DNA Profiling

Discovery – Sir Alec Jeffreys

• Discovered in 1984 by Dr. Alec Jeffreys at the university of Leicester

• Was knighted for his discovery

Its Uses

• Identification of remains

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 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 (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)

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 to1 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?

Growth

Growth

Replication fork

DNA polymerase

New strandDNA polymerase

Replication fork

Original strand

New strand

Helicase Helicase

SSBP

SSBP

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

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 co-worker 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