Forensic DNA: Use, Abuse, Promise, and Peril William M. Shields

DNA Identification Where does DNA come from? Where does DNA come from? 1/2 from mom 1/2 from mom 1/2 from dad 1/2 from dad What is it? What is it? “Blue print” of life “Blue print” of life How is DNA different among us? How is DNA different among us? Common vs Different Common vs Different What does “DNA” mean? What does “DNA” mean? Deoxyribonucleic Acid Deoxyribonucleic Acid

Cell Types Where can DNA be found? Cell Blood Sweat Hair Roots Saliva Various Tissue Semen SAME

Nucleus Where is DNA in the body? Cell

Nuclear DNA Where are the types of DNA found in a cell? Mitochondrial DNA Cell

Maternal Chromosome Paternal Chromosome Nucleus Where is DNA in the body?

Where is DNA packaged in the body? Chromosome DNA

A =Adenine T =Thymine G =Guanine C =Cytosine Units Double Helix G A T C DNA- What does it look like?

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

Types of objects where DNA may be found  Blood Stains  Semen Stains  Chewing Gum  Stamps & Envelopes  Penile Swabs  Plant Material  Sweaty Clothing  Bone  Hair  Fingernail Scraping  Saliva  Animal Material

Where DNA Evidence is Found

Blood Hair Roots Saliva Sweat Tissue Chemical DNA Isolation of DNA

Semen stain Chemical Remove Epithelial DNA Differential Isolation of DNA Different Chemical Sperm DNA Semen stain Epithelial DNA Sperm DNA

DNA Solution AmplificationAmplification (making copies)

T G C A T T A G C A T A G T A G A A T C A T C T Heat Step one of a single cycleDENATURE

T A C T A T T C T T A T C A A T A G G Step two of a single cycleANNEAL

Step three of a single cycle T A A A T A G G T A C T T A T C T A C T T C A A A G G G G T T T T A GEXTEND

1 Cycle 2 Cycles 3 Cycles 4 Cycles 5 Cycles 28 CyclesAmplificationAmplification DNA PCR (Polymerase Chain Reaction)

Analysis of amplified DNA Amplified DNA Profile

Brief History of Forensic DNA Typing Ray White describes first polymorphic RFLP marker Ray White describes first polymorphic RFLP marker Alec Jeffreys discovers multilocus VNTR probes Alec Jeffreys discovers multilocus VNTR probes first paper on PCR first paper on PCR FBI starts DNA casework FBI starts DNA casework first STR paper first STR paper FSS starts UK DNA database FSS starts UK DNA database 1996 – First mtDNA case 1996 – First mtDNA case FBI launches CODIS database FBI launches CODIS database

DNA Use in Forensic Cases Most are rape cases or murders Most are rape cases or murders Looking for match between evidence and suspect Looking for match between evidence and suspect Must compare victim’s DNA profile Must compare victim’s DNA profile Mixtures must be resolved DNA is often degraded Inhibitors to PCR are often present Challenges

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

Sample Obtained from Crime Scene or Paternity Investigation Biology DNA Extraction DNA Extraction DNA Quantitation DNA Quantitation 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

Progression of DNA Typing Markers RFLP RFLP multilocus VNTR probes multilocus VNTR probes single locus VNTR probes (P 32 and chemiluminescence) single locus VNTR probes (P 32 and chemiluminescence) PCR PCR DQ-alpha (reverse dot blot) DQ-alpha (reverse dot blot) PolyMarker (6 plex PCR; dots for SNPs) PolyMarker (6 plex PCR; dots for SNPs) D1S80 (AMP-FLPs) D1S80 (AMP-FLPs) singleplex STRs with silver staining singleplex STRs with silver staining multiplex STRs with fluorescent dyes multiplex STRs with fluorescent dyes Mitochondrial DNA sequencing Mitochondrial DNA sequencing Multiplex Y-STR with fluorescent dyes Multiplex Y-STR with fluorescent dyes

Blood Hair Roots Saliva Sweat Tissue Chemical DNA Extraction of DNA

RFLP Analysis Enzymes break DNA into restriction fragments Enzymes break DNA into restriction fragments Measurements taken of fragments that vary in length across people (length polymorphism) because they contain VNTRs Measurements taken of fragments that vary in length across people (length polymorphism) because they contain VNTRs can produce extremely low random match probabilities can produce extremely low random match probabilities requires relatively large fresh samples (>50 ng DNA) requires relatively large fresh samples (>50 ng DNA) slow and expensive slow and expensive

Which Suspect, A or B, cannot be excluded from the class of potential perpetrators of this assault?

PM+DQA1 Test PCR-based PCR-based Extremely sensitive(1ng DNA) Extremely sensitive(1ng DNA) degraded samples degraded samples faster and cheaper than RFLP faster and cheaper than RFLP Statistics less impressive, particularly with mixed samples Statistics less impressive, particularly with mixed samples Possible Problems: interpretation is subjective and can be difficultinterpretation is subjective and can be difficult mixtures difficult to interpretmixtures difficult to interpret statistical characterization of mixed samples is trickystatistical characterization of mixed samples is tricky

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

Short Tandem Repeats (STRs) 1. CTTA with silver- stained gel PCR-based PCR-based 3 loci for identification plus sex-typing 3 loci for identification plus sex-typing Easier interpretation of mixtures Easier interpretation of mixtures

Short Tandem Repeats (STRs) 2. Gel-based systems with Fluorescent Detection

Short Tandem Repeats (STRs) 3. Capillary Electrophoresis AmpFlstr Profiler Plus Groups of amplified STR products are labeled with different colored dyes (blue, green, yellow) Groups of amplified STR products are labeled with different colored dyes (blue, green, yellow) Electrophoresis and detection occur in computer-controlled capillary device (ABI Prism 310 Genetic Analyzer) Electrophoresis and detection occur in computer-controlled capillary device (ABI Prism 310 Genetic Analyzer)

Short Tandem Repeats (STRs) 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

Short Tandem Repeat AGAT 6 4 DNA Profile =4,6 TCTA 7 5 DNA Profile =5,7 TCTA STR

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

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

DNA Quantitation using Slot Blot AMEL D3 TH01 TPOX Penta D Penta E FGA D21 D18 CSF D16 D7 D13 D5 VWA D8 PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis Blood Stain Overview of Steps Involved in DNA Typing Genotyping by Comparison to Allelic Ladder

Calculation of DNA Quantities in Genomic DNA Important values for calculations: 1 bp = 618 g/molA: 313 g/mol; T: 304 g/mol; A-T base pairs = 617 g/mol G: 329 g/mol; C: 289 g/mol; G-C base pairs = 618 g/mol 1 genome copy = ~3 x 10 9 bp = 23 chromosomes (one member of each pair) 1 mole = 6.02 x molecules Standard DNA typing protocols with PCR amplification of STR markers typically ask for 1 ng of DNA template. How many actual copies of each STR locus exist in 1 ng? 1 genome copy = (~3 x 10 9 bp) x (618 g/mol/bp) = 1.85 x g/mol = (1.85 x g/mol) x (1 mole/6.02 x molecules) = 3.08 x g = 3.08 picograms (pg) Since a diploid human cell contains two copies of each chromosome, then each diploid human cell contains ~6 pg genomic DNA  1 ng genomic DNA (1000 pg) = ~333 copies of each locus (2 per 167 diploid genomes)

Short Tandem Repeats (STRs) the repeat region is variable between samples while the flanking regions where PCR primers bind are constant AATG 7 repeats 8 repeats AATG Primer positions define PCR product size Fluorescent dye label

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

Chemistry Involved Injection Injection electrokinetic injection process electrokinetic injection process importance of sample preparation (formamide) importance of sample preparation (formamide) Separation Separation capillary capillary POP-4 polymer POP-4 polymer buffer buffer Detection Detection fluorescent dyes with excitation and emission traits fluorescent dyes with excitation and emission traits virtual filters (hardware/software issues) virtual filters (hardware/software issues)

Sample Tube DNA - - Electrokinetic Injection Process Electrode Capillary DNA - - Q is the amount of sample injected r is the radius of the capillary c s is the sample concentration E is the electric field applied t is the injection time s is the sample conductivity b is the buffer conductivity  ep is the mobility of the sample molecules  eo is the electroosmotic mobility Rose et al (1988) Anal. Chem. 60: Q = s  r 2 c s (  ep +  eo )Et b

Separation Issues Run temperature o C helps reduce secondary structure on DNA and improves precision Run temperature o C helps reduce secondary structure on DNA and improves precision Electrophoresis buffer -- urea in running buffer helps keep DNA strands denatured Electrophoresis buffer -- urea in running buffer helps keep DNA strands denatured Capillary wall coating -- dynamic coating with polymer Capillary wall coating -- dynamic coating with polymer Polymer solution -- POP-4 Polymer solution -- POP-4

DNA Separation Mechanism + - DNA - Size based separation due to interaction of DNA molecules with entangled polymer strands Polymers are not cross-linked (as in slab gels) “Gel” is not attached to the capillary wall Pumpable -- can be replaced after each run Polymer length and concentration determine the separation characteristics

ABI 310 Filter Set F WAVELENGTH (nm) FAM JOENEDROX Laser excitation (488, nm) Laser excitation (488, nm) Normalized Fluorescent Intensity Fluorescent Emission Spectra for ABI Dyes

Sample Detection CCD Panel Color Separation Ar+ LASER (488 nm) Fluorescence ABI Prism spectrograph Capillary or Gel Lane Size Separation Labeled DNA fragments (PCR products) Detection region Principles of Sample Separation and Detection

15,1616,1720,2312,1430,30X,Y13.2,15 Evidence Area 1Area 2Area 3Area 4Area 5 AREAS OF DNA SAMPLE SexArea 6 Ref.Std.2 Ref.Std.1 15,1616,1720,2312,1430,30X,Y13.2,15 14,1517,1823,2413,1330,30X,X15,1914,1517,1823,2413,1330,30X,X15,19

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

PERKIN-ELMER’S PROFILER+ AND COFILER STATE OF TENNESSEE VERSUS TAYLOR LEE SMITH

JUST THE FACTS: NOT A MIXTURE? 1. Sperm Fraction: Eight of thirteen loci have a total of nine alleles not found in either the victim or the suspect. 2. Suspect Known: Eight of thirteen loci have a total of 12 different alleles not found in the sperm fraction “mixture”. 3. Victim Known: Ten of thirteen loci have a total of 11 different alleles not found in the sperm fraction “mixture”.

COINCIDENCE OR EVIDENCE? The likelihood ratios for producing homozygous genotypes at four of thirteen STR loci* with DNA from a single individual versus a mixture of DNA from two individuals. Theta = 0.03 Theta = 0.05 Theta = 0.03 Theta = 0.05 African American1 in278,000,0001 in43,000,000 Likelihood Ratio16,6006,500 Caucasian1 in183,000,0001 in27,500,000 Likelihood Ratio13,5005,200 Hispanic1 in15,000,000 1 in 3,700,000 Likelihood Ratio 3,9001,990 *Observed Sperm fraction genotypes: vWA=16, TPOX=8, D5S818=12, and D16S539=10).

The Future of Forensic DNA CODIS SNP’s & Chips

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

13 CODIS Core STR Loci with Chromosomal Positions CSF1PO D5S818 D21S11 TH01 TPOX D13S317 D7S820 D16S539D18S51 D8S1179 D3S1358 FGA VWA AMEL

STR Analysis by Hybridization on Microchips