buried for many years if you have living relatives whose DNA you can compare it to.

42. MtDNA and evolutionary biology • MtDNA mutates at a relatively constant rate of 2-4% every million years. • Allows scientists to trace gene frequency changes over time. • “Eve hypothesis” allowed scientists to trace a majority of people now living on Earth to a common female ancestor from ancient Africa

44. Other applications of DNA fingerprinting • Distinguishing between the North American and Asian strains of herb ginseng. • The different strains putatively have different therapeutic effects • Asians want NA strain • Americans want Asian strain • DNA RFLP analysis can distinguish between the two (used in this case as a means of monitoring quality control/quality assurance)

45. DNA evidence has shown that the majority of bison herds have some domestic livestock as ancestors. • No outward (phenotypic) evidence that this is the case, however.

46. Careers in DNA testing • Laboratory technicians • must be able to work very meticulously • forensic science technicians must pass a test to demonstrate these skills before being let loose at a crime scene • sometimes have to perform their sample manipulation in a “clean room” • Requirements • B.S. degree in biology, biochemistry or molecular biology or a specialized Associate’s degree in biotechnology and laboratory experience. • Good writing skills (lab notebook entries) • Good math and communication skills