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DNA in Court

DNA in Court . Using Short Tandem Repeats (STR’s) in DNA Typing . Structure of DNA. Polymer (very large molecule) Monomers (individual units) are nucleotides Adenine ( A ), Guanine ( G ), Cytosine ( C ), Thymine ( T ) Monomers join linearly to form a strand

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DNA in Court

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  1. DNA in Court Using Short Tandem Repeats (STR’s) in DNA Typing

  2. Structure of DNA • Polymer (very large molecule) • Monomers (individual units) are nucleotides • Adenine (A), Guanine (G), Cytosine (C), Thymine (T) • Monomers join linearly to form a strand • Two strands wrap around each other to form the DNA double-helix • DNA SEQUENCE is simply the order of the nucleotides (A,T,G & C) along a strand

  3. Complementary base pairing and stacking in DNA

  4. Terms Used • PCR - polymerase chain reaction • technique for making many copies of identical DNA from template DNA • STR - short tandem repeat • short DNA sequence that repeats • example: ATCG repeated 5 times • ATCGATCGAT CGATCGATC • hundreds of loci (particular sites on the chromosomes) are known to contain STR’s; their lengths (# repeats) varies among people

  5. RFLP - restriction fragment length polymorphism • DNA pieces that vary in length from one individual to the next • VNTR - variable number tandem repeat • sequences that repeats one right after another but can be of any length • CODIS - combined DNA index system • 13 particular STR’s (loci) chosen by the FBI as the standard set for forensic analysis • Allele - STR with a particular number of repeats • Markers - another word for loci

  6. Brief History of Forensic DNA Typing • 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

  7. DNA Use in Forensic Cases • Most are rape cases (>2 out of 3) • Looking for match between evidence and suspect • Must compare victim’s DNA profile Challenges • Mixtures must be resolved • DNA is often degraded • Inhibitors to PCR are often present

  8. Human Identity Testing • Forensic cases -- matching suspect with evidence • Paternity testing -- identifying father • Historical investigations • Missing persons investigations • Mass disasters -- putting pieces back together • Military DNA “dog tag” • Convicted felon DNA databases

  9. DNA Quantitation PCR Amplification of Multiple STR markers DNA Extraction Separation and Detection of PCR Products (STR Alleles) Sample Genotype Determination Comparison of Sample to test Samples from selected individuals Generation of Case Report with Probability of Random Match If match occurs, comparison of DNA profile to population databases Steps in DNA Sample Processing Sample Obtained from Crime Scene or Paternity Investigation Biochemistry Technology Genetics

  10. Sources of Biological Evidence • Blood • Semen • Saliva • Urine • Hair • Teeth • Bone • Tissue

  11. chromosome cell nucleus Double stranded DNA molecule Target Region for PCR Individual nucleotides DNA in the Cell

  12. 5’ 3’ 5’ 3’ Starting DNA Template 3’ 5’ 3’ 5’ Separate strands (denature) Forward primer 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ Make copies (extend primers) Reverse primer Add primers (anneal) DNA Amplification with the Polymerase Chain Reaction (PCR)

  13. Original DNA target region Thermal cycle Thermal cycle Thermal cycle PCR Copies DNA Exponentially through Multiple Thermal Cycles In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created

  14. AATG Short Tandem Repeats (STRs) 7 repeats 8 repeats the repeat region is variable between samples while the flanking regions where PCR primers bind are constant Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another

  15. 170 bp 195 bp TCAT repeat unit Different primer sets produce different PCR product sizes for the same STR allele

  16. Over 10 Markers Can Be Copied at Once Sensitivities to levels less than 1 ng of DNA Ability to Handle Mixtures and Degraded Samples Different Fluorescent Dyes Used to Distinguish STR Alleles with Overlapping Size Ranges Multiplex PCR

  17. Available Kits for STR Analysis • Kits make it easy for labs to just add DNA samples to a pre-made mix • 13 CODIS core loci • Profiler Plus and COfiler (PE Applied Biosystems) • PowerPlex 1.1 and 2.1 (Promega Corporation) • Increased power of discrimination • CTT (1994): 1 in 410 • SGM Plus™ (1999): 1 in 3 trillion • PowerPlex ™ 16 (2000): 1 in 2 x 1017

  18. capillary Syringe with polymer solution Injection electrode Autosampler tray Outlet buffer Inlet buffer ABI Prism 310 Genetic Analyzer

  19. Analysis by Gel Electrophoresis • PCR of samples (thermocyler) • Electrophoresis and staining • DNA is a charged molecule and will move toward the positive electrode when exposed to an electric field • gels (agarose and polyacrylamide) stabilize convection and allow for separation based on molecular weight • staining (ethidium bromide or coomassie blue)

  20. FBI’s CODIS DNA Database Combined DNA Index System • Used for linking serial crimes and unsolved cases with repeat offenders • Launched October 1998 • Links all 50 states • Requires >4 RFLP markers and/or 13 core STR markers • Current backlog of >600,000 samples

  21. 13 CODIS Core STR Loci with Chromosomal Positions TPOX D3S1358 TH01 D8S1179 D5S818 VWA FGA D7S820 CSF1PO AMEL D13S317 AMEL D16S539 D18S51 D21S11

  22. Human Identity Testing with Multiplex STRs AmpFlSTR® SGM Plus™ kit DNA Size (base pairs) D3 TH01 amelogenin D8 VWA D16 D19 D21 D18 D2 FGA probability of a random match: ~1 in 3 trillion Two different individuals Results obtained in less than 5 hours with a spot of blood the size of a pinhead D3 amelogenin D19 D8 D16 VWA D21 D18 D2 FGA TH01 Simultaneous Analysis of 10 STRs and Gender ID

  23. Microvariant allele STR genotyping is performed by comparison of sample data to allelic ladders

  24. 45 40 35 30 Caucasians (N=427) 25 Frequency Blacks (N=414) 20 Hispanics (N=414) 15 10 5 0 6 7 8 9 9.3 10 Number of repeats STR Allele Frequencies TH01 Marker *Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34

  25. Possible Results • Match - profile matches to high probability • Exclusion - no matches on profile • Inconclusive - part of profile matches • crime lab reports may state that a particular suspect “cannot be excluded” • statistical data should be presented (FBI)

  26. Recent Case • Rape case - possible multiple assailants • Small amount of sample from victim’s garment • 13 CODIS loci examined • Victim’s profile matched • One suspect’s profile matched

  27. Other alleles present not matching the victim or the first suspect • Some alleles matched each of 2 other suspects. • Crime lab report statement: “Other alleles were found for TH01, DS81179, TPOX and DS21S11. Mr. Doe and Mr. Smith could not be excluded as donors of these alleles.”

  28. What is a juror to think?? Did Mr. Doe and Mr. Smith participate in the rape?

  29. My Analysis of the Crime Report • Statement too vague • Exactly which alleles were found?? • Which ones matched Mr. Doe and which ones matched Mr. Smith? • Statistical analysis completely missing!!!!!!!!!!!!

  30. Conclusion • STR’s are a powerful tool for forensic investigators. • The FBI CODIS is systematizing analysis • Is becoming more accepted as evidence in court. • Jurors must make informed decisions

  31. An educated public = Better justice for all

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