Download Chapter 8 DNA Fingerprinting and Forensic Analysis

Chapter 8
DNA Fingerprinting and Forensic
Analysis
• Forensic science is the application of
science to law
– previous technologies used
• photography, video cameras
• fingerprinting
– new technologies
• DNA fingerprints
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
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
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
• 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.
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
• 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.
Step 3
PCR using primers targeting STRs at
different loci
• PCR amplify STRs using target sites on
chromosome
Step 3
PCR amplification of DNA
1 strand
of DNA
Heat to
denature
doublestranded
DNA
Design primers that anneal to STR locus
Amplify all the regions of the chromosome
where the STRs exist.
STR locus
STR locus
PCR allows you to make
millions of copies of the STR
region from a single copy of
DNA you recovered from crime
scene.
• 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
Restriction Fragment Length Polymorphism
For 1 STR sequence at 1 locus
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-X-G-G
well
C-C-X-X-G-G
well
Gel
electrophoresis
• 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
Different
STRs at
other loci
STR1
STR2
STR3
Do any of the
individuals
compare with
forensic sample?
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.
• 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.
Dot Blot
biotin
C-G-T-A
Probe made
from sequence
obtained from
forensic sample
G-C-A-T…….
Single strand of HLA gene amplified DNA from sample
Binding of probe to
complementary DNA
biotin
biotin
C-G-T-A
G-C-A-T…….
C-G-T-C
Probe 3
Binding
takes place
No binding
takes place
Wash away unreacted probe
and add biotin-reactive enzyme
Colorless substrate
Strepavidin
(colorless
enzyme)
Colored product (spot lights up)
biotin
C-G-T-A
G-C-A-T…….
Dot Blot
• 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
biotin
biotin
biotin
C-G-T-A
Probe 1
C-G-T-T
Probe 2
C-G-T-C
Probe 3
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
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
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
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.
stopped
• Need lucky break or a lead that gets you to
the perpetrator or the crime.
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
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?
• DNA fingerprint can be used to refute
erroneous evidence that would otherwise
convict an innocent suspect
• DNA evidence can expose a faulty eyewitness
– 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
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.
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
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.
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
Common Technical Problems
-
• Band shifting
– Add DNA
samples from
crime scene and
suspects
Gel is not of uniform porosity
+
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
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
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
– DNA extraction and fingerprinting.
No longer necessary
with PCR technology
PCR amplification,
then DNA fingerprinting
Mother’s STRs
STRs of suspected Father
Is the suspect the father?
Offspring STRs
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.
• 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.
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 Diamond’s “Guns Germs and Steel”
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)
• 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.
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
– Salaries $31K to $45K entry level depending on
location.