Download Extracting DNA

“The capacity to
blunder slightly is the
real marvel of DNA.
Without this special
attribute, we would still
be anaerobic bacteria and
there would be no music.”
—Lewis Thomas, Physician, author
DNA Analysis
Students will learn:
 That DNA is a long-chain polymer found
in nucleated cells, which contains genetic
information.
 That DNA can be used to identify or
clear potential suspects in crimes.
 How DNA is extracted and
characterized.
 How to apply the concepts of PCR and
STRs to characterize DNA.
 The role that statistics plays in
determining the probability that two people
would share a DNA profile
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DNA Analysis
Students will be able to:

Explain that DNA is a long molecule, tightly
packed in the form of a chromosome with
genetic material wrapped around it.
 Isolate and extract DNA from cells.
 Describe the PCR process
 Describe STR testing and calculate probabilities
of identity using STR.
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Historical Information

1953---James Watson and Francis Crick—
discovered the configuration of the DNA molecule

1980---Ray White describes first polymorphic
RFLP marker

1985---Alec Jeffreys isolated DNA markers and
called them DNA fingerprints

1983---Kary Mullis developed PCR testing

1988—FBI starts DNA casework

1991—first STR research paper published

1994---DNA Identification Act initiates
database/CODIS

1998—FBI launches NDIS/CODIS database
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People of Historical
Significance
James Watson, Francis Crick, and Maurice Wilkins
jointly received the Nobel Prize in 1962 for their
determination of the structure of DNA. What is
interesting about this fact is that Rosalind Franklin had
as much to do with the discovery as the other three men
with her work with X-ray crystallography. She died of
cancer and could not be honored for her work.
Kary Mullis received the 1993 Nobel Prize for his 1983
discovery of PCR, a way to quickly make copies of DNA
in the lab.
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General DNA Information
 Double helix—two coiled DNA strands
 Composed of nucleotides—units containing a sugar
molecule (deoxyribose), a phosphate group and a
nitrogen-containing base
 In humans, the order of these bases is 99.9% the
same.
 Four bases
 Adenine
 Cytosine
 Guanine
 Thymine
 Bases always pair A to T and G to C
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Where Is DNA Found?
 DNA is found in all nucleated body
cells—white blood cells, semen, saliva,
urine, hair root, teeth, bone, tissue
 Abundant in cheek cells
 Red blood cells have no nuclei, and
therefore, no nuclear DNA
 DNA obtained from blood comes from
white blood cells
 Genes are portions of DNA that code for
specific proteins
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Extracting DNA

Break open the cell
 Release DNA from
the proteins that
package it in the cell
 Isolate the DNA
from the extraction
solution by
precipitation
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DNA
Extraction
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DNA Typing


DNA typing is a method in which DNA
is characterized by
length differences
or
sequence differences.
Only 0.1 % 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.
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Non-Coding Regions
 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. These
show great variety between people
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Uses of DNA Profiling
 To identify potential suspects
 To exonerate individuals
 To identify source of biological evidence for
reconstruction of crime
 To link or exclude individuals
 To identify crime and casualty victims
 To establish paternity
 To match organ donors
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DNA TYPING
 RFLP—Restriction Fragment Length
Polymorphism (fingerprinting)
 PCR—Polymerase Chain Reaction
 STR—Short Tandem Repeats
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POLYMORPHISM

MANY
FORMS
 These are regions of DNA where individuals
differ
• They can differ by LENGTH
• RFLP
• STR
• They can differ by SEQUENCE or ORDER
• Dot blot
• Sequencing
• SNP
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RFLP—Restriction Fragment
Length Polymorphisms
Restriction enzymes are used to cut DNA into
smaller fragments that can then be separated
and characterized for identification
 Isolate—separate DNA from the cell
 Cut—using restriction enzymes to make shorter
base strands
 Sort—by size using electrophoresis
 Probe selected regions of DNA
 Analyze—the specific alleles for identification
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RFLP



The first DNA typing used for forensic
work
Advantage: Very discriminating
Disadvantage: Requires a large
sample
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Electrophoresis
Pipette the DNA.
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Electrophoresis
Load DNA into the
gel wells.
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Electrophoresis
 Run the gel.
 Observe and
compare bands
of DNA.
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PCR—Polymerase
Chain Reaction
PCR is a technique used for making
copies of a defined segment of a DNA
molecule. This can be valuable when
the amount of evidence is minimal.
Millions of copies of DNA can be made
from a single speck of blood.
The PCR Song
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The Brains Behind PCR

Kary Mullis

This link takes you to the Nobel Prize
site which has a great deal of
information about PCR, as well as an
interview of Dr. Mullis.
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PCR—Polymerase Chain
Reaction Procedure
 Mix DNA, polymerase, nucleotides
 Heat to denature...separate strands
 Cool to anneal the primer...target area of
interest (loci)
 Heat to extend...copy
 Repeat 30 times for about one billion
copies!
 Each cycle takes about 2 minutes and
doubles the number of copies
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Test your PCR knowledge
Play this game:
PCR Mystery Game:
The Eye of the Donkey

More on PCR:
See power of PCR multiplication
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Advantages of PCR
 Tiny amounts of DNA may be tested
 Degrade DNA may be tested.
 Large numbers of copies of specific DNA
sequences at different regions of DNA (loci)
can be amplified simultaneously with multiplex
PCR reactions.
 Commercial kits are now available for easy
PCR reaction setup and amplification.
Contaminant DNA, such as fungal and
bacterial sources, will not amplify because
human-specific primers are used. However,
human contamination can be a problem.
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Electrophoresis
 A technique used to separate DNA
fragments.
 An electrical current is moved through a
gel substance causing molecules to sort
by size.
 The smaller, lighter molecules will move
the furthest on the gel.
 The fragments can be visualized for
characterization.
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Short Tandem Repeats
(STR)
STR is a method of DNA typing.
STR’s are locations (loci) on the chromosome
that contain short sequences of 2 to 5 bases
that repeat themselves in the DNA molecule.
The advantages of this method are that it
provides greater discrimination, requires
less time, a smaller sample size, and the
DNA is less susceptible to degradation.
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Short Tandem Repeats
(STR) Procedure
 Extract the DNA
 Amplify the sample by means of PCR
 Separate by electrophoresis
 Examine the distance the STR migrates
to determine the number of repeats
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STR Gels
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Short Tandem Repeats
(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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Short Tandem Repeats
(STR)
 Each person has two STR types at each
locus, 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 probable person.
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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 Example
Determining Probability



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Databases determine 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.
Follow the product rule for
independently inherited traits.
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DNA Interactive
The website below has a STR
animation demonstration. Click on
human identification, profiling and then
on the third circle called Today’s DNA
Profiling to see the demonstration.
http://www.dnai.org/d/index.html
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Three Possible Outcomes
 Match—The DNA profile appears the
same. Lab will determine the frequency.
 Exclusion—The genotype comparison
shows profile differences that can only be
explained by the two samples originating
from different sources.
 Inconclusive—The data does not
support a conclusion as to whether the
profiles match.
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Types of DNA
Nuclear
Mitochondrial
 found in the nucleus
 found in the cytoplasm
 constitutes 23 pair of
 is inherited only from
chromosomes inherited
mother
from both parents
 each cell contains
 each cell contains only
hundreds to thousands
one nuclei
of mitochondria
 can be found in skeletal
remains, degrade, or
low quantity samples
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Mitochondrial DNA
 Analysis of mDNA is more:
 rigorous
 time consuming
 costly than nucleic testing of DNA
 mDNA is constructed in a circular or loop
 37 genes are involved in mitochondrial
energy generation
 Is used when nuclear DNA typing is not
possible
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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
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The Future
 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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People in the News
Sir Alec Jeffreys is credited with DNA profiling using
RFLP. In September of 1984 after years of work, he
saw his first series of blots on an X-ray. The
technique was first used in forensics, when in 1985
he was asked by police to confirm the rape
confession of 17 year old Richard Buckland, who was
denying a rape of another young woman. he DNA
from Buckland and the DNA taken from the victims
eliminated him as a suspect. Jefferys then used
samples from other suspects to later convict Colin
Pitchfork whose DNA did match.
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More about DNA
For additional information about DNA
and some famous cases, check out
Court TV’s Crime Library at:
www.crimelibrary.com/criminal_mind/forensics/dna/1.html
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