DNA3 - DNA
 Jeanne)e
M.
Perr,
Ph.D.
...

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Unformatted text preview: DNA
 Jeanne)e
M.
Perr,
Ph.D.
 Forensic
Biology
 •  Biology
rela:ng
to,
used
in,
or
appropriate
 for
courts
of
law
 •  The
purpose
is
to
detect,
iden:fy
and
 classify
HUMAN
blood
and
body
fluids
from
 a
crime
scene,
missing
person,
mass
 disasters
 •  Forensic
biology
can
also
extend
to
other
 species
(i.e.,
dog,
cat,
deer,
etc.)
as
well
as
 plants,
microbes,
and
insects
 What
is
DNA?
 •  DNA
=
Deoxyribonucleic
acid
 •  Carries
gene6c
informa6on
 •  Arranged
along
chromosome
 •  Double‐stranded
helix
 •  Made
of
units
called
nucleo6des
 •  Sugar
molecule
 •  Phosphorous
group
 •  Nitrogen
containing
base
 The
DNA
Alphabet Four
Bases:
 
 
A
=
Adenine

 
 
G
=
Guanine

 
 
C
=
Cytosine

 
 
T
=
Thymine
 Complementary
base
pairing:
 
 
A≅T
(3x)
 
 
G=C
(2x)
 
 What
is
a
DNA
marker?
 DNA
markers
are

short‐cuts
to
genomic
 sequence
informa:on 
 Gene:c
polymorphisms
(differences)
 that
can
dis:nguish
one
individual/group
 from
another
 Resolu:on
that
is
obtained
depends
on
the
choice
of
 marker(s),
choice
of
technique
and
the
popula:on
 database
used
for
comparison/matching
 Human
Iden:ty
Tes:ng
 •  •  •  •  •  •  •  Forensic
cases
‐‐
matching
suspect
with
evidence
 Paternity
tes:ng
‐‐
iden:fy
father
 Historical
inves:ga:ons
 Missing
persons
inves:ga:ons
 Mass
disasters
‐‐
puVng
pieces
back
together
 Military
DNA
“dog
tag”
 Convicted
felon
DNA
databases
 Types
of
DNA
Markers

 •  Length
polymorphisms
 –  VNTR:
Variable
number
tandem
repeats;
locus‐specific;
non‐PCR
 based
 –  RFLP:
Restric6on
fragment
length
polymorphisms;
 experimentally
derived
 •  non‐PCR
and
PCR‐based
 –  STR:
Short
tandem
repeats;
interspersed
throughout
 •  PCR‐based
 •  Sequence
polymorphisms
 –  SNP:
Single
nucleo6de
polymorphisms;
base
changes
at
a
 par<cular
sequence
site
 DNA
Typing
 1985:
Sir
Alec
Jeffreys
at
Leicester
University
(UK)

 
Portions
of
the
DNA
structure
of
certain
genes
are
as
unique
to
 each
individual
as
fingerprints
 
Called
this
process
of
isolating
and
reading
DNA
markers,
“DNA
 fingerprinting”.
 This
was
later
developed
by
the
forensic
community
and
 others
into
a
technology
called
DNA
profiling
or
typing.

 DNA
typing
can
link
biological
evidence
(blood,
semen,
hair,
 saliva,
etc)
to
a
single
individual!!! 
 

 Jeffrey’s
Work
 •  Jeffreys'
DNA
method,
which
is
o\en
called
DNA
fingerprin:ng,
was
 first
put
to
use
when
he
was
asked
to
help
in
a
disputed
immigra:on
 case
to
confirm
the
iden:ty
of
a
Bri:sh
boy
whose
family
was
originally
 from
Ghana.
The
case
was
resolved
when
the
DNA
results
proved
that
 the
boy
was
closely
related
to
the
other
members
of
the
family,
and
 Jeffreys
saw
the
relief
in
the
mother's
face
when
she
heard
the
results.
 •  DNA
fingerprin:ng
was
first
used
as
a
police
forensic
test
to
iden:fy
 the
rapist
and
killer
of
two
teenagers,
Lynda
Mann
and
Dawn
 Ashworth,
who
were
both
murdered
in
Narborough,
Leicestershire
in
 1983
and
1986
respec:vely.
Colin
Pitchfork
was
iden:fied
and
 convicted
of
murder
a\er
samples
taken
from
him
matched
semen samples
taken
from
the
two
dead
girls.
 •  Another
early
achievement
was
to
confirm
the
iden:ty
for
German
 prosecutors
of
the
Nazi
Dr.
Menele,
who
had
died
in
1979,
by
 comparing
DNA
obtained
from
a
femur
bone
of
his
exhumed
skeleton,
 and
DNA
from
his
widow
and
son,
in
a
similar
way
to
paternity
tes:ng.
 DNA
Typing
Part
I:
RFLPs
 •  Not
all
of
DNA
molecule
codes
for
the
produc6on
of
proteins
 •  In
the
non‐coding
regions
of
DNA,
por6ons
of
the
DNA
 molecule
contain
sequences
of
lePers
that
repeat
numerous
 6mes,
so
called
tandem
repeats
 •  Up
un6l
the
mid‐1990’s
forensic
scien6sts
focused
on
 characterizing
these
repeat
segments,
called
Restric6on
 Fragment
Length
Polymorphisms
(RFLPs)
 Polymorphism - Length ATCG ATCG ATCG ATCG ATCG ATCG Variable
Number
of
Tandem
Repeats
 Restriction
Enzyme
Digest 
 ‐Experimentally
Derived
Lengths
 G C G C G C G C G C G C G C G C Separation of Fragments on Agrose Gels VNTR
Pattern
of
Bands
(DNA
bar
code)

 DNA
is
‘probed’
with
oligonucleotide
matching
the
repeat
 For
N
different
alleles,
the
total
possible
genotypes
is
given
by:
 RFLPs
 •  Match
the
“band”
sets

 
to
determine
a
match.

 •  For
each
loci
used,

 
use
mul:plica:on
rule.
 Restriction
Fragment‐Length
 Polymorphisms
for
Forensics 
 Five
RFLP
probes
provide
almost
exclusive
identity
 (~
1
in
109
individuals)
 RFLP
requires
a
minimum
of
25
ng
of
relatively
 undegraded
DNA
 Discrimination
power:
5
RFLP
probes
gave
high
 discriminatory
power
BUT
SLOW
process
 POP
quiz!!!
 Interpretation???
 Which
Suspect,

 A
or
B,
cannot
 be
excluded
from
 potential

perpetrators
 of
this
assault?
 DNA
Typing
II:
STRs
 •  Since
the
mid‐1990’s
Short
Tandem
 Repeats
(STRs)
have
taken
over
as
the
 preferred
method
of
DNA
typing
 •  An
STR
is
a
region
of
a
DNA
molecule
that
 contains
repea:ng
sequences
of
3‐7
bases.

 •  The
en:re
strand
of
an
STR
is
very
short:
 less
than
400
base
in
length.

 •  Because
they
are
short,
they
are
ideal
 candidates
for
mul:plica:on
by
PCR
 (polymerase
chain
reac:on)
 DNA
replica:on
 DNA
replica6on
 starts
with
unwinding
of

 the
strand
 Then
free
nucleo6des
 bind
and
assemble
along
 the
free
strands
with
the
 aid
of
enzymes
called
polymerases
 
 
 

 DNA
replica:on
ex
vivo
 •  Polymerase
Chain
Reac:on
(PCR)
 –  A
technique
for
replica:ng
or
copying
a
por:on
of
 a
DNA
strand
outside
a
living
cell
(ex
vivo).
This
 technique
leads
to
millions
of
copies
of
the
DNA
 strand.
 Kary
Mullis,
inventor
of
PCR
 Nobel
Prize
in
Chemistry,
1993
 PCR
 • Initialization
step
(94‐96°C
for
1‐9
minutes)
 • Denaturation
step
(94‐98°C
for
20‐30
seconds)
 • melting
DNA
template
and
primers
by
disrupting
 the
hydrogen
bonds
yielding
single
strands
of
DNA
 • Annealing
step
(50‐65°C
for
20‐40
seconds)
 • allowing
annealing
of
the
primers
to
the
single‐ stranded
DNA
 • Extension/Elongation
(75‐80°C)
 • DNA
polymerase
synthesizes
a
new
DNA
strand
 complementary
to
the
DNA
template
strand
by
 adding
dNTP's
that
are
complementary
to
the
DNA
 strand
 • Final
elongation
(70‐74°C
for
5‐15minutes)
 • after
the
last
PCR
cycle
to
ensure
that
any
 remaining
single‐stranded
DNA
is
fully
extended
 • Final
hold
(4‐15°C
for
an
indefinite
time)
 • may
be
employed
for
short‐term
storage
of
the
 reaction
 The PCR Process 5’ 3’ 3’ 5’ Starting DNA Template Forward primer 3’ Separate strands (denature) Add primers 5’ (anneal) 5’ 3’ 3’ 5’ 3’ Reverse primer Make copies (extend primers) Repeat Cycle, Copying DNA Exponentially PCR
Copies
DNA
Exponen6ally
through
Mul6ple
 Thermal
Cycles 
 Original
DNA
target
region
 Thermal
cycle
 In
32
cycles
at
100%
efficiency,
1.07
billion
 copies
of
targeted
DNA
region
are
created 
 Short
Tandem
Repeats
(STRs)
 Fluorescent
dye
 label 
 AATG 
 AATG 
 AATG 
 7
repeats
 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
 Primer
posi<ons
define
PCR
product
size
 Overview
of
Steps
Involved
in
STR
Human
DNA

Typing 
 TH01 
 D3 
 AMEL 
 D7 
 D5 
 VWA 
 D13 
 
 D21 D8 
 TPOX 
 CSF 
 D16 
 D18 
 FGA 
 Penta
D 
 Penta
E 
 Blood
Stain
 PCR
Amplification
with
Fluorescent
STR
Kits
and
 Separation
with
Capillary
Electrophoresis
 DNA
Quantitation
 using
Slot
Blot
 Genotyping
by
Comparison
to
Allelic
Ladder
 Multiplex
PCR
 •  •  •  •  Over
10
Markers
Can
Be
Copied
at
 Once
 Sensi:vi:es
to
levels
less
than
1
ng
of
 DNA
 Ability
to
Handle
Mixtures
and
 Degraded
Samples
 Different
Fluorescent
Dyes
Used
to
 Dis:nguish
STR
Alleles
with
 Overlapping
Size
Ranges
 AmpFlSTR® Identifiler™ 6FAM (blue) D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 VIC (green) D19S433 VWA TPOX D18S51 NED (yellow) AMEL D5S818 FGA PET (red) GS500 LIZ size standard LIZ (orange) Gene:c
Marker
for
Gender
 •  Amelogenin
gene
 –  Present
on
both
X
and
Y
chromosomes
 –  Shorter
by
6
base
pairs
in
X
than
Y
 –  Females
have
___
band(s),
Males
have
____
 band(s).

 Mul:plexing
and
Frequency
of
 Occurrence
 What
if
you
have
a
mix
of
male
DNA?
 •  STR
pa)erns
may
be
complex
and
overlapping
 –  (2
bands
per
marker)
x
(#
of
males)
 •  Y‐chromosome
STRs
(Y‐STRs)
 –  Only
one
band
per
STR
marker,
so
simpler
pa)ern
 –  Originates
only
from
a
male
donor
of
DNA
 Mitochondrial
DNA
 •  Mitochondria

 –  Located
outside
nucleus
 –  power
plants
of
our
bodies

 –  >100,000
in
each
cell
 –  Each
contain
several
loops
of
DNA
(mtDNA)
 •  mtDNA
analysis
 –  When
nuclear
DNA
is
not
available
 –  More
sensi:ve,
but
more
:me
consuming,
costly
 –  Two
highly
variable
regions
are
analyzed
 •  HV1
and
HV2
 mtDNA
 Advantage:
Can
use
a
small
quantity
of
DNA
as
mtDNA
exists
as
 more
than
one
copy
per
cell
 Disadvantage:
Maternally
inherited
so
a
mother
and
all
her
 children
will
have
the
same
profile
 Mixture
of
mitochondria
within
a
 single
cell
 Mixture
of
multiple

mtDNA
 sequence
types
in
a
given
 individual
 Leads
to
false
exclusions
 Collec:on
and
Preserva:on
of
DNA
 Evidence
 •  Before
any
collec6on,

 –  Notes,
sketches,
photographs
 •  Wear
latex
gloves!!
 •  No
plas6c
bags!!

Paper
bags
or
well‐ven6lated
mini‐box
(such
as
a
 swab
box)
 •  Light
and
heat
are
DNA’s
enemies!
Refrigerate
samples.
 Collec:on
of
reference
DNA
 •  Buccal
swab
 Develop
Investigative
Leads
 CODIS
Laboratories
 A
National
Network
of
DNA
Profiles
 Seattle Augusta Missoula Waterbury Ea Albany st La St. Paul en ns rid ing West. CO Me Suffolk CO Milwaukee Bethlehem ville Madison Gree Pierre Pikes Chi Pa nsbu cag ines rg Fairview W. Trenton o ville Heights Wa Wilmingto Lowell Charlest n on Qu shingto Springfield Cincinnati antic n, D Indianapolis C o Kansas City R St. Louis Frankfort ichmo Norfo lk nd Topeka Louisvile Knoxville Cl ev ela nd (2) Portland Cheyenne Reno Berkeley Oakland Salt Lake City Denver Great Bend Jefferson City Raleigh Flagstaff Wichita Los Angeles Nashville (2) S Scottsdale Little Rock Orange CO. Huntsville Phoenix Columbia Oklahoma City Birmingham Decatur Mesa San Diego Lubbock Garland Tucson Montgomery Dallas Jacksonville Ft. Worth (2) Tallahassee Mobile Austin (2) Orlando Tampa Ft. Lauderdale Houston (3) Anchorage San Antonio Miami ern an B ardin o 2003 Offender Profiles Forensic Profiles 1,493,536 70,931 2004 2,038,514 93,956 2005 2,826,505 126,315 2006 3,977,433 160,582 2007 5,372,773 203,401 2008 6,539,919 248,943 Investigations Aided Forensic Hits 11,220 3,004 20,788 5,147 30,455 7,071 43,156 9,529 62,059 11,750 80,948 14,122 National State Total Offender Hits 1,151 7,118 8,269 1,864 11,991 13,855 2,855 18,664 21,519 4,276 28,163 32,436 6,508 43,305 49,813 8,479 58,304 66,783 Innocence
Project
 Samuel
Scott
 
 Year
of
Incident:
1986
 Jurisdiction:
Georgia
 Sentence:
Life
+
 Year
of
Exoneration:
2002
 Sentence
Served:
15
years
 The
Innocence
Project
at
the
Benjamin
N.
Cardozo
School
of
Law,
created
by
Barry
C.
 Scheck
and
Peter
J.
Neufeld
in
1992,
is
a
non‐profit
legal
clinic.
This
Project
only
handles
 cases
where
post
conviction
DNA
testing
of
evidence
can
yield
conclusive
proof
of
 innocence.
 
 Exonera:ons
 •  About
25%
of
the
DNA
examina:ons
 conducted
by
the
FBI
laboratory
since
1989
 have
excluded
suspects
iden:fied
by
police
as
 the
source
of
the
DNA
evidence
collected
from
 the
crime
scene.

 http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm#Presentations DNA
in
Forensics
 Convicts
&
exonerates
 June
5,
2005:159
EXONERATED
 Reality
vs
Hollywood
 
 CSI‐MIAMI Questions?

 ...
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This note was uploaded on 03/06/2011 for the course CHS 3501 taught by Professor Perr during the Spring '09 term at FIU.

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