DNA%20Profiling%20and%20Forensic%20Analysis

1
DNA Profiling 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 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 profileStep 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
Types of Blot Tests
24
Characterization Southern blot hybridization
-transfer of DNA from a gel to a membrane (e.g.,
nitrocellulose, nylon) -developed by Edwin
Southern
25
Characterization Northern blot hybridization
X RNA
X
x salt
X RNA
-transfer of RNA from a gel to a membrane (e.g.,
nitrocellulose, nylon) -reveals mRNA size (and
approximate protein size), tissue- and
organ- specific expression, and kinetic patterns
of expression
26
Characterization Western blotting
X Protein
Enzyme reaction or
X
React with Antibody X
x Buffer requires electric current
X
-transfer of protein from a gel to a membrane
(e.g., nitrocellulose, nylon) -requires the use
of an electric current to facilitate transfer
27
Characterization Eastern blotting
-biochemical technique used to analyze protein
post transitional modifications (PTM) such as
lipids, phosphomoieties and glycoconjugates. It
is most often used to detect carbohydrate
epitopes -can be considered an extension of the
biochemical technique of Western blotting
28
DNA fingerprinting in practice

• Rape cases often sample a victims vagina for
  sperm in order to get a fingerprint of the rapist
• Victims vaginal 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 vaginal cells.
• They used this knowledge to separate DNA from
  sperm and vaginal cells

29
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
30
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
31

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

32
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

33
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?

34

• 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

35
Meeting Legal Standards

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

36
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

37
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.

38
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

39
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

40
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.

41
No longer necessary with PCR technology
PCR amplification, then DNA fingerprinting
42
Mothers STRs
Offspring STRs
STRs of suspected Father
-
Is the suspect the father?
43
Tracing genealogy 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.

44
(No Transcript)
45

• 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.

46
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

47
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.