topics in (nano) biotechnology medical forensics and dna sleuthing lecture 9 7th november, 2006 phd...
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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)
• 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
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”
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?
• 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
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
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:
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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”
• 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