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

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

Where can DNA be found? SAME Cell Cell Types Blood Hair Roots Saliva Sweat What does DNA mean? DNA stands for deoxyribonucleic acid. This is just a big name for describing what DNA is made up from. Where can DNA be found? DNA is found in virtually all of our cells. Some examples of where cells are found are blood, hair roots, saliva, sweat, semen and various tissues. All the DNA from one individual will be the same no matter what cells are looked at. Thus, the DNA from blood will be the same as that from muscle. Semen Various Tissue

Where is DNA in the body? Cell Nucleus Where does DNA come from? DNA comes from your mother and father. Half from each. This DNA comes from your parents in the form of a Chromosome. A chromosome is the way the DNA is packaged. Like you would place something in a box to keep it all together. The chromosome would be the box and the DNA would be inside the chromosome.

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

Where is DNA in the body? Nucleus Paternal Chromosome Maternal Where does DNA come from? DNA comes from your mother and father. Half from each. This DNA comes from your parents in the form of a Chromosome. A chromosome is the way the DNA is packaged. Like you would place something in a box to keep it all together. The chromosome would be the box and the DNA would be inside the chromosome. Maternal Chromosome Paternal Chromosome

Where is DNA packaged in the body? Chromosome DNA Where does DNA come from? DNA comes from your mother and father. Half from each. This DNA comes from your parents in the form of a Chromosome. A chromosome is the way the DNA is packaged. Like you would place something in a box to keep it all together. The chromosome would be the box and the DNA would be inside the chromosome.

DNA- What does it look like? Double Helix Units A =Adenine G A T C T =Thymine G =Guanine DNA: - double stranded molecule - made of four bases - Sugar phosphate backbone - blue print of life and is the same in all nucleated cells - combination of the bases that determines a persons genetic make up - everybody’s genetic make up is different except for identical twins - inherited 1/2 from mother and 1/2 from father C =Cytosine

Sources of Biological Evidence Blood Semen Saliva Urine Hair Teeth Bone 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 does DNA come from? DNA comes from your mother and father. Half from each. This DNA comes from your parents in the form of a Chromosome. A chromosome is the way the DNA is packaged. Like you would place something in a box to keep it all together. The chromosome would be the box and the DNA would be inside the chromosome.

Where DNA Evidence is Found

Isolation of DNA Blood Hair Roots Saliva Sweat Tissue Chemical DNA The isolation process consists of taking part of the evidence say from a bloodstain or vaginal swab and placing it into a tube. I will then add some chemicals that will break open the cells and allow me to get the DNA. In the case of a vaginal swab sample I will continue to get the DNA from the sperm by adding a different chemical. Since sperm have a different outer coating I am able to separate the female cells from the male cells.

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

Amplification (making copies) DNA Solution

DENATURE Step one of a single cycle G T A C Heat T G C A

ANNEAL Step two of a single cycle A T G T A C

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

PCR (Polymerase Chain Reaction) Amplification PCR (Polymerase Chain Reaction) 5 Cycles 28 Cycles DNA 4 Cycles 3 Cycles 2 Cycles 1 Cycle The amplification or copy making of the DNA is what makes PCR so useful. I can start out with a small amount of DNA and after a bunch of cycles have a large amount of DNA.

Analysis of amplified DNA Profile

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 1996 – First mtDNA case 1998 - FBI launches CODIS database

DNA Use in Forensic Cases Most are rape cases or murders 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

Human Identity Testing Forensic cases -- matching suspect with evidence Paternity testing -- identifying father Historical investigations-Czar Nicholas, Jesse James 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 Extraction Quantitation 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

Extraction of DNA Blood Hair Roots Saliva Sweat Tissue Chemical DNA The isolation process consists of taking part of the evidence say from a bloodstain or vaginal swab and placing it into a tube. I will then add some chemicals that will break open the cells and allow me to get the DNA. In the case of a vaginal swab sample I will continue to get the DNA from the sperm by adding a different chemical. Since sperm have a different outer coating I am able to separate the female cells from the male cells.

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

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

An Example Forensic STR Multiplex Kit 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 bp 300 bp 200 bp Size Separation Color Separation D3 FGA vWA 5-FAM (blue) D13 D5 D7 NED (yellow) A D8 D21 D18 JOE (green) GS500-internal lane standard ROX (red)

Overview of Steps Involved in DNA Typing 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 DNA Quantitation using Slot Blot Genotyping by Comparison to Allelic Ladder

Calculation of DNA Quantities in Genomic DNA Important values for calculations: 1 bp = 618 g/mol A: 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 109 bp = 23 chromosomes (one member of each pair) 1 mole = 6.02 x 1023 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 109 bp) x (618 g/mol/bp) = 1.85 x 1012 g/mol = (1.85 x 1012 g/mol) x (1 mole/6.02 x 1023 molecules) = 3.08 x 10-12 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) Fluorescent dye label AATG AATG 7 repeats 8 repeats the repeat region is variable between samples while the flanking regions where PCR primers bind are constant Primer positions define PCR product size

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

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

Electrokinetic Injection Process Q is the amount of sample injected r is the radius of the capillary cs 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: 642-648 Q = s r2cs(ep + eo)Etb Capillary Electrode Sample Tube DNA- - DNA- -

Separation Issues Run temperature -- 60 oC helps reduce secondary structure on DNA and improves precision Electrophoresis buffer -- urea in running buffer helps keep DNA strands denatured Capillary wall coating -- dynamic coating with polymer 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

Fluorescent Emission Spectra for ABI Dyes 5-FAM JOE NED ROX ABI 310 Filter Set F 100 80 60 Normalized Fluorescent Intensity 40 20 520 540 560 580 600 620 640 WAVELENGTH (nm) Laser excitation (488, 514.5 nm)

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

Evidence Area 1 Area 2 Area 3 Area 4 Area 5 AREAS OF DNA SAMPLE Sex Area 6 15,16 16,17 20,23 X,Y 12,14 30,30 13.2,15 Ref.Std.2 Ref.Std.1 15,16 16,17 20,23 12,14 30,30 X,Y 13.2,15 14,15 17,18 23,24 13,13 X,X 15,19 14,15 17,18 23,24 13,13 30,30 X,X 15,19

Human Identity Testing with Multiplex STRs Simultaneous Analysis of 10 STRs and Gender ID AmpFlSTR® SGM Plus™ kit Two different individuals DNA Size (base pairs) amelogenin D19 D3 D8 TH01 VWA D21 FGA D16 D18 D2 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

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 African American 1 in 278,000,000 1 in 43,000,000 Likelihood Ratio 16,600 6,500 Caucasian 1 in 183,000,000 1 in 27,500,000 Likelihood Ratio 13,500 5,200 Hispanic 1 in 15,000,000 1 in 3,700,000 Likelihood Ratio 3,900 1,990 *Observed Sperm fraction genotypes: vWA=16, TPOX=8, D5S818=12, and D16S539=10).

Why the Y Chromosome? Applications forensic investigations (98% of violent crime by men) genealogical purposes evolutionary studies Advantages to Human Identity Testing male component isolated without differential extraction paternal lineages Needs population studies to evaluate diversity of haplotypes robust assay for accurate characterization of Y markers

Prinz et al. 1997 (Forensic Sci Int, vol. 85, pp. 209-218) Y STR Multiplex Assay Prinz et al. 1997 (Forensic Sci Int, vol. 85, pp. 209-218) 100 bp 400 bp 300 bp 200 bp DYS19 389I 389II 390 Primer Amounts Dye Y19 0.25 M JOE Y389 0.125 M FAM Y390 0.25 M JOE “Quadruplex I”

Mitochondrial DNA What is mtDNA Typing? Database and statistical issues

A Mitochondrial Exclusion

A Mitochondrial Inclusion

Mitochondrial Inconclusive?

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 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 TH01 D8S1179 D5S818 VWA FGA D7S820 CSF1PO AMEL D13S317 AMEL D16S539 D18S51 D21S11

Cold Hits and Solved Cases On August 25, 1979, an 8-year old girl was brutally raped and murdered in San Pablo, CA. Semen was collected from the body and placed in an evidence room, where it sat for 22 years. Through this program, a DNA profile was made and submitted to the state and federal databases. This resulted in a “cold hit” identifying Joseph Cordova Jr. as the suspect. Cordova was a habitual child molester who at the time of the DNA analysis was incarcerated in a Colorado prison. Cordova was subsequently charged with molesting, raping and murdering the 8-year old girl. On November 8, 2000, a 12 year old girl, was kidnapped off of the street in Rancho Cordova, CA, and driven to Feather River in Sutter County where she was sexually assaulted and then killed. Nine months later, Justin Weinberger was stopped for a traffic violation in New Mexico. A check by police revealed that Weinberger was wanted on a federal warrant for child pornography. He was detained and voluntarily provided a DNA sample. Analysis of that DNA sample resulted in a match with evidence identifying Weinberger as the suspect in this case. Weinberger was subsequently extradited to California where he was tried and convicted of the murder of the 12-year old girl.

STR Analysis by Hybridization on Microchips