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Forensic DNA Typing or Did you kill (rape

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How DNA can 'definitively' say. Adapted from: National Institutes of Science & Technology. http://www.cstl.nist.gov/div831 ... 1988 - FBI starts DNA casework ... – PowerPoint PPT presentation

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Title: Forensic DNA Typing or Did you kill (rape


1
Forensic DNA Typing or Did you kill (rape) that
person? How DNA can definitively say. Adapted
from National Institutes of Science Technology
  • http//www.cstl.nist.gov/div831/strbase/intro.htm

2
Brief History of Forensic DNA Typing
  • 1980 - Ray White describes first polymorphic RFLP
    marker (Restriction Fragment Length Polymorphism
    alleles).
  • Different RFLP for different people
  • 1985 - Alec Jeffreys discovers multilocus VNTR
    (variable number of tandem repeats) probes.
  • 1985 - first paper on PCR
  • 1988 - FBI starts DNA casework
  • 1991 - first STR paper (renaming of VNTR could
    be larger repeats, STR 4-6 bps. now using mostly
    4 bases )
  • 1995 - FSS (Forensic Science Service-UK) starts
    UK DNA database
  • 1998 - FBI launches CODIS (Combined DNA
    Information Service) Now FBI use 13 loci PCR
    identifies it in the quadrillions except for
    identical. Except for police mistakes, its done
    deal.

3
RFLPs Sickle Cell hemoglobin Case 1 Screening
for the sickle-cell gene
  • Sickle cell disease is a genetic disorder in
    which both genes in the patient encode the amino
    acid valine (Val) in the sixth position of the
    beta chain (betaS) of the hemoglobin molecule.
    "Normal" beta chains (betaA) have glutamic acid
    at this position.
  • The only difference between the two genes is the
    substitution of a T for an A in the middle
    position of codon 6.
  • This
  • converts a GAG codon (for Glu) to a GTG codon for
    Val and
  • abolishes a sequence (CTGAGG, which spans codons
    5, 6, and 7) recognized and cut by one of the
    restriction enzymes.         

4
Brief History of Forensic DNA Typing
  • 1980 - Ray White describes first polymorphic RFLP
    (Restriction Fragment Length Polymorphism)
    markerdetect to transferring to membrane. Probe
    w southern blot (radiological). Diff. RFLP for
    dif. People. Single rflp
  • 1985 - Alec Jeffreys discovers multilocus VNTR
    (variable number of tandem repeats) probes (stat.
    very impressive identical 4-6 bp that are spec. 7
    and 9 repeat, one from mom and dad, on chrom. 1-
    nowadays use pcr- but flanking sequence that is
    unique to chromo1)). Jeffreys almost ident.
    Typing. Now use PCR.
  • 1985 - first paper on PCR (Kerry Mullis)
  • 1988 - FBI starts DNA casework
  • 1991 - first STR paper ( renaming of VNTR could
    be larger repeats, STR 4-6 bps. now using mostly
    4 bases )
  • 1995 - FSS (Forensic Science Service-UK) starts
    UK DNA database
  • 1998 - FBI launches CODIS (Combined DNA
    Information Service) database. Now FBI use 13
    loci PCR identifies it in the quadrilians
    except for identical.

5
DNA Use in Forensic Cases
  • Most are rape cases (gt2 out of 3)
  • Looking for match between evidence and suspect
  • Must compare victims DNA profile

Challenges
  • Mixtures must be resolved
  • DNA is often degraded (stored wet- have mold,
    nuclease)
  • Inhibitors to PCR are often present

6
Human Identity Testing
  • Forensic cases -- matching suspect with evidence
  • Paternity testing -- identifying father
  • Historical investigations
  • Missing persons investigations
  • Mass disasters -- putting pieces back together
  • Military DNA dog tag
  • Convicted felon DNA databases

7
Steps in DNA Sample Processing
Sample Obtained from Crime Scene or Paternity
Investigation
Biology
Technology
Genetics
8
Sources of Biological Evidence
9
DNA in the Cell
10
DNA Amplification with the Polymerase Chain
Reaction (PCR)
11
PCR (Polymerase Chain Reaction) Copies DNA
Exponentially through Multiple Thermal Cycles
Oligos
DNA Poly. dUTP
1 copy
2 copies
4 copies
In 32 cycles at 100 efficiency, 1.07 billion
copies of targeted DNA region are created
12
Short Tandem Repeats (STRs) (say chromo 3)
Identical in all people
7 repeats
Identical in all people
8 repeats
the repeat region is variable between samples
while the flanking regions where PCR primers bind
are constant
Homozygote both alleles are the same
length Heterozygote alleles differ and can be
resolved from one another
13
Diff. PCR primers sets, can amplify the same
region. Different companies sell different kits.
TCAT repeat unit
Different primer sets produce different PCR
product sizes for the same STR allele
14
Choosing which STRs Significant statistical
variation but not too many. Freq. that are
measured in pop. Loc 1 -10. Loc 2 10 locus
12 -1/100. Random match with 13 primers 1/1013.
Variation Among STRs
15
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

Most rxns require 2 PCR (tubes) 7 or 8 primer
pairs in one tube need total of about 2 tubes
for 13 different STRs.
20-25 per rxn in lab. 150 incl labor. Cost for
forensic up to 1000.
16
An Example Forensic STR Multiplex Kit
17
Available Kits for STR Analysis
  • Kits make it easy for labs to just add DNA
    samples to a pre-made mix
  • 13 CODIS core loci
  • Profiler Plus and COfiler (PE Applied Biosystems)
  • PowerPlex 1.1 and 2.1 (Promega Corporation)
  • Increased power of discrimination
  • CTT (1994) 1 in 410
  • SGM Plus (1999) 1 in 3 trillion
  • PowerPlex 16 (2000) 1 in 2 x 1017

18
ABI Prism 310 Genetic Analyzer
5 min from inj. to output.
19
Close-up of ABI Prism 310 Sample Loading Area
See Technology section for more information on CE
20
D tells chromosome 21happens to be downs
syndrome. 2 peaks cause heterozygotic
Amelogenin amel protein that happens to be on sex
chromosome, tooth enamel top 2 peaks x and y.
(universal that two diff. people.) Bottom only 1
peak cause they have two X chromosomes.
21
STR Allele Frequencies
TH01 Marker
Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34
22
FBIs CODIS DNA Database
  • Combined DNA Index System --all 50 states can
    upload their convicted felony and seq. of
    unsolved cases. In Florida to convicted felon.
  • Used for linking serial crimes and unsolved cases
    with repeat offenders
  • Launched October 1998
  • Links all 50 states
  • Requires gt4 RFLP markers
  • and/or 13 core STR markers
  • Current backlog of gt600,000 samples

23
13 CODIS Core STR Loci with Chromosomal Positions
TPOX
D3S1358
TH01
D8S1179
D5S818
VWA
FGA
D7S820
CSF1PO
AMEL
D13S317
AMEL
D21S11
D16S539
D18S51
24
The End
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