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TOPICS IN (NANO) BIOTECHNOLOGY Medical Forensics and DNA Sleuthing Lecture 9

PhD Course . TOPICS IN (NANO) BIOTECHNOLOGY Medical Forensics and DNA Sleuthing Lecture 9. 19th May, 2006. DNA is the Fingerprint of the 21st Century.

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TOPICS IN (NANO) BIOTECHNOLOGY Medical Forensics and DNA Sleuthing Lecture 9

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  1. PhD Course TOPICS IN (NANO) BIOTECHNOLOGY Medical Forensics and DNA Sleuthing Lecture 9 19th May, 2006

  2. DNA is the Fingerprint of the 21st Century “[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)

  3. 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.

  4. DNA Data Banks: 21st Century Crime Fighting 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 preventing serious crime.

  5. 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

  6. 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

  7. Markers Used (Biology) High RFLP Multi-Locus Probes Multiplex STRs RFLP Single Locus Probes Power of Discrimination (Genetics) PolyMarker D1S80 mtDNA single STR DQ ABO blood groups Low Slow Fast Speed of Analysis (Technology)

  8. 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

  9. 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)

  10. 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

  11. DNA Quantitation PCR Amplification of Multiple STR markers DNA Extraction Separation and Detection of PCR Products (STR Alleles) Sample Genotype Determination Comparison of Sample Genotype to Other Sample Results Generation of Case Report with Probability of Random Match If match occurs, comparison of DNA profile to population databases DNA Sample Processing Sample Obtained from Crime Scene or Paternity Investigation Biology Technology Genetics

  12. Polymorphisms • Most of our DNA is identical to DNA of others. However, there are inherited regions of our DNA that can vary from person to person. Variations in DNA sequence between individuals are termed"polymorphisms". • Sequences with the highest degree ofpolymorphismare very useful for DNA analysis in forensics cases. This activity is based on analyzing the inheritance of a class of DNA polymorphisms known as"Short Tandem Repeats",or simplySTRs.

  13. 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…

  14. 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

  15. DNA sample PCR (amplifying polymorphic regions) DNA profile Forensic analysis (matching suspect with evidence, Paternity testing…) Combined DNA Index System • CODIS STR – the core of the United States national database. A database of 13 STRs.

  16. Sources of Biological Evidence For DNA extraction Blood Semen Saliva Urine Hair Teeth Bone Tissue

  17. DNA and Forensics • 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)

  18. 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

  19. 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

  20. DNA Fingerprinting • 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

  21. 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

  22. Electrophoresis results

  23. 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”

  24. An actual DNA fingerprint

  25. 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?

  26. II. Forensic Pathology - Examples

  27. DNA and Forensics • 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

  28. 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)

  29. III. Microbial Identifications

  30. 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

  31. Infectious disease spread and historical origins • 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

  32. IV. Fossilized DNA from Archeological Samples – Molecular Paleontology

  33. 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

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

  35. 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?)

  36. V. Mitochondrial DNA  Use in Evolutionary Analyses

  37. Human Genome 23 Pairs of Chromosomes + mtDNA http://www.ncbi.nlm.nih.gov/genome/guide/ 1 2 3 4 5 6 7 8 9 10 11 12 Mitochondrial DNA 13 14 15 16 17 18 19 20 21 22 X Y Located in cell nucleus Autosomes 2 copies per cell Located in mitochondria (multiple copies in cell cytoplasm) mtDNA 16,569 bp Sex-chromosomes Nuclear DNA 3.2 billion bp 100s of copies per cell

  38. 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)

  39. A. Characteristics of mitochondrial DNA • 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

  40. 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

  41. 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”

  42. VI. Case Studies

  43. Grand Duchess Anastasia Nicolaievna

  44. Grand Duchess Anastasia Nicolaievna • 1918 the Romanov family were assassinated • 1920a 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

  45. DNA analysis in paternity testing

  46. DNA analysis in paternity testing

  47. DNA analysis in criminal testing

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