The ABC s of DNA. Jerome F. Buting BUTING, WILLIAMS & STILLING, S.C. Wisconsin State Public Defender Annual Conference November 12, 2015

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1 The ABC s of DNA Jerome F. Buting BUTING, WILLIAMS & STILLING, S.C. Wisconsin State Public Defender Annual Conference November 12, 2015 Now what? The State has DNA evidence. Discovery Checklist Demand copy of Crime Lab s complete file, including: Chain of custody Computer printouts Lab worksheets Protocols in effect at time of testing Contamination log or record of anomaly or adventitious events Lab Sheets or Bench Notes Bench notes drawing Are the DNA results good or bad for your defense? exclude client from important evidence? exclude victim from important evidence? DNA profile for a third party? Third party: opportunity, access, and motive Theory of Defense Does inculpatory DNA really matter? Consent defense Or, absence of DNA where it would otherwise be expected is exculpatory Suppression motion? Trial defense of DNA: Yes, it s my DNA, but not because I did the crime... it was transferred to the scene or the weapon by another individual the real perpetrator.

2 Touch DNA The Basics of DNA Genetic Blueprint Directs the development of cells Inherited from Parents Can also be used to identify or exclude individuals Understanding the Language of DNA Nuclear DNA Mitochondrial DNA STR Alleles Loci Where Do We Find DNA? 1. Nuclear DNA is found in the nucleus of cells. 2. Mitochondrial DNA outside nucleus All cells except red blood cells, which have neither a nucleus or mitochondria. (DNA from blood comes from white blood cells). Chromosomes Each chromosome has DNA and proteins that code for different genes, traits, diseases, etc. Certain traits linked to person s sex, and thus are passed on through the #23 sex chromosome Alleles The way chromosomes are paired, we have two copies of every sequence in our DNA Each copy of a particular part of the sequence is called an allele. Most have two alleles for any genetic marker, or locus (position on chromosome/dna). Human Chromosomes Humans have 46 chromosomes, arranged in 23 pairs Numbered chromosomes 1-22 are called autosomes Benign polymorphisms = Junk DNA

3 These are the only areas of the DNA strand that are used in forensics. Unlike functional, DNA which code for cancer or other diseases, benign sections supposedly don t reveal any sensitive information about one s health It May Not Be Junk Scientists assume the area of DNA they are looking at has no hereditary linkage to each other This assumption plays a crucial role in statistical probabilities What DNA Is Used In Testing? Nuclear DNA, also known as Autosomal DNA most common, generally called STR DNA Mitochondrial DNA = mtdna Y-Chromosome, called Y-STR DNA Short Tandem Repeats: STR STR s currently in vogue for forensics The short tandem repeat is a short stretch of sequence (between 1-5 base pairs) that repeats several times without other nucleotides intervening between the repeats. 13 STR Original Markers used by FBI in CODIS Bodily Fluid Presumptive Tests Blood phenolphthalein Semen microscopic exam for sperm PSA P30 protein Saliva amylase enzyme What is Touch DNA? DNA that is transferred via an unknown method when object is handled or touched. By definition, it is not DNA transferred by blood, semen or saliva. Unclear whether transmitted by skin cells, or oils, or some other method. The Source of Touch DNA Sloughed off dead skin cells? Average human sheds 400,000 skin cells per day

4 - - there is usually no intact nucleus in dead skin cells yet nuclear DNA fragments have been found in experiments on touch DNA Sweat Or Oils? DNA transmitted via sweat, but the amounts vary greatly Some theorize DNA finds it way to skin surface through sebaceous oils. Transferred Via Saliva? People who habitually touch their face or mouth load their fingers with DNA from saliva, then transferred to object touched Yet not always positive presumptive tests for saliva. Why Does The Body s Source Of DNA Matter? Because it may have been deposited in an innocent manner, unrelated to crime hypothesis consistent with innocence? (reasonable doubt J.I.) Type of substrate Rough surface (grip of gun) better than smooth Fabric Vehicle How long can touch DNA stay on object? Could defendant s DNA be there because he was at scene at a time unrelated to crime? Raymond, J.J., et al, Trace DNA (2009): Outdoors up to two weeks Baggie in a drug case - full DNA profile 55 days after seizure Laptop keyboard - 62 days after seizure. Transference Secondary transfer of DNA 1. person to person to object: I shake your hand, transfer my DNA, you pick up gun, swab of your hands shows my DNA 2. person to object to person: I pick up knife, transfer my DNA, you pick up same knife, swab of your hands shows my DNA 3. person to object to object: I use bath towel, towel wrapped around gun, my DNA found on gun

5 Non-intimate contact transfer One study: transfer of female volunteer s DNA to both the underwear and penis of male volunteer even though no direct contact of female to those areas occurred. Scenario involved 1 minute of face touching, 3 minutes of handholding, followed by urination by the male Who handled the object last? Not possible to tell. Example: one person s DNA prevalent on a car key doesn t mean that person handled it last. Nor does it mean the key is more likely that person s key than a minor contributor. DNA/STR Typing Procedures Extraction Quantitation Amplification Interpretation Statistical Analysis How is evidence tested for DNA? Extraction Removing possible DNA-containing material from evidence: Swab Cut Put in tube with reagent Dissolve in buffer solution Extracting DNA Numerous steps (possible errors or contamination) takes several hours, including cook time at 132F Differential Extraction of Sperm and Non-Sperm Cells Two stage process, each with many steps Sperm cells harder to break open than epithelial cells(skin, saliva, vaginal fluids) Wash with weaker detergent to break open epithelial cells and release DNA Centrifuge into separate pellets or fractions (sperm and non-sperm, sometimes called male and female fractions) Differential Extraction (continued) Re-wash only the sperm fraction with stronger detergent to break open sperm cells and release sperm-cell DNA Result is supposed to be all sperm, i.e. male Always some epithelial cells (i.e., female?) wash over, but should be very few. Quantitation

6 Prepare plate with numerous wells or capillary tubes Run samples from numerous different cases at same time Computerized process, but room for human error setting up wells and instrument Measures amount or strength of human DNA in each tube or well, analyst selects certain ones to amplify DNA Lab Techs Applied Science Bio Robots Amplification PCR method amplifies by making many copies of each DNA strand DNA flows through gel by electrophoresis, which separates DNA because larger pieces move slower and smaller pieces move faster and are thus separated farther in the gel Gel Electrophoresis The ABI 310 Genetic Analyzer: SIZE, COLOR & AMOUNT ABI 310 Genetic Analyzer: Capillary Electrophoresis Electropherogram from GeneMapper Software Interpretation Lots of subjective decisions made at this stage Analyst reviews the GeneMapper printout Double checks the peak heights threshold Decides whether an apparent peak is just an artifact, like stutter or whether it s a real allele Enlarged GeneMapper Result Peak Detection Thresholds Different Labs have different peak height detection thresholds: Wisconsin and ASCLAD : above 150 RFU s can be used for inclusion, between requires caution, used for exclusion only NOTE: analyst can change height of peak on printout by changing RFU parameters Example Example enlarged

7 Example of handwritten notations Contaminated Control MC12 Mixtures Hottest area of debate in the field right now Many scientists are split on how to compute probability when there are more than two people Computerized probabilistic genotype analysis (TrueAllele) Statistical Analysis Final step before writing report Generally considered pretty straightforward if the DNA is single source (but that is now changing) More complicated if mixture of DNA is found Many scientists challenge use of probability equations in mixture cases Product Rule Use of product rule is why they get astronomical probabilities: like one in one trillion. Product rule only valid if genetic markers are independently inherited, i.e. no linkage Independent vs. Linked Genes that code for legs, eyes and ears in humans are linked so we get two of each Genes that determine eye color are independent of genes that code for hair color. The Loci Tested in Forensic DNA Have No Known Purpose Found in region of DNA known to be highly variable, but no one has a clue why they are there; for what purpose they are coded. So can t really be certain they aren t ever linked. Resources DNA Transfer DNA transfer within forensic exhibit packaging: potential for DNA loss and relocation, Forensic Sci. Int. Genet. 6 (2012) The retention and transfer of spermatazoa in clothing by machine washing, Can. Soc. Forensic Sci. J. Vol 29 (1996). DNA Transfer: review and implications for casework, Forensic Sci Int. Genetics, Vol 7 (July 2013)

8 Resources - Probabilities Inside the Cell: The dark side of forensic DNA (Nation Books, 2015), Erin Murphy (NYU Law professor) The dark side of DNA databases, TheAtlantic.com, Oct 8, 2015 State v. Wakefield, 47 Misc.3d 8509 N.Y.S.3d N.Y. Slip Op Feb 9, 2015 (allowing TrueAllele, by Cybergenetics, as evidence under Frye test, which employs automated computerized probabilistic genotype analysis)