1. Chapter 13 DNA 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 Jefferys first developed a method to test for individuality from chromosomal material after the Human Genome Project mapped human chromosomes.

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 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 fingerprint Step 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. Step 2 DNA fingerprinting is a comparative process: DNA extraction
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 3 PCR 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. Step 3 PCR amplification of DNA
1 strand
of DNA
Heat to
denature
double-
stranded
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 at other loci

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

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

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

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

23. What do we end up with?
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
Scene DNA
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
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 evidence can expose a faulty eye-witness
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