TOPICS IN (NANO) BIOTECHNOLOGYMedical Forensics and

DNA SleuthingLecture 9

7th November, 2006

PhD Course

“[The] use of DNA evidence can revolutionize the way crime is fought.

Not since fingerprints has law enforcement had such a powerful ally.”

(Los Angeles Times, 01/27/02)

DNADNA is the Fingerprint of the is the Fingerprint of the 21st Century21st Century

For almost a 100 years, people arrested for criminal offenses have provided

fingerprints, palm prints and mug shots during the typical police station booking

process.

These traditional identification tools have assisted law enforcement in solving crime

by identifying criminals.

With the emergence of DNA data banks, law enforcement can now use DNA not just to assist in establishing

guilt of a known suspect, but in solving crimes and ultimately ultimately

preventing serious crimepreventing serious crime..

DNA Data Banks:DNA Data Banks:21st Century Crime Fighting21st Century Crime Fighting

Definitions

• Gene– Heritable particle controlling some phenotype– Piece of DNA that codes for a protein– Piece of DNA that gets transcribed– Piece of DNA that has ANY function

• Genotype– The set of genes in an individual

• Phenotype– The physical or biochemical expression of the genotype

• Alleles are variants of a gene

• Gene with multiple alleles = polymorphic

Basis of DNA Profiling

The genome of each individual is unique (with the exception of identical twins) and is inherited from parents

Probe subsets of genetic variation in order to differentiate between individuals (statistical probabilities of a random match are used)

DNA typing must be performed efficiently and reproducibly (information must hold up in court)

Current standard DNA tests DO NOT look at genes – little/no information about race, predisposal to disease, or phenotypical information (eye color, height, hair color) is obtained

Speed of Analysis (Technology)

Power of Discrimination

(Genetics)

Low

High

Slow Fast

Markers Used (Biology)

Markers Used (Biology)

RFLPSingle Locus Probes

RFLPMulti-Locus Probes

ABO blood groups

Multiplex STRs

DQsingle STR

D1S80mtDNA

PolyMarker

Definitions

• Gene with two alleles, A and a

• Homozygote – individual with only one type of allele, i.e., aa, or AA

• Heterozygote – individual with more than one type of allele, i.e., aA

A. Identification of individuals based on DNA matching techniques

• Useful in identifying individuals in a variety of situations– Criminology and Forensics

• Forensic pathology/Law enforcement

– Paternity testing• Determine if person is related to another More accurate

than blood type testing

– Diagnostics

– Microbial strain identification • Contaminating sources

– Evolutionary studies (mitochondria)

History of DNA forensics

• 1980 - Ray White describes first polymorphic RFLP marker

• 1985 - Alec Jeffreys discovers multilocus VNTR

probes

• 1985 - first paper on PCR

• 1988 - FBI starts DNA casework

• 1991 - first STR paper

• 1995 - FSS starts UK DNA database

• 1998 - FBI launches CODIS database

Sample Obtained from Crime Scene or

Paternity Investigation

Biology

DNAExtraction

DNAExtraction

DNAQuantitation

DNAQuantitation

PCR Amplificationof Multiple STR

markers

PCR Amplificationof Multiple STR

markers

TechnologySeparation and Detection of

PCR Products(STR Alleles)

Sample Genotype

Determination

GeneticsComparison of Sample

Genotype to Other Sample Results

Comparison of Sample Genotype to Other

Sample Results

If match occurs, comparison of DNA profile to population databases

If match occurs, comparison of DNA profile to population databases

Generation of Case Report with Probability

of Random Match

Generation of Case Report with Probability

of Random Match

DNA Sample Processing

Polymorphisms

• Most of our DNADNA is identical to DNADNA of others.

However, there are inherited regions of our DNADNA that

can vary from person to person. Variations in DNADNA

sequence between individuals are termed

"polymorphisms"."polymorphisms".

• Sequences with the highest degree of polymorphismpolymorphism are very useful for DNADNA analysis in forensics cases. This activity is based on analyzing the inheritance of a class of DNADNA polymorphisms known as "Short "Short Tandem Repeats",Tandem Repeats", or simply STRsSTRs.

What are : STR – Short Tandem Repeat

• Short sequences of DNA.

• The repeats’ length: 2 - 5 base pairs.

• Repeats randomly several time across the genome.

A repeat of 2 base pairs: CACACACACA

A repeat of 3 base pairs: CAGCAGCAGCAGCAG

AND SO ON…

STR Polymorphisms

• Polymorphisms – variations in DNA sequences.• Polymorphism in STRs – different number of

copies of the repeat element.

An example . . .

STR sequence: ggtt ggttggtt

ggttggttggttggttggtt

CCombined ombined DDNA NA IIndex ndex SSystemystem

• CODIS STR – the core of the United States national database. A database of 13 STRs.

DNA sample

Forensic analysis (matching suspect with evidence, Paternity testing…)

DNA profile

PCR (amplifying polymorphic regions)

Sources of Biological Evidence

BloodSemenSalivaUrineHairTeethBoneTissue

For DNA extraction

• Gene probes developed by Alec J. Jeffreys– Based on repeating sequences called VNTRs (variable

number tandem repeats)

• Non-coding sequences found in variable numbers between different individuals at different locations in the genome

• As number of repeats increases, so does the length of the sequence

• Sequences are made up of “minisatellites”, ranging from 2-100 nucleotides long (usually 14-100)

DNA and Forensics

– Example: (GGAAG)n make up minisatellites– Each variant = VNTR allele– Some loci have many alleles– Restriction enzymes can be used to cut these

VNTRs – generating RFLPs– Combined with RFLPs a fingerprint is

generated that is unique for that person

• Can be combined with PCR when sample size is very small (e.g. single hair)

– 1 ng DNA can be detected (0.000,000,001g) 1 hair has 10ng

DNA and Forensics

• Imagine two alleles with frequencies 0.2 and 0.4 respectively

• What is the probability of an individual having allele 1 and allele 2?

= (0.2 x 0.4) + (0.4 x 0.2) = 0.16

16% of individuals have alleles 1 and 2

DNA and Forensics

• Combine multiple loci• Number of individuals with matching genotype

becomes very low• Add 9 other loci with similar allele frequencies• Genotype frequency = 0.1610

• Must reduce the expected frequency of matching genotype to less than 1

• Individual with matching genotype, must be the same individual as left the DNA sample

DNA Fingerprinting

B. Making a DNA fingerprint – the procedure

• Isolation of DNA– Specimen obtained and DNA extracted

• Blood, hair, cells, semen• Purification of DNA

• Cutting, sizing and sorting– Digestion of DNA with restriction enzymjes

• DNA cut into fragments at sites that flank the RFLP or the VNTR• Separation of fragments by agarose gel electrophoresis

• Transfer of DNA to nylon– Southern blotting

• Probing– Probe with radioactive DNA probes– Autoradiograph

• DNA fingerprint– Matches can place suspects at the crime scene or exonerate them from a

crime

Electrophoresis results

Methods to analyze fragment migration

• Ethidium bromide – mark locations on the gel and create bands visible under

ultraviolet light at the end of the fragments

• Southern Blot – melts the DNA fragments and then blots them on

nitrocellulose paper– A hybridization reaction is performed in which a radioactive

genetic probe of a specific tandem repeat is added to the melted DNA in the gel

– The radioactive probe adheres to matching patterns in the gel and an x-ray of the gel shows bands for where the probe is, producing a “fingerprint”

An actual DNA fingerprint

C. Limitations• But frequencies are not absolutely accurate because

databases are limited

• Contamination at crime scene or from victim

• Sloppy lab management

• Contaminated lab reagents

• Civil rights issues– Establish degree of probable cause before testing DNA?– Can blood collected for other reasons be used?

II. Forensic Pathology - Examples

• A. To single out suspects form a large panel of suspects

• B. Determine statistical likelihood that DNA at crime scene matches suspect’s DNA

• C. To identify body remains burnt or decomposed beyond recognition– War victims– Airplane disasters

DNA and Forensics

• D. To verify the identify of the “Unknown Soldier” (1998)– Remains exhumed from Arlington National Cemetery– Compared with female believed to be his mother– Identified as Air Force pilot shot down over Vietnam

in 1972

• Identification of remains of Czar Nicholas II of Russia – Died in 1918 buried in a mass grave– PCR of bone DNA compared to DNA of living family

descendents (including Prince Philip of GB)

DNA and Forensics

III. Microbial Identifications

Infectious disease spread and historical origins

• Mycobacterium tuberculosis probes– Lung tissue of Peruvian mummy (~AD 1000)– Results confirm presence of TB in Americans prior

to European invasion• M. leprae

– Confirmation of leper colony in Israel reported in biblical times

• Swine flu epidemic results in 30 million deaths

• Influenza virus generated by genetic shift (mixing of human and swine viral strains multisegmented RNA virus)

• Lung samples obtained from WWI soldiers whose lungs were paraffin-preserved at the Armed Forces Institute of Pathology– Viral genes compared to swine flue genes match found

• 1997: Similar technique links death of boy to Avian flu outbreak in Hong Kong– Matched to flu virus of a chicken– Thousands of chickens were destroyed

Infectious disease spread and historical origins

IV. Fossilized DNA from Archeological Samples – Molecular Paleontology

A. Data from prehistoric DNA can provide important clues pertaining to:

• Kinships

• Gene pools

• Migratory patterns

• Rates of evolution

• Taxonomic relationships – subspecies versus separate species– South African’s extinct quagga (subspecies of plains zebra)– Saber-tooth tiger DNA shows closer relationship to “Great

Cats” (tigers) than cats– Neanderthals not on the direct of succession to modern Homo

sapiens

• Egyptian mummy – 2400 years old– 1985: 3400 bp sequences

• Other examples:– 5500 year old bone

• PCR-based analysis in 1989– 18 million year old magnolia leaves

• 1991: Obtained from Idaho– 30 million year old fossil bee and fossil termite

• 1992: Preserved in amber

• Data from prehistoric DNA can provide important clues pertaining to:

– Bacillus spp. – 25 million years old• 1994: From bee intestinal material

– Dinosaur bones – 65 and 80 million years old• 1993-1994• Controversial – Need to perform comparison –

homology studies with reptiles and birds• Results didn’t support claims (contaminated?)

• Data from prehistoric DNA can provide important clues pertaining to:

V. Mitochondrial DNA Use in Evolutionary Analyses

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1 2 3 4 5 6 7 8 9 10 11 12

13 14 15 16 17 18 19 20 21 22 X Y

Human Genome 23 Pairs of Chromosomes + mtDNA

Sex-chromosomes

mtDNA

16,569 bp

Autosomes

Mitochondrial DNA

Nuclear DNA

3.2 billion bp

Located in cell nucleus

Located in mitochondria

(multiple copies in cell cytoplasm)

2 copies per cell

100s of copies per cell

A. Characteristics of mitochondrial DNA

• Inherited from mother only – Can trace evolutionary links through maternal lines

• Mitochondrial DNA = small (15-18kbp), double-stranded, circular

• Numerous mitochondria per cell (500-1000 mitochondrial genomes/cell)

• Encodes genes for:– Enzymes for energy metabolism– Small rRNA– Amino acid synthesis– Cytochromes and cytochrome oxidases involved in electron transport– Both strands used to encode product, sometimes overlapping

• No rearrangement during meiosis

• Subject to random mutation, just like nuclear DNA– Mutations that affect restriction sites can be identified like RFLP

• Rate of mutation found to be 2-4% every million years

A. Characteristics of mitochondrial DNA

B. How to investigate mutations and relate to rate of mutations

• Mitochondrial sequences compared across geographical regions

– 147 females from:• Europe• Asia• Africa• Australia• New Guinea

C. Mitochondrial studies comparing geographically distant populations

• Results: Most DNA closely homologous indicated relatively recent divergence

– But African women most diverse• Conclusion: African women have lived longer

because number of mutations were greater and took longer to be acquired

• Estimated time to accumulate mutations = ~140,000-280,000 years– “Mitochondrial Eve”

VI. Case Studies

Grand Duchess Anastasia Nicolaievna

• 1918 the Romanov family were assassinated• 1920 a woman jumped off a bridge in Berlin. She

was rescued and taken to a hospital. She had no ID and refused to give her identity

• Later, she insisted to be Anastasia• Many supported or denied Anderson• 1938-1970 German court: no evidence!• 1970 She married an American• 1977, Forensic expert: she is Anastasia• Recent DNA fingerprint proved that she was not

Anastasia

Grand Duchess Anastasia Nicolaievna

DNA analysis in paternity testing

DNA analysis in paternity testing

DNA analysis in criminal testing