Presentation on theme: "Forensic DNA Typing or Did you kill (rape…) that person? How DNA can “definitively” say. Adapted from: National Institutes of Science & Technology"— Presentation transcript:
Forensic DNA Typing or Did you kill (rape…) that person? How DNA can “definitively” say. Adapted from: National Institutes of Science & Technology http://www.cstl.nist.gov/div831/strbase/intro.htm
Brief History of Forensic DNA Typing 1980 - Ray White describes first polymorphic RFLP marker (Restriction Fragment Length Polymorphism [alleles]). Different RFLP for different people 1985 - Alec Jeffreys discovers multilocus VNTR (variable number of tandem repeats) probes. 1985 - first paper on PCR 1988 - FBI starts DNA casework 1991 - first STR paper (renaming of VNTR– could be larger repeats, STR 4-6 bp’s. now using mostly 4 bases ) 1995 - FSS (Forensic Science Service-UK) starts UK DNA database 1998 - FBI launches CODIS (Combined DNA Information Service) Now FBI use 13 loci: PCR identifies it: in the quadrillions – except for identical. Except for police mistakes, it’s done deal.
RFLP’s: Sickle Cell hemoglobin Case 1: Screening for the sickle-cell gene Sickle cell disease is a genetic disorder in which both genes in the patient encode the amino acid valine (Val) in the sixth position of the beta chain (beta S ) of the hemoglobin molecule. "Normal" beta chains (beta A ) have glutamic acid at this position.valine hemoglobinglutamic acid The only difference between the two genes is the substitution of a T for an A in the middle position of codon 6.codon This converts a GAG codon (for Glu) to a GTG codon for Val and abolishes a sequence (CTGAGG, which spans codons 5, 6, and 7) recognized and cut by one of the restriction enzymes.
Brief History of Forensic DNA Typing 1980 - Ray White describes first polymorphic RFLP (Restriction Fragment Length Polymorphism) marker—detect to transferring to membrane. Probe w southern blot (radiological). Diff. RFLP for dif. People. Single rflp 1985 - Alec Jeffreys discovers multilocus VNTR (variable number of tandem repeats) probes (stat. very impressive identical 4-6 bp that are spec. 7 and 9 repeat, one from mom and dad, on chrom. 1- nowadays use pcr- but flanking sequence that is unique to chromo1)). Jeffreys almost ident. Typing. Now use PCR. 1985 - first paper on PCR (Kerry Mullis) 1988 - FBI starts DNA casework 1991 - first STR paper ( renaming of VNTR– could be larger repeats, STR 4-6 bp’s. now using mostly 4 bases ) 1995 - FSS (Forensic Science Service-UK) starts UK DNA database 1998 - FBI launches CODIS (Combined DNA Information Service) database. Now FBI use 13 loci: PCR identifies it: in the quadrilians – except for identical.
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 Mixtures must be resolved DNA is often degraded (stored wet- have mold, nuclease) Inhibitors to PCR are often present Challenges
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
Sample Obtained from Crime Scene or Paternity Investigation Biology DNA Extraction DNA Extraction DNA Quantization DNA Quantization PCR Amplification of Multiple STR markers PCR Amplification of Multiple STR markers Technology Separation and Detection of PCR Products (STR Alleles) Sample Genotype Determination Genetics Comparison of Sample Genotype to Other Sample Results If match occurs, comparison of DNA profile to population databases Generation of Case Report with Probability of Random Match Steps in DNA Sample Processing
Sources of Biological Evidence Blood Semen Saliva Urine Hair Teeth (useful in fires). Bone (there are cells. Decalcify it. 100,000 year old- has DNA. Has Dinosaur!) Tissue All felony arrests- cheek swab.
DNA in the Cell Target Region for PCR chromosome cell nucleus Double stranded DNA molecule Individual nucleotides
Make copies (extend primers) Starting DNA Template 5’ 3’ 5’ 3’ Add primers (anneal) 5’ 3’ 5’ Forward primer Reverse primer DNA Amplification with the Polymerase Chain Reaction (PCR) Separate strands (denature) 5’ 3’
In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created PCR (Polymerase Chain Reaction) Copies DNA Exponentially through Multiple Thermal Cycles Original DNA target region Heat Cool Heat DNA Poly. dUTP Oligo’s 1 copy 2 copies Cool 4 copies Heat …
Short Tandem Repeats (STRs) (say chromo 3) the repeat region is variable between samples while the flanking regions where PCR primers bind are constant 7 repeats 8 repeats AATG Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another Identical in all people
170 bp 195 bp Different primer sets produce different PCR product sizes for the same STR allele TCAT repeat unit Diff. PCR primers sets, can amplify the same region. Different companies sell different kits.
Choosing which STRs: Significant statistical variation – but not too many. Freq. that are measured in pop. : Loc 1 - 10%. Loc 2 – 10%; locus 1+2 -1/100. Random match with 13 primers 1/10 13. Variation Among STRs
Multiplex PCR 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 Most rxns: require 2 PCR (tubes) 7 or 8 primer pairs in one tube– need total of about 2 tubes for 13 different STRs. $20-$25 per rxn in lab. $150 incl labor. Cost for forensic up to $1000.
An Example Forensic STR Multiplex Kit D3FGAvWA 5-FAM (blue) D13 D5 D7 NED (yellow) AD8D21D18 JOE (green) GS500-internal lane standard ROX (red) AmpFlSTR ® Profiler Plus™ Kit available from PE Biosystems (Foster City, CA) 9 STRs amplified along with sex-typing marker amelogenin in a single PCR reaction 100 bp 400 bp300 bp200 bp Size Separation Color Separation
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 10 17
ABI Prism 310 Genetic Analyzer capillary Syringe with polymer solution Autosampler tray Outlet buffer Injection electrode Inlet buffer 5 min from inj. to output.
Close-up of ABI Prism 310 Sample Loading Area Autosampler Tray Sample Vials Electrode Capillary See Technology section for more information on CE
amelogenin D19 D3 D8 TH01 VWA D21 FGA D16 D18D2 amelogenin D19 D3 D8 TH01 VWA D21 FGA D16 D18 D2 Two different individuals DNA Size (base pairs) Results obtained in less than 5 hours with a spot of blood the size of a pinhead probability of a random match: ~1 in 3 trillion Human Identity Testing with Multiplex STRs Simultaneous Analysis of 10 STRs and Gender ID AmpFlSTR ® SGM Plus™ kit D tells chromosome 21—happens to be down’s syndrome. 2 peaks cause heterozygotic Amelogenin amel protein that happens to be on sex chromosome, tooth enamel– top: 2 peaks: x and y. (universal that two diff. people.) Bottom only 1 peak cause they have two X chromosomes.
STR Allele Frequencies Caucasians (N=427) Blacks (N=414) Hispanics (N=414) TH01 Marker * Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34 Number of repeats Frequency
FBI’s CODIS DNA Database Combined DNA Index System -- all 50 states can upload their convicted felony and seq. of unsolved cases…. In Florida to convicted felon. 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