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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 (betaS) of the hemoglobin molecule. "Normal" beta chains (betaA) have glutamic acid at this position. The only difference between the two genes is the substitution of a T for an A in the middle position of codon 6. 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. 1nowadays 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 Challenges •Mixtures must be resolved •DNA is often degraded (stored wet- have mold, nuclease) •Inhibitors to PCR are often present 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 Steps in DNA Sample Processing Sample Obtained from Crime Scene or Paternity Investigation Biology DNA Quantization DNA Extraction PCR Amplification of Multiple STR markers Technology Separation and Detection of PCR Products (STR Alleles) Comparison of Sample Genotype to Other Sample Results Sample Genotype Determination Genetics If match occurs, comparison of DNA profile to population databases Generation of Case Report with Probability of Random Match 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 chromosome cell nucleus Double stranded DNA molecule Target Region for PCR Individual nucleotides DNA Amplification with the Polymerase Chain Reaction (PCR) 5’ 3’ 5’ 3’ 3’ 3’ 5’ 5’ Starting DNA Template Separate strands (denature) Forward primer 5’ 3’ 5’ 3’ Make copies Add primers (extend primers) 5’ (anneal) 3’ 3’ 5’ Reverse primer PCR (Polymerase Chain Reaction) Copies DNA Exponentially through Multiple Thermal Cycles Original DNA target region 1 copy Heat Oligo’s DNA Poly. dUTP Cool Heat Cool 2 copies Heat 4 copies In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created … Short Tandem Repeats (STRs) (say chromo 3) Identical in all people AATG 7 repeats Identical in all people 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 Diff. PCR primers sets, can amplify the same region. Different companies sell different kits. 195 bp 170 bp TCAT repeat unit Different primer sets produce different PCR product sizes for the same STR allele Variation Among STRs 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 1/1013. 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 AmpFlSTR® Profiler Plus™ Kit available from PE Biosystems (Foster City, CA) 200 bp Color Separation 100 bp Size Separation D3 A vWA D8 D5 FGA 300 bp 400 bp 5-FAM (blue) D21 D18 JOE (green) D13 D7 NED (yellow) ROX (red) GS500-internal lane standard 9 STRs amplified along with sex-typing marker amelogenin in a single PCR reaction 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 ABI Prism 310 Genetic Analyzer 5 min from inj. to output. capillary Syringe with polymer solution Injection electrode Outlet buffer Autosampler tray Inlet buffer Close-up of ABI Prism 310 Sample Loading Area Electrode Capillary Sample Vials Autosampler Tray See Technology section for more information on CE D tells chromosome 21—happens to be down’s syndrome. 2 peaks cause heterozygotic Human Identity Testing with Multiplex STRs 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. AmpFlSTR® SGM Plus™ kit Two different individuals DNA Size (base pairs) amelogenin D19 D3 D8 TH01 VWA D21 D16 D18 D2 FGA probability of a random match: ~1 in 3 trillion amelogenin D3 D19 D8 VWA TH01 Results obtained in less than 5 hours with a spot of blood the size of a pinhead D16 D21 FGA D18 Simultaneous Analysis of 10 STRs and Gender ID D2 STR Allele Frequencies 45 40 TH01 Marker Frequency 35 30 Caucasians (N=427) Blacks (N=414) Hispanics (N=414) 25 20 15 10 *Proc. Int. Sym. Hum. ID 5 (Promega) 1997, p. 34 0 6 7 8 9 9.3 Number of repeats 10 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 13 CODIS Core STR Loci with Chromosomal Positions TPOX D3S1358 D8S1179 D5S818 FGA CSF1PO TH01 VWA D7S820 AMEL D13S317 D16S539 D18S51 D21S11 AMEL The End