Download DNA

Historical Information
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In 1953, James Watson and Francis Crick
discovered the configuration of the DNA molecule
In 1980, Ray White described first polymorphic
RFLP marker
In 1985, Alec Jeffreys isolated DNA markers and
called them DNA fingerprints
In 1985, Kary Mullis developed PCR testing
In 1988, the FBI starts DNA casework
In 1991, the first STR paper was written
In 1998, the FBI launches CODIS database.
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General DNA Information
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There are approximately 60 trillion cells in the body.
Each of these cells contain strands of chromosomes
Chromosomes are made of genes
A gene is the basic unit of heredity
Each gene is composed of DNA
DNA stands for deoxyribonucleic acid
DNA is a polymer which is a large molecule made by
linking repeating units
General DNA Information
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The repeating units of DNA are nucleotides
These nucleotides contain a sugar molecule (deoxyribose),
phosphate group and a nitrogen-containing base
There are four nitrogen-containing bases:
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Adenine
Cytosine
Guanine
Thymine
The bases always pair A to T and G to C
The only way that the base pairings can be properly
aligned is in a double-helix configuration
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General DNA Information
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The order in which the base pairs are arranged
determines the role and function of a DNA
molecule
The average human chromosome has DNA that
contains 100 million base pairs
DNA directs the production of proteins
A protein is a polymer made of amino acids
There are twenty known amino acids that are used
to make thousands of proteins
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General DNA Information
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DNA replication is the process of creating a new
strand of DNA from an existing strand of DNA
 The process begins with the unwinding of the
double helix followed by the “unzipping” of the
two strands
 Next, free nucleotides pair with each single strand
to create two identical double helix molecule
 Many enzymes and proteins are involved in this
process
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DNA Body Locations
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Can be found in all body cells--blood, semen,
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
Collection and Preservation of
Evidence
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DNA may be extracted from one cell so the
collection of even the smallest item is crucial
 All biological evidence should be photographed
close up and recorded in notes and sketches
 All body fluids and biological evidence must be
collected with a minimum of human contact
 Gloves should be changed frequently during the
collection process
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Collection and Preservation of
Evidence
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All clothing from both the victim and suspect
should be collected and bagged separately
Paper bags must be used for the collection of
biological evidence
A substrate control must be collected. This is a
sample from an unstained portion of the object
from which biological evidence has been collected
DNA evidence should be compared to the suspects
DNA
Blood and buccal cell samples should be collected
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Collection and Preservation of
Evidence
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If an individual is not available to give a
DNA sample, items should as a toothbrush,
razor and comb may be collected and
swabbed
Small items should be collected with clean
forceps. A new pair of forceps should be
used for each item
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At the Laboratory
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Once the evidence has been checked into the lab,
the technicians will need to extract the DNA from
the objects
 Contamination must be avoided so each piece of
evidence will be examined separately before
another piece is brought into the area
 After DNA has been extracted, the process of
DNA typing begins
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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.
Non-Coding Regions
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3 percent of the human DNA sequences
code for proteins
97 percent is non-coding and is repetitive;
repeating the same sequence over and over
50 percent of the human genome has
interspersed repetitive sequences
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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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The following are different ways to type DNA:
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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
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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 which causes
the strands to separate (unzip).
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 cooled down 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.
 Commercial kits are now available for easy PCR reaction
setup and amplification.
Problem: Contaminant DNA, such as fungal and bacterial
sources, will not amplify because human-specific primers
are used.
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
STR
Probability of Matching Previous STR
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
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+
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
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0.112
FGA
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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
The Future
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Greater automation of the DNA typing process
 Use of SNP’s--single nucleotide polymorphism
which measures a one nucleotide change or
difference from one individual to another. More
sites are needed to differentiate between
individuals (30 to 50 SNPs to attain the
frequencies of the 13 STR loci), but it can be done
with robots and automation.
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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.
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