Lecture 2: DNA Analysis

Speaker 1: DNA Analysis with Wayne Walker

• Works at indian river crime lab in ft. pierce, Fl

  • Cover “treasure coast”

• A serologist

• Walker’s background

  • Graduated UCF in 2007

  • BS in forensics (biochem) & minor in chem and micro bio

  • Interned at FDLE in drug chem section

  • Went forensic tech -> analyst

• Cocaine is branded

• What does a forensic biologist do?

  • Examine evidence to find bio material, then develop DNA profile from any bio evidence found

• Sources of biological evidence

  • Blood, semen, saliva, urine, hair, teeth, bone, tissue, epithelial cells

• Limitations of DNA testing

  • Temperature, sunlight, humidity

  • Age of stain

  • Contamination

  • Material stain is on

• Conventional Serology

  • Identify body fluids through visual exam-> presumptive testing -> confirmatory testing

    • Small amounts of sample are typically used in analysis b/c you should only use as much as you need so you have some leftover

    • In military, if you're going to use all of the sample you have to tell prosecution & defense attorneys so they can come and witness you use all the evidence

  • Evaluate hair for DNA potential

• Tests for blood

  • Presumptive: Kastle-meyer

    • Take a piece of filter paper and rub it on blood, if it turns pink positive- if not, negative

  • Confirmatory: Hematrace

    • Used to detect if blood is human (or ferret,,,?)

    • Only done as an extra step to check

• Tests for Semen

  • Presumptive: acid phosphatase

    • Place filter paper and watch colour again, same as kastle meyer but turns red

    • Requires confirmatory test as many things can cause false positive (pre pubescent vaginal fluid, pregnant women vaginal fluid, feminine products)

  • Confirmatory: p-30

    • Microscopic exam

• Tests for saliva

  • Presumptive: amylase plate

    • Agarose gel with starch tests for presence of alpha amylase from saliva

  • Also presumptive: SALIgAE

    • Proprietary…

    • Saliva makes it turn yellow

• Tests for hair

  • Presumptive: visual examination

    • Look at medulla to determine if its human- ours is amorphous, animals have other distinct patterns

    • Anagen hair is suitable for DNA (has lots of pigment, attached at the root)

    • Telogen hair is NOT suitable for DNA (not much pigment, not attached at the root; already dead and removed itself from your root)

• What is DNA?

  • Double helix that makes up your genome

  • Compressed into chromosomes

    • 23 pairs found on humans, with the last pair being made up of a chromosome from each parent

• Why do we use it?

  • Same in all cell types

  • Two copies: one from each parent

  • Stays the same through your whole life and is unique to everyone

  • Stable molecule

• Types of DNA testing

  • Nuclear testing (STR & PCR based)

  • Mitochondrial testing (maternally inherited)

  • Y-STR testing (on y chromosome, paternally inherited)

• STRs

  • 2-7 BP long, side by side repeats

  • Same thing over and over

  • CAT CAT CAT CAT

  • 21 assigned loci; don't tell you about person’s genetics

• DNA Analysis

  • Stain is ID’d

  • Cutting or swabbing taken placed into tube

  • 5 step process begins:

    • 1. Extraction

      • Add heat and silica beads to attract DNA, press silica beads to magnet once DNA is attached

      • Can be done either:

        • Manually

        • Robotics (B3K/automated)

          • DNA IQ

          • Prepfiler

      • Differential extraction done to extract sperm specifically, making two samples from one

    • 2. Quantification

      • Estimate of amount of DNA in sample

      • Useful in quality control and analysis

      • Done through Real time PCR (qPCR) 

    • 3. Amplification

      • Done with PCR!

      • Repeated 29 times

      • Done with commercial kits like Globalfiler & Powerplex fusiona

    • 4. Separation

    • 5. Analysis of Data

  • 1 nanogram of DNA is ideal for amplication

  • Three possible outcomes:

    • Inclusion

      • Individual cannot be excluded as a potential contributor of DNA to evidence sample

    • Exclusion

      • Individual is eliminated as a potential contributor of DNA to the evidence sample

    • Inconclusive

      • DNA profiling results are not detailed enough to know or not

  • Once person is included…

    • Apply likelihood ratio: LR= Pr(E/Hp)/ Pr (E/ Hd)

      • E= evidence DNA profile

      • Hp= prosecutors hypothesis- DNA belongs to POI

      • Hd= defense hypothesis- DNA belongs to someone else

    • If LR> 1, Hp is supported

    • If LR<1, Hd is supported

    • If Lr=1, it doesn't support either

  • Ex. 

    • E is 15,15

    • POI is 15,15

    • Frequency of 15 allele is .3

    • Remember hardy-weinberg (for homozygote, p^2+p(1-p) )

    • Applying formula: LR= 1/ hardy weinberg= 1/.0963= 10.38

    • Bc the evidence profile is approximately 10 times more likely to be observed if it originated from POI than if it originated from someone else.

  • Probabilistic Genotyping

    • Use bio modeling, statistical theory, computer algorithms, & probability distributions to infer genotypes & calculate LRs

    • Genotype weights are primary output

    • Multiple PG software available

      • Ex. STRmix

• Paternity Statistics

  • For one obligate paternal allele: 2p-p^2

  • For two: 2(p+q)-(p+q)^2

  • Parentage index is approximately 1 billion

• CODIS

  • FBI established DNA database containing DNA profiles from convicted offenders, forensic crime scene samples & missing persons

  • Allows searches of profiles between states

• Want to look at some cases?

  • Case #1

    • Store owner robbed and tied up, suspects were wearing blue masks that they dropped on the way out

    • Masks provided techs with full DNA profiles of each suspect

  • Case #2

    • Stolen vehicle- steering wheel swabbed

    • Provided 4 person mix

  • Case #3

    • Multiple burglaries with similar MO

    • First time burglarizing occupied home

    • Pizza box found on counter

      • Suspect took a bite from a piece and then left the crust

    • Suspect ID’d who was near scene of multiple of the burglaries

Lecture 2: Case Studies

• Case #1: Bullet Lead Analysis

  • First used after kennedy assasination

  • Used chemistry to link CSI bullets to those posessed by suspects

    • Theory: each batch of lead had a unique elemental makeup

  • In 2004, ruled unreliable and misleading b/c of how overstated connection between bullets was

  • FBI responded by saying “sir yes sir”, but never went back to remedy any cases that may have been a mistrial bc of inclusion of bullet lead analysis as evidence

  • Brought to light by metallurgist William Tobin, a whistleblower

    • Found that bullets made in the same batch did not always match

  • In 2007, identified 250 cases of concern (12 of which were reversed)

    • FBI finally admitted to proposing erroneous testimony

  • FBI used faulty statistical calculations to declare bullets matched even when they were slightly off, and overstated significance of match

    • Basically claimed that if error bars were close enough to overlapping, should be considered statistically similar

• Case #2: Microscopic Hair Comparison

  • Another flawed FBI testimony!

  • Found 26/28 of examiners overstated comparison match done by hair examiners

  • Came to light from a Washington Post Report

  • FBI did acknowledge immediately that there was an error, and that they didn’t consider confirmation bias, prosecutorial bias and outcome bias

    • But claimed it wasnt their fault because there wasnt enough hair research