DNA Profiling

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DNA Profiling
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Discovery Sir Alec Jeffreys

• Discovered in 1984 by Dr. Alec Jeffreys at the
  university of Leicester
• Was knighted for his discovery

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Its Uses

• Identification of remains

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The Angel of Death Josef Menegle

• 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
  Mengeles wife and son, it was concluded that it
  was more than 99.94 certain that the skeleton
  was Mengeles.

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Paternity Cases
2.
1.

• Whos your daddy?

1.
2.
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Homicide or RapesOJ Simpson
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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 girls
  underpants tested against Bloodsworths DNA
• He was exonerated

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Exoneration
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DNA Profiling

• I didnt 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.

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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 doesnt 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

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Potential Sources of DNA

• Blood (White blood cells)
• Semen (Sperm cells)
• Hair with roots (Hair follicle cells)
• Skin, dandruff (Skin cells)
• Sweat stains (Skin cells sloughed off)
• Vaginal fluids (Mucosal surfaces)
• Nasal secretions (Mucosal surfaces)
• Urine (Mucosal surfaces)
• Feces (Digestive system cells)

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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!

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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

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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 everyones DNA is different, enzymes cut
  in different places
• The resulting samples contain DNA fragments of
  different size (Restriction Fragment Length
  Polymorphisms)

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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

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RFLP Autoradiograph
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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)

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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

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How do we get so much variation? Recall
inheritance patterns...
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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

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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

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Extracting DNA

• Break open the cells
• Mortar pestle
• Lysis buffer
• Centrifugation
• Micro pestles

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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

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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

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Biology 101 How does DNA Replication Occur in
Cells?
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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)

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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

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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

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Where do the data go?

• CODIS Combined DNA Index System

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CODIS

• Uses 13 loci
• Terameric repeats
• All forensic laboratories that use the CODIS
  system can contribute to a national database.
• Only Mississippi doesnt 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

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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

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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

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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 co-worker bragging
  they had given a DNA sample for another man
• Police obtain DNA from Colin Pitchfork and obtain
  a perfect match

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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

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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?

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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

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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