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Forensic DNA Analysis

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Forensic DNA Analysis Powered By Docstoc
					Forensic DNA
Analysis
   SFS3f. Compare short tandem repeat
   patterns (STR) and relate to identifying
   the DNA of an individual.
   SFS3g. Explain the use of the DNA
   database for DNA profiling.
EQ
n   What is DNA?
n   How is DNA evidence analyzed by
    Criminalist & how has it become
    useful to criminal investigations
    over the past quarter century?
What is DNA?
n   DNA “fingerprinting” is a common
    way to ID people by their unique
    genetic code.
n   DNA is a double stranded
    molecule located in the nucleus of
    a cell; Every cell in an organism
    contains the same DNA.
n   Characteristics of DNA varies btwn
    individuals w/in a species & btwn
    species.
What is DNA?
        n   DNA- deoxyribonucleic
            acid.
        n   Long-chain molecule
            made up of four bases
            that pair together & held
            together by hydrogen
            bonds.
        n   A=T; G=C
        n   In humans- order of
            bases are 99.9% the
            same.
What is DNA?

           n   DNA is contained
               in chromosomes
           n   Chromosomes
               contain DNA &
               associated proteins
               (which allow DNA
               to
               condense/wind/coil
               up tightly)
What is DNA?
n   Genes are
    sequences of
    DNA nucleotides
n   Genes are
    located on
    chromosomes
n   Alleles: different
    types of the same
    gene (dominant,
    recessive, co-
    dominant, etc).
Forensic DNA analysis seeks
nucleotide variation in DNA
1.    1. Point mutations from errors in
      DNA
     replication
        F   – RFLP (Restriction Fragment
            Length Polymorphism) analysis
1.   2. Variable number tandem
     repeats (VNTRs)
        F   – STR (Short tandem repeat)
            analysis
Why is there nucleotide variation between
the DNA of individuals of the same
species?
    n   All the DNA
        nucleotides in a cell
        must be copied
        (replicated) prior to
        cell division.
    n   Random copying
        errors are made
        during DNA
        replication.
    n   DNA replication
        copies DNA
        nucleotides
         u Every human cell
           contains 3.4 billion
        nucleotide pairs.
DNA replication
Objectives of Forensic
    DNA Testing
    n   To link an individual to a crime
        scene/criminal act.
    n   To exonerate suspects.
    n   To identify victims of mass
        disasters.
    n   To determine Paternity.
    Specimen Samples used
       for Forensic DNA
            Testing
n   Blood (what components?)
n   Semen
n   Saliva
n   Bones
n   Skin scrapings
n   Hair follicular tag
Main Procedures for
DNA Fingerprinting:
n   Isolation of the DNA to separate
    the DNA from the cell.
n   Cutting w/ a restriction enzyme to
    make shorter base strands.
n   Sorting the segments by size,
    using electrophoresis
n   Analyzing the resulting print by
    identifying specific alleles/ genes.
DNA Fingerprinting
Analysis:
n   RFLP (Restriction Fragments
    Lengths polymorphism)-use
    restriction enzymes to cut the DNA in
    fragments that are many diff lengths
    and shapes. The length vary from
    person to person.
    n   Restriction Enzyme-recognize a specific
        sequence of bases & cut the DNA
        molecule at specific points.
    n   Electrophoresis-a procedure that sorts/
        separates DNA fragments by size.
Early 1980s: Restriction Fragment
Length Polymorphism (RFLP)
                    n   Genetic variation in the distance
                        between restriction enzyme sites
                    n   Template DNA digested by
                        enzymes, electrophoresed,
                        detected via Southern blotting
Sir Alec Jeffreys
                    n   Power of discrimination in the
                        range of 106-108 for a six probe
                        analysis
Mechanisms
for RFLPs
    The Catch:
n   RFLP testing requires a relatively
    large amount of HMW DNA
    (~50ng = thousands of cells)
n   Not ideal for forensic evidence, in
    which small, degraded samples
    are common
                   2. PCR To The Rescue!
                   n   Polymerase Chain Reaction =
                       molecular Xeroxing
                   n   Three temperature phases, carried
                       out in a Thermal Cycler, replicate/
                       copy or “amplify” the desired DNA
Dr. Kary Mullis
Eccentric Genius       fragment(s)
    PCR (cont’d)
n   Works with lower quantity (1-2ng),
    lower quality samples
n   Power of discrimination goes from
    102-106...not good enough for
    databasing
3. The Current Method of Choice:
Autosomal Short Tandem Repeats
             (STR)
         n   Non-coding, tetranucleotide
             sequences which vary greatly from
             person to person in the number of
             repeating units
         n   Requires <1ng of DNA to type 13-
             15 STR loci
         n   Power of discrimination ranges
             from 1014-1023. World population
             is 109 so bring on the database!
Applied Biosystems 310 Genetic Analyzer
      The Process In a Nutshell
Amplified DNA samples are injected
into a capillary. Fluorescent tags on
the DNA fragments are excited by a
laser as they pass a window in the
capillary, the fluorescence is recorded
by a camera, and this signal is
converted into a “peak” by the
computer software.
        STR data


X, Y,
   XY
STR data (cont’d)
                STR data (cont’d)




“The DNA profile obtained from Item 25(S) matches the
DNA profile of the suspect. The combination of genetic
marker types exhibited by Item 25(S) and the suspect
occurs in approximately one in one hundred quadrillion
(1017) individuals…”
    How are these astronomical figures derived?

The product rule: combined probability of a series of independent
events is determined by multiplying the probabilities of each event.
STR loci are inherited independently (unlinked)
Homozygous loci: p2 (same allele inherited from mother and father)
Heterozygous loci: 2pq (either allele could be inherited from either
parent)
p(17)2 x 2p(15)q(17) x 2p(23)q(26)….
(.223)2 x 2(.083)(.25) x 2(.14)(.02) = .000013, which is equivalent to a
probability of one in 76,000 using just 3 of the 13 loci!
             STR Artifacts
-A (“minus A”): Incomplete addition of
nucleotide ‘A’ by DNA polymerase;
results in a peak that is one base pair
smaller than allele peak.
               STR Artifacts

Stutter: Slippage of DNA polymerase;
results in a peak that is four base pairs
(one repeat unit) smaller than allele peak.
               STR Artifacts

Pull-up: Incomplete filtration of spectral
overlap in fluorescent detection system.
Pull-up
                   DNA Mixtures




When more than one source of DNA is detected in a sample,
assignment of genotypes becomes more difficult.
       Degraded/Trace DNA Samples




Larger alleles “drop-out” when template DNA is low in
quantity or quality, reducing certainty of genotypes.
The Combined DNA Index System
           (CoDIS)
         n   A database of DNA profiles from
             violent felons and crime scene
             samples
         n   Laws concerning who is eligible for
             the database vary from state to
             state
         n   Database currently contains about
             2,038,470 felons and 93,956 crime
             scene profiles (19,00 hits so far)
The Mystical Power of CoDIS

       n   Extremely powerful investigative
           tool, linking crimes, and pulling
           suspects out of thin air!
       n   Can prevent, as well as solve
           crimes!
The Dark Side of CoDIS
 (What the FBI doesn’t want you to know.)

         n   DNA mixtures and degraded DNA
             profiles have lead to spurious
             matches
         n   Stringent laws explicitly permit
             databasing innocent people
         n   Adding arrestees to database
             violates presumption of innocence
         n   However, the prosecution rate on
             case to offender matches is
             shockingly low! (~10%)
PCR
n   Isolate the DNA from crime scene evidence to
    get a pure sample and cut target strands.
n   Mix the DNA samples, primer, DNA polymerase
    enzyme & nucleotides in tube. Place in PCR
    chamber.
n   Heat the dsDNA to separate to 95°C.
n   Cool to 50°C so that primers can attach to
    ssDNA.
n   Reheat to 72 °C so that DNA polymerase can
    attach to end of primer.
n   The DNA polymerase will now add
    complementary nucleotides to the ssDNA. Two
    new strands are made.
n   Repeat cycle until desired # of DNA is copied.
Electrophoresis
n   Isolate pure sample & cut the DNA into
    fragments.
n   Make agarose gel.
n   Add DNA with a buffer solution to wells in gel.
n   Place the gel in the electrophoresis plate so
    that a current passes through.
n   The DNA will migrate from one end to the next
    based on size.
n   Stain the gel with Ethidium Bromide when the
    electrophoresis is done.
n   Place under UV light to observe band.
n   Analyze/ compare bands for size comparison.
REview
 Take this scenario:             What system used by the FBI
     A woman gets out of           compiles DNA of known
     bed, watches TV, talks        violent offenders from all
     on the phone, pets her        over the nation and can be
     cat, and then shoots her      used to match DNA with a
     husband. She hides the        sample found at a crime
     gun and runs away.            scene?
     Which object would be      n   A. IAFIS          B. IBIN
     most useful to the            C. SICAR         D. CODIS
     forensic serologist in        E. DNAW
     finding out who the
     woman was?
n    A. the cat
n    B. the phone
n    C. the TV
n    D. the TV remote
n    E. none listed
Review
Review: Who’s DNA is the
   unknown?
  Suspect A Unknown Suspect B
Review
In this gel, the base-pair
    lengths are listed to
    the right. Which end
    of the DNA fingerprint
    was plugged to the
    NEGATIVE terminal
    during
    electrophoresis?

    A. Top
n   B. Bottom
    C. Left
    D. Right
Review
 In a talk-show scandal, it
     is revealed that one of
     three siblings has a
     different father.
n    GASP! Using the DNA
     fingerprint of just the
     mother, daughter and
     two sons, which
     sibling has a
     DIFFERENT father.
      A. Daughter
      B. Son 1
      C. Son 2
      D. all have the same
           father—go figure!
Review
Which suspect's
  DNA matches
  the felon's DNA
  left at the crime
  scene?
n A. suspect 1
  B. suspect 2
  C. suspect 3
  D. none listed
Review
Whose the daddy? This
   mother is trying to
   decide between two
   men who desperately
   want to support her
   and her newborn
   baby. Both want to be
   a part of the baby's
   life, because they love
   the mother so much.
   Who gets the honor
   and privilege?
n  A. Dad 1
n  B. Dad 2
n  C. neither man
   D. both
REview
Compare the sperm
  DNA to the two
  suspects and the
  boyfriend. Which
  male is most
  likely the
  assailant?
  A. suspect 1
  B. suspect 2
  C. boyfriend
  D. none listed
Review
In this DNA fingerprint,
    the blood sample
    taken from a crime
    scene is not the
    victim's and so is
    assumed to be the
    perpetrator. Which
    individual is the best
    suspect?
    A. Bob       B. Sue
    C. John      D. Lisa
    E. none
Review
There was a mix-up
   at the hospital.
   Which of these
   children belong
   to the parents?
n  A. All of the
   children
n  B. Children 2, 3
   &6      C.
   Children 1 & 5
   D. Children 2 & 4

				
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