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Title: Chapter 8 DNA Fingerprinting and Forensic Analysis


1
Chapter 8DNA 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 persons 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
How do we distinguish one persons DNA from
another?
  • We do not need to sequence the entire 3 billion
    base pairs of a persons DNA to distinguish it
    from another persons 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

5
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

6
  • The uniqueness of an individuals 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.

7
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

8
  • 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.

9
Step 3PCR using primers targeting STRs at
different loci
  • PCR amplify STRs using target sites on chromosome

10
Step 3 PCR amplification of DNA
STR locus
Design primers that anneal to STR locus Amplify
all the regions of the chromosome where the STRs
exist.
STR locus
11
PCR allows you to make millions of copies of the
STR region from a single copy of DNA you
recovered from crime scene.
12
  • Since the of times sequence is repeated is
    different for each person, fragment size will be
    different.
  • This is done for 13 different STR sequences at
    this one locus
  • Differences occur among individuals at each of
    the 13 loci on the chromosome where the STRs
    occur
  • This allows for a lot of variation

13
Restriction Fragment Length Polymorphism
For 1 STR sequence at 1 locus
14
  • If you do this for 13 different repeat sequences
    at 13 different loci on the chromosome, each
    person produces a different band pattern when the
    fragments are separated by gel electrophoresis

15
Different STRs atother loci
STR1 STR2 STR3
Do any of the individuals compare with forensic
sample?
16
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.

17
  • Dot blotting (continued)
  • Because every gene amplified by PCR has the same
    length, we dont 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.

18
Dot Blot
Probe made from sequence obtained from forensic
sample
Single strand of HLA gene amplified DNA from
sample
19
Binding of probe to complementary DNA
Binding takes place
No binding takes place
20
Wash away unreacted probeand add biotin-reactive
enzyme
Colorless substrate
Strepavidin (colorless enzyme)
Colored product (spot lights up)
21
Dot Blot
  • A visual signal is produced when the different
    probes anneal (bind) to the complementary
    sequence in the DNA sample

Probe 1
Crime scene PCR amplified DNA on each spot
22
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

Suspect DNA
Scene DNA
23
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
    rapists sperm cells make it difficult to get a
    fingerprint of the rapists 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

24
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
25
Always need suspect DNA
  • When suspects DNA doesnt 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.

stopped
26
  • Need lucky break or a lead that gets you to the
    perpetrator or the crime.

27
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

28
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.
  • Is this a good idea?

29
  • 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

30
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.

31
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. Expert witnesses used in this case.
  • Marx standard-court must be able to understand
    and evaluate scientific evidence. A university
    professor may be brought in to give a lecture of
    the concept.
  • Daubert standard requires special pretrial
    hearings for scientific evidence. Scientific
    procedure must be described in a peer-reviewed
    journal

32
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.

33
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. These people could become
    suspects based on this DNA
  • 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

34
Common Technical Problems
  • Band shifting
  • Add DNA samples from crime scene and suspects


35
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
36
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

37
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 childs parents before birth
  • collect fetal cells from amniotic fluid
  • DNA extraction and fingerprinting.

38
No longer necessary with PCR technology
PCR amplification, then DNA fingerprinting
39
Mothers STRs
Offspring STRs
STRs of suspected Father
-
Is the suspect the father?
40
Tracing geneology through mothers 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 mothers mtDNA is the same as her
    mothers MtDNA, and so on, back through the
    maternal bloodline.

41
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42
  • 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 buried corpse
    that has been buried for many years if you have
    living relatives whose DNA you can compare it to.

43
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
  • Followed human migration and dispersal from that
    location to other parts of the world.
  • Jared Diamonds Guns Germs and Steel

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 Associates degree in
    biotechnology and laboratory experience.
  • Good writing skills (lab notebook entries)
  • Good math and communication skills
  • Salaries 31K to 45K entry level depending on
    location.
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