Download DNA Analysis

DNA
Profiling
Discovery – Sir Alec Jeffreys
• Discovered in
1984 by Dr. Alec
Jeffreys at the
university of
Leicester
• Was knighted for
his discovery
What is it?
• What do you know about DNA Profiling?
– How does it work?
– From what sources can DNA for profiling be
obtained?
– How reliable is it?
– How is enough DNA obtained when little is
available from the crime scene?
– What, exactly, is a DNA profile or fingerprint?
• Take 5 minutes with your lab partner and
discuss these questions
Its Uses
• Identification of remains
The Angel of Death: Josef Mengele
• Josef Mengele was a Nazi war criminal
notorious for grotesque human experiments that
he carried out at the Auschwitz concentration
camp.
• After the Second World War he fled from the
Allies and escaped to South America. The
fugitive succeeded in living out the rest of his
days without being caught.
• In 1985 investigators went to the cemetery of
Nossa Senhora do Rosario in the small
Brazilian town of Embu to dig up the skeleton of
a man who had been drowned in a swimming
accident six years previously.
• Using DNA extracted from blood provided by
Mengele’s wife and son, it was concluded that it
was more than 99.94% certain that the skeleton
was Mengele’s.
Paternity Cases
• Who’s your daddy?
1.
2.
1.
2.
Homicide or Rapes:
OJ Simpson
Exoneration
• Kirk Bloodsworth
– Convicted in 1985 for
the rape and
strangulation of a 9-year
old girl and sent to death
row
– In 1992, defense
attorneys were
successful in having a
dime-sized semen stain
on the girl’s underpants
tested against
Bloodsworth’s DNA
– He was exonerated
Exoneration
DNA Profiling
• “I didn’t understand the DNA stuff at all. To
me, it was just a waste of time. It was way out
there and carried absolutely no weight with
me at all.”
•
Post-trial commentary from a juror in the O.J. Simpson trial: V. Bugliosi,
Outrage (New York: Dell Publishing, 1996).
• “In a forensic setting, ... an innocent suspect
has little to fear from DNA evidence, unless
he or she has an evil twin.”
•
N. Risch & B. Devlin, “On the Probability of Matching DNA Fingerprints”
(1992) 255 Science.
DNA Analysis
A New Technique –- Why Use It?
• DNA Analysis is useful because:
– The DNA contains “detectable” patterns unique to each
individual
– DNA is a robust molecule, and is stable under most (but not all)
environmental conditions
– DNA can be isolated from a wide range of biological samples
likely present at a crime scene
– The source of DNA doesn’t matter – it is the same in all sources
(blood, semen, sputum, skin etc)
– Rapid advances in technology allow the precise patterns to be
detected even with very small samples (a blood spot, single hair
follicle, lip-prints on a glass, physical fingerprints, saliva/skin on a
cigarette butt etc)
– Methods are fast and relatively cheap
– Data are complied in databases, and are easily searched
Potential Sources of DNA
•
•
•
•
•
•
•
•
•
Blood
Semen
Hair with roots
Skin, dandruff
Sweat stains
Vaginal fluids
Nasal secretions
Urine
Feces
(White blood cells)
(Sperm cells)
(Hair follicle cells)
(Skin cells)
(Skin cells sloughed off)
(Mucosal surfaces)
(Mucosal surfaces)
(Mucosal surfaces)
(Digestive system cells)
How Does It Work?
• Biology 101:
– Every cell in your body contains the same set of DNA
(except sperm/eggs)
– DNA is unique to each individual: even though we
share 99.9% of our genome in common with other
humans, 0.1% of 3 billion nucleotides is a significant
and detectable level of difference (1 out of every 1000
nucleotides)
– Most variation exists in non-coding (viz. “junk DNA”)
regions.
– Mutations in the non-coding are tolerated and can
accumulate with no effect on the organism
– The challenge: find the differences!
The 2 Main Types of DNA Profiling
• Restriction Fragment Length Polymorphisms (RFLPs)
– Restriction from the enzymes that cut the DNA (restriction
enzymes)
– Fragment for the fragments produced by the cutting
– Length and Polymorphisms for the different sized fragments
produced (polymorphic = many forms)
• Short Tandem Repeats (STRs)
– Short because the differences are short – usually 1-4
nucleotides in length
– Tandem because they occur one after the other
– Repeats because they are repeats of the same DNA sequence
– e.g. ACTG-GCC-GCC-GCC-GCC-ATCGACC = 4 tandem
repeats of GCC
RFLPs
• DNA is cut by molecular “scissors” – enzymes
which recognize particular sequences of
nucleotides
• These enzymes identify short sequences of
DNA, then snip it
• Because everyone’s DNA is different, enzymes
cut in different places
• The resulting samples contain DNA fragments of
different size (Restriction Fragment Length
Polymorphisms)
RFLP: Electrophoresis
• DNA is visualized using electrophoresis
• Negatively charged DNA moves through a gel with a
current
• Smaller DNA moves faster than larger DNA fragments
RFLP: Autoradiograph
How unique are these profiles?
• The probability of 2 people having exactly
the same DNA profile is between
1 in 5 million to
1 in 100 billion
(greater than the population of humans on
earth)
• This number becomes even larger if you
consider more regions of DNA
• Thus, the odds that the DNA evidence
from a crime scene will match your DNA
profile is astronomically small (unless you
have an evil identical twin)
STRs
• Much of the process of collecting STR data has
been automated, including gel electrophoresis
• To collect and analyze STR evidence, copies of
the variables regions of the DNA are amplified
(millions of copies are made)
• The DNA is then fed through a machine that
reads the DNA by size – a laser scans and
detects the stained DNA samples as they
electrophorese through the machine
How do we get so much variation? Recall inheritance patterns...
How do we get so much variation? Recall inheritance patterns...
• In this
example,
there are 4
types of
offspring
possible for
the parents
with their
genotypes;
• 6,8
• 6,2
• 3,8
• 3,2
Analyzing the DNA
• Although DNA is relatively stable, it does
denature or get destroyed through enzyme
action, from bacteria or through oxidation
• Therefore, samples should be collected soon
and preserved (usually in a buffer and by
freezing) if possible
• Care should also be taken not to cross
contaminate during collection
• Blood is also a potential pathogen, so care must
be taken to avoid exposing yourself to blood
borne viruses like Hep B, tuberculosis or HIV
Extracting DNA
1. Break open the cells
–
–
–
–
Mortar pestle
Lysis buffer
Centrifugation
Micro pestles
2. Quantify DNA
• This is important for 2 reasons:
– It is a standard or control (i.e. important
for Daubert challenges) – one needs to
argue that the same amount of DNA is
used in each lab, by each lab
technician and every time sample is
processed
– The amount has been optimized for
subsequent reactions – so it ensures
optimal results
• Quantification is done by some form
of fluorescence – tagging DNA with
a fluorescent tag, and the more
DNA there is, the brighter it will be
3. Amplifying the DNA of Interest
• Because most tissue samples from a crime
scene contain very little DNA, the goal is amplify,
or make many copies of the DNA of interest
• In STR analysis, you want to amplify the DNA
containing the tandem repeats and only this
DNA
• The process used is called Polymerase Chain
Reaction (PCR)
• PCR Machines, or thermocyclers, use repeated
cycles of heat and cooling to replicate the DNA
using many of the same enzymes found in cells
which facilitate DNA replication
Biology 101 – How does DNA Replication Occur in Cells?
SSBP
DNA
polymerase
New strand
Helicase
Helicase
Growth
DNA
polymerase
Growth
Replication
fork
Replication
fork
SSBP
New strand
Original
strand
PCR
• Ingredients:
– dNTPs (nucleotides)
– Buffer (to keep the pH and salt levels
constant)
– Taq polymerase (heat stable DNA
polymerase)
– Primers (short strands of DNA flanking the
gene(s) of interest – they initiate DNA
replication)
PCR
• Typical PCR reaction:
– 1 minute 95 ºC to denature DNA (does what
helicase does)
– 1.5 minutes 60-65 ºC (allows primers to
anneal)
– 1 minute 72 ºC (allows Taq to add dNTPs)
• This cycle is repeated 30-40 times
produced millions of copies of the genes
or sequences of interest
STR PCR (lots of acronyms)
• The procedure is the same for STR analysis, but recall
that each chromosome may have different numbers of
STRs
• The maternally and paternally inherited chromosomes
usually have different numbers of inserts, so the result
will be a 50/50 mix of amplified DNA with different
repeats
• For example, if you have 6 repeats from your mother and
2 from your father, you will amplify 2 different sized
pieces of DNA – one larger than the other
• In STR PCR, several different STR primers amplifying
several areas of interest simultaneously
How to amplify DNA...
How to amplify DNA...
Where do the data go?
• CODIS – Combined DNA Index System
CODIS
• Uses 13 loci
• Terameric repeats
• All forensic laboratories that use the CODIS system can
contribute to a national database.
• Only Mississippi doesn’t participate
• The Forensic Index contains DNA profiles from crime
scene evidence.
• The Offender Index contains DNA profiles of individuals
convicted of sex offenses (and other violent crimes) with
many states now expanding legislation to include other
felonies.
– Forensic Profiles in NDIS: 119,782
– Convicted Offender Profiles in NDIS: 2,643,409
A Sample Profile
• By combining the frequency information for all
13 CODIS loci, the frequency of this profile
would be 1 in 7.7 quadrillion Caucasians
Case Study: The First Use of DNA Evidence
• Two teenage girls raped and murdered in Leicestershire,
England
• Semen from the victims indicated a male with Type A
blood and a rare enzyme = 10% of the local male
population
• A local boy, Richard Buckland, confesses upon
interrogation
• Police use DNA fingerprinting to confirm, but DNA
profiles of Buckland and crime scene DNA do not match
• Ironically, Buckland becomes the first person exonerated
by DNA evidence
Case Study: The First Use of DNA Evidence
• Police request DNA samples from all
adult males in 3 nearby villages (5000
men)
• 6 months later – no results!
• A year later, police are informed by a
bakery worker that they overheard a coworker bragging they had given a DNA
sample for another man
• Police obtain DNA from Colin Pitchfork
and obtain a perfect match
The Result?
• In 1988, Colin Pitchfork was tried and
convicted and sentenced to life in prison
for the double rape and homicide based in
large part to the DNA evidence
As the technology gets smarter, so too do the criminals
• A physician in Canada eludes authorities for years
• Accused of drugging and sexually assaulting patients,
DNA profiles from semen samples from the assaulted
women do not match Dr. Schneeberger
• Blood was drawn on 3 occasions in 1992, 1993 and
1996, but never came back as a match
• Finally police obtain blood from a finger prick, swabbed
the inside of his cheek and took hair samples
• The results matched the DNA from the semen of the
victims
• How did he get away with it?
As the technology gets smarter, so too do the criminals
• On the previous 3 occasions, blood was drawn
from the same arm
• The last time the blood was drawn, the technician
stated that the blood looked brown and “old”
• Schneeberger had surgically implanted a piece of
rubber tubing in his arm and filled it with stored
blood from a patient
Some Phraseology
• Recall from general biology the heirarchy of structure of DNA:
– Humans carry 2 copies of the DNA in their cells (diploid). The exception
is sperm and eggs which contain one copy (haploid)
– The DNA is organized into chromosomes – long strands of DNA
– On the chromosomes, genes (sequences of DNA that code for a
protein) are found. The location of the gene on the chromosome is its
locus (plural: loci).
– Much of the DNA is non-coding (junk DNA) and even in protein coding
genes, there may be sequences that are cut out (introns) before they
are used to make a protein. The remaining sequences are the exons.
– Genes are sequences of DNA – there are only 4 building blocks of DNA
(A,T,G and C), so the genes are actually sequences of these
nucleotides. The length and order of nucleotides determines the type of
protein that is produced by that gene.
– Differences exist between individuals largely in the non-coding DNA
(introns and junk DNA). DNA profiles detect and exploit these
differences