Download DNA

Historical Information
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James Watson and Francis Crick--1953 discovered
the configuration of the DNA molecule
Ray White--1980 describes first polymorphic
RFLP marker
Alex Jeffreys--1985 isolated DNA markers and
called them DNA fingerprints
Kary Mullis--1985 developed PCR testing
1988--FBI starts DNA casework
1991--first STR paper
1998--FBI launches CODIS database.
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General DNA Information
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Double helix--two coiled DNA strands
Composed of nucleotides--unit
containing a sugar molecule
(deoxyribose), phosphate group and a
nitrogen-containing base
Four bases
•
•
•
•
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Adenine
Cytosine
Guanine
Thymine
Bases always pair A to T and G to C
DNA Body Locations
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Can be found in all body cells--blood, seme, saliva,
urine, hair, teeth, bone, tissue
 Most abundant in our buccal (cheek) cells
 Blood is 99.9% red blood cells that have no nuclei;
and therefore, no nuclear DNA
 DNA obtained from blood comes from white blood
cells
DNA TYPING
DNA typing is a method in which DNA is
converted into a series of bands that
ultimately distinguishes each individual.
Only one-tenth of a single percent of DNA
(about 3 million bases) differs from one
person to the next. Scientists use these
regions to generate a DNA profile of an
individual.
General DNA Typing Process
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Obtain a DNA sample.
Cut the DNA into small pieces at certain
chromosomes landmarks where there is
variation from person to person.
Load and run electrophoresis. This
separates the pieces by length.
Visualize the bands.
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DNA TYPING
“Fingerprinting”
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RFLP-- Restriction Fragment Length
Polymorphism
 PCR-- Polymerase Chain Reaction
 STR--Short Tandem Repeats
 Mitochondrial--use of maternal DNA in the
mitochondria
RFLP--Restriction Fragment
Length Polymorphisms
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Fragments are cut from the sequence of bases by a
restriction enzyme.
 The enzyme find its combination, bonds at one
end and dissolves through the DNA at the other.
 Fragments are loaded into a gel and run by
electrophoresis.
 DNA is extracted from the gel by blotting it into a
nylon membrane.
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RFLP--Restriction Fragment
Length Polymorphisms
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Radioactive phosphorus-32 probes are added to
the membrane which bond to the precise DNA
fragments making them radioactive.
 Then the membrane is placed over standard X-ray
film where the radiation emitted from the P-32
gradually exposes the film and shows the DNA
bands.
This process takes about 10 weeks to complete.
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PCR--Polymerase Chain
Reaction
PCR is a technique for making many copies of a
defined segment of a DNA molecule. It looks at six
different inherited traits, each controlled by a
specific gene. Every gene has at least two
alternative forms called alleles. An individual
receives one allele from mother and one from father.
If the alleles are the same, the individual is said to
be homozygous for the trait; if the two alleles are
different, the individual is heterozygous.
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PCR--Polymerase Chain
Reaction
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Heat the DNA strands to 94 degrees.
Add primers to the separated strands.
The primers combine with the DNA strands.
Add DNA polymerase and a mixture of free
nucleotides to the separated strands.
The tube is heated up to 72 degrees which
results in the rebuilding of a double-strand
of DNA.
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PCR--Polymerase Chain
Reaction
The outcome is a doubling of the number
DNA strands. Heating, cooling, and strand
rebuilding is repeated typically 25 to 30
times, yielding more than one million
copies of the original DNA molecule. Each
cycle takes less than two minutes from start
to finish.
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Advantages of PCR
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Minute amounts of DNA template may be used
from as little as a single cell.
DNA degraded to fragments only a few hundred
base pairs in length can serve as effective
templates for amplification.
Large numbers of copies of specific DNA
sequences can be amplified simultaneously with
multiplex PCR reactions.
Contaminant DNA, such as fungal and bacterial
sources, will not amplify because human-specific
primers are used.
Commercial kits are now available for easy PCR
reaction setup and amplification.
Short Tandem Repeats (STR)
STR is the latest method of DNA typing. STR’s
are locations (loci) on the chromosome that
contain short sequences of 3 to 7 bases that
repeat themselves with the DNA molecule.
This method’s advantages include a higher
discrimination than RFLP, less time, smaller
sample size, and less susceptible to
degradation.
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Short Tandem Repeats STRs
Counting the number of repeats by size comparison
Largest
AGCT AGCT AGCT AGCT
4 Repeats
AGCT AGCT AGCT
3 Repeats
AGCT AGCT
2 Repeats
AGCT
1 Repeat
Smallest
Short Tandem Repeats (STR)
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Extract the gene TH01 from the sample. (TH01
has seven human variants and a repeating
sequence of A-A-T-G)
 Amplify the sample by means of PCR
 Separate by electrophoresis
 Examine the distance the STR migrates to
determine the number of times TH01 repeats
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Short Tandem Repeats (STR)
Each person has two STR types for TH01--one
inherited from each parent.
By continuing the process with additional STRs from
other genes, you can narrow down the probability
of DNA belonging to only one possible person.
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STR
STR typing is visualized by peaks shown on a
graph. Each represents the size of the DNA
fragment.
The possible alleles are numbered for each
loci.
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Profiler Plus Allelic Ladders
VWA
D3S1358
AMEL
D8S1179
D5S818
FGA
D21S11
D13S317
D18S51
D7S820
COfiler Allelic Ladders
D3S1358
AMEL
D16S539
TH01
TPOX
CSF1PO
D7S820
My STR’s
Probability of Matching My STR’s
STR Allele Frequencies
45
40
TH01 Marker
Frequency
35
30
Caucasians (N=427)
Blacks (N=414)
Hispanics (N=414)
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20
15
10
*Proc. Int. Sym. Hum. ID
5
(Promega) 1997, p. 34
0
6
7
8
9
9.3
Number of repeats
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Electrophoresis
An electrical current moves through a
substance causing molecules to sort by size.
Smaller, lighter molecules will move the
furthest on the gel.
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Electrophoresis
Pipette the DNA.
Electrophoresis (cont.)
Load DNA into the gel
wells.
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Electrophoresis (cont.)
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Run the gel.
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Observe and compare
bands of DNA.
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Capillary Electrophoresis
(CE)
Fill with Polymer
Solution
Argon Ion
Laser
50-100 m x 27 cm
-
+
Burn capillary
window
DNA Separation occurs in
minutes...
Inlet
(cathode)
5-20 kV
Outlet
(anode)
Data Acquisition and Analysis
Three Possible Outcomes
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Match--The DNA profile appears the same.
Lab will determine the frequency.
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Exclusion – The genotype comparison shows
profile differences that can only be explained
by the two samples originating from different
sources.
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Inconclusive – The data does not support a
conclusion as to whether the profiles match.
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Types of DNA
Nuclear
Mitochondrial
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found in the nucleus
constitutes 23 pair of
chromosomes inherited
from both parents
each cell contains only
one nuclei
found in the cytoplasm
 is inherited only from
mother
 each cell contains
hundreds to thousands of
mitochondria
 can be found in skeletal
remains
Nuclear DNA is present in the head of the sperm. Mitochondrial DNA is
present in the tail. At conception, the head of the sperm enters the egg and
unites with the nucleus. The tail falls off, losing the father’s mitochondrial
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DNA.
Mitochondrial DNA
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Analysis of mtDNA is more:
• rigorous
• time consuming
• costly than nucleic testing of DNA
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mtDNA is constructed in a circular or loop
 37 genes are involved in mitochondrial energy
generation
 Is best used when nuclear DNA typing is not
possible
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Determining Probability
Databases are established by which one has
determined how often a particular allele on
a loci appears in a given population. By
increasing the number of alleles on different
loci the probability of having two people
with the exact combination becomes
astronomical.
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Probability of a Random Match Using 13 CODIS STR Markers
STR Marker #Alleles Random match probability (FBI Caucasian)
CSF1PO
11
0.112
FGA
19
0.036
TH01
7
0.081
TPOX
7
0.195
VWA
10
0.062
D3S1358
10
0.075
D5S818
10
0.158
D7S820
11
0.065
D8S1179
10
0.067
D13S317
8
0.085
D16S539
8
0.089
D18S51
15
0.028
D21S11
20
0.039
Product
0.000000000000001683
One in
594,059,679,247,540
1 in 594 trillion
Probability Numbers:
What do they mean?
1,000,000
1,000,000,000
1,000,000,000,000
1 x 1015
1 x 1018
1 x 1021
1 x 1024
1 x 1027
1 x 1030
1 x 1033
million
billion
trillion
quadrillion
quintillion
sextillion
septillion
octillion
nonillion
decillion
Present Work in DNA
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CODIS--Computer Based DNA Information
System
• A data base of DNA profiles of individuals convicted of
sex crimes and other violent crime
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TWGDAM--The Working Group for DNA
Analytical Methods
• Wrote the standards for DNA analysis that are part of a
national crime laboratory accreditation program
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Innocence Project
• Group of college law students work on cases where
DNA may prove innocence (or guilt)
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FBI’s CODIS DNA Database
Combined DNA Index System
 Used for linking serial crimes and
unsolved cases with repeat offenders
 Launched October 1998
 Links all 50 states
 Requires >4 RFLP markers
and/or 13 core STR markers
DNA--The Search for Innocence
DNA implicates the guilty and exonerates the innocent--R. Saferstein
Over the years many innocent people have been
wrongly convicted with no way of proving their
innocence. Since the late 1980’s, DNA
technology has been available. People, as a result,
have been reopening cases and testing the
evidence for DNA. Some have been exonerated
because their DNA did not match.
In this unit you will research a case in which DNA
was used to convict or vindicate the suspect. You
will work with a partner and will present your
findings to the class using PowerPoint.
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DNA Tree at the
St. Louis Science Center
Forensic Science classes
of Fall 2001 made
DNA ornaments to
decorate one of 14
theme Christmas trees
at the St. Louis
Science Center.
WORDS OF WISDOM
Keep in mind a famous
quote from Albert
Einstein, “ If we knew
what we were doing, it
would not be called
research, would it?”
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