Download DNA - Ms Futch

What do these items have to do
with one another?
Deoxyribonucleic Acid (DNA)
Forensic Science
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Possible Sources of DNA
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Skin
Sweat
Blood
Mucus
Saliva
Tissue
Semen
Urine
Hair (root)
Ear Wax
Vaginal or rectal cells
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Introduction to DNA
• Like fingerprints, DNA is unique to each
individual  individual evidence!
• DNA can definitively link a suspect to a victim or
crime scene.
• The primary unit is called a gene
• Each gene contains DNA that controls our genetic
traits
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Structure of DNA
double
helix
• DNA (deoxyribonucleic acid) is a molecule
comprised of repeating units called nucleotides
– A nucleotide consists of
• Sugar-phosphate backbone
– Deoxyribose sugar
– Phosphate
• Nitrogenous base
– adenine, guanine, cytosine, thymine
• Adenine bonds only to thymine, and
guanine bonds only to cytosine
• The order of bases is our genetic code!
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The Bases
• The bases on each strand [adenine (A), guanine
(G), cytosine (C), and thymine (T)] are properly
aligned in a double-helix configuration, which is
two strands of DNA coiled together.
• As a result, adenine pairs with thymine and
guanine pairs with cytosine.
• This concept is known as complementary base
pairing.
• The order of the bases is what distinguishes
different DNA strands.
Web-Extra 9-1
• Understand the structure of DNA and
follow the step-wise construction of the DNA
molecule.
Source: National Institute of Justice, U.S. Department of Justice, Principles of Forensic
DNA for Officers of the Court, 2006.
Structure of DNA
• The long strands of DNA are coiled upon
themselves into chromosomes
– When paired, chromosomes resemble the letter X
• Humans have 23 pairs of chromosomes,
including 2 sex chromosomes
– Females are XX
– Males are XY
DNA at Work
• DNA directs the production of proteins, which are made by
combining amino acids.
• The sequence of amino acids in a protein chain determines
the shape and function of the protein.
• Each group of three nucleotides in a DNA sequence codes
for a particular amino acid.
– Example: G-A-G codes for the amino acid glutamine,
while C-G-T codes for alanine.
• If a nucleotide is “changed,” for example a T is substituted
for A and G-A-G becomes G-T-G, the “wrong” amino acid
is placed in the protein (in this case: glutamine is replaced
with valine).
• As a result, the protein may not function correctly and this
is the basis for many diseases and health issues.
DNA Replication
• DNA duplicates itself prior to cell division.
• DNA replication begins with the unwinding of the
DNA strands of the double helix.
• Each strand is now exposed to a collection of free
nucleotides that will be used to recreate the double
helix, letter by letter, using base pairing.
• Many enzymes and proteins, such as DNA
polymerases, are involved in unwinding the DNA,
keeping the DNA strands apart, and assembling
the new DNA strands.
DNA Replication con’t
• Polymerase chain reaction (PCR) is a technique for
replicating small quantities of DNA or broken pieces of
DNA found at a crime scene, outside a living cell.
• The ability to multiply small bits of DNA now
means that sample size is no longer a limitation in
characterizing DNA recovered at a crime scene.
Recombinant DNA
• Recombinant DNA relies on the ability of certain
chemicals, known as restriction enzymes, to cut DNA into
fragments that can later be incorporated into another DNA
strand.
• Restriction enzymes can be thought of as highly specialized
scissors that cut a DNA molecule when it recognizes a
specific sequence of bases.
• Once a portion of the DNA strand has been cut out with
the aid of a restriction enzyme, the next step in the
recombinant DNA process is to insert the isolated DNA
segment into a foreign DNA strand, usually that of a
bacterium.
• As the bacteria multiply rapidly, copies of the altered DNA
are passed on to all descendants.
DNA Typing
• DNA typing (a.k.a. DNA Fingerprinting) was
developed by British geneticist Sir Alec Jeffreys
in 1984.
• This technique converts DNA into readable bands
on a gel
With these bands, we
can compare suspect
and crime scene DNA,
or child and possible
father, etc.
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DNA Typing
• Portions of the DNA molecule contain sequences of bases
that are repeated numerous times, known as tandem
repeats.
• To a forensic scientist, these tandem repeats offer a means
of distinguishing one individual from another through
DNA typing.
• Tandem repeats seem to act as filler or spacers between the
coding regions of DNA.
• What is important to understand is that all humans have
the same type of repeats, but there is tremendous variation
in the number of repeats each of us have.
• Web-Extra 9-2 An Animated Demonstration
of Gel Electrophoresis
• Polymerase Chain Reaction (PCR)
https://www.youtube.com/watch?v=2KoLnIw
oZKU
Restriction Fragment Length
Polymorphisms (RFLP)
• Length differences associated with relatively long repeating
DNA strands are called restriction fragment length
polymorphisms (RFLP) and form the basis for one of the
first DNA typing procedures.
• Typically, a core sequence consists of 15 to 35 bases in
length and repeats itself up to a thousand times.
• The key to understanding DNA typing lies in the
knowledge that numerous possibilities exist for the number
of times a particular sequence of base letters can repeat
itself on a DNA strand.
A Positive RFLP Test
• Once the DNA molecules have been cut up by a restriction
enzyme, the resulting fragments are sorted out by
electrophoresis.
• The smaller DNA fragments will move at a faster rate on
the gel plate than the larger ones.
• The fragments are then transferred to a nylon membrane
in a process called Southern blotting.
• To visualize the RFLPs, the nylon sheet is treated with
radioactive probes containing a base sequence
complementary to the RFLPs being identified (a process
called hybridization).
A Positive RFLP Test
• Next, the nylon sheet is placed against X-ray film and
exposed for several days.
• When the film is processed, bands appear where
radioactive probes stuck to fragments on the nylon sheet.
• A typical DNA fragment pattern will show two bands (one
RFLP from each chromosome).
• When comparing the DNA fragment patterns of two or
more specimens, one merely looks for a match between the
band sets.
• A high degree of discrimination can be achieved by using a
number of different probes and combining their
frequencies.
Short Tandem Repeats
(STRs)
• A common method of DNA typing
• There are locations (loci) on a chromosome that
contain short segments of 3 – 7 bases that repeat
themselves
• With the technology of PCR one can extract and
amplify a combination of different STR’s.
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Short Tandem Repeats
• The latest method of DNA typing, short tandem
repeat (STR) analysis, has emerged as the most
successful and widely used DNA profiling
procedure.
• STRs are locations on the chromosome that
contain short sequences that repeat themselves
within the DNA molecule.
• They serve as useful markers for identification
because they are found in great abundance
throughout the human genome.
STR Advantages
• STRs normally consist of repeating sequences of 3 to 7
bases in length, and the entire strand of an STR is also
very short, less than 450 bases in length.
• This means that STRs are much less susceptible to
degradation and may often be recovered from bodies or
stains that have been subjected to extreme decomposition.
• Also, because of their shortness, STRs are ideal candidates
for multiplication by PCR, thus overcoming the previously
mentioned limited-sample-size problem often associated
with crime-scene evidence.
The Power of STR
• What makes STRs so attractive to forensic
scientists is that hundreds of different types
of STRs are found in human genes.
• The more STRs one can characterize, the
smaller will be the percentage of the
population from which a particular
combination of STRs can emanate.
Standardizing STR Testing
• Currently, U.S. crime laboratories have
standardized on 13 STRs for entry into a national
database (CODIS).
• A high degree of discrimination and even
individualization can be attained by analyzing a
combination of STRs (multiplexing) and
determining the product of their frequencies.
• With STR, as little as 125 picograms of DNA is
required for analysis.
• This is 100 times less than that normally required
for RFLP analysis.
Short Tandem Repeats
• https://www.youtube.com/watch?v=DbR9x
MXuK7c
Collecting and Packaging
Biological Evidence
• Photograph evidence first
• Wear gloves at all times
• Package each stained article separately
in paper or a well-ventilated box (to
avoid bacterial or fungal growth)
• Remove dried blood using a sterile
swab moistened with distilled water
• Store biological evidence in the
refrigerator or a cool location until it is
delivered to the lab
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DNA Sequencing: PCR
Polymerase Chain Reaction
(PCR)
• Polymerase chain reaction is the outgrowth of
knowledge gained from an understanding of how DNA
strands naturally replicate within a cell.
• A technique for making many copies of a
specific piece of DNA to be analyzed forensically
– Can amplify very minute quantities of DNA millions
of times!
• This method works by cycling
through different temperatures
• A device called a thermocycler controls the
temperatures, allowing for fast and accurate
copying of DNA
Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR)
https://www.youtube.com/watch?v=2KoLnIw
oZKU
https://www.youtube.com/watch?v=JRAA4C2
OPwg
Polymerase Chain Reaction (PCR)
The steps of PCR
• Denaturation:
– Extracted and purified DNA is heated to “unzip”
(separate) the double helix
– This is done at high temperature, about 94°C
Polymerase Chain Reaction (PCR)
The steps of PCR
• Annealing:
– Short template pieces called “primers” bind with
the separated strands for new DNA to build upon.
– This occurs at ~65°C
Polymerase Chain Reaction (PCR)
The steps of PCR
• Extension/Elongation:
– Taq Polymerase, a DNA building enzyme, adds free
nucleotides from the surrounding solution onto the
template primers
– In this way, new strands are built out of the original
2 separated stands
– This happens at 72°C
new DNA
strands
Polymerase Chain Reaction (PCR)
• Each step only requires a few minutes
• The thermocycler machine cycles through
these temperatures for several hours
• Each cycle doubles the number of copied
DNA strands
PCR is specific to your
“region of interest.” Different
primers will selectively
amplify different genes
Electrophoresis
PCR and RFLP
• PCR technology cannot be applied to RFLP
DNA typing.
• The RFLP strands are too long, often
numbering in the thousands of bases.
• PCR is best used with DNA strands that are
no longer than a couple of hundred bases.
PCR Advantages
• One advantage in moving to shorter DNA strands is that
they would be expected to be more stable and less subject
to degradation brought about by adverse environmental
conditions.
• The long RFLP strands tend to readily break apart under
the adverse conditions not uncommon at crime scenes.
• PCR also offers the advantage in that it can amplify
minute quantities of DNA, thus overcoming the limited
sample size problem often associated with crime scene
evidence.
Electrophoresis
• DNA can be visualized through the
process of electrophoresis
• In the lab, DNA molecules are cut by
restriction enzymes into fragments of
various sizes. Restriction enzymes cut
at specific sequences throughout the
DNA.
• The resulting fragments are forced to
move along a gel-coated plate under
the influence of an electrical potential
Electrophoresis
• The DNA molecule has a
slight negative charge, so
it is attracted to the
positive end of an
induced electric field.
• DNA fragments are
separated by size
– Smaller fragments move
farther along the gel
Electrophoresis
• After the fragments have
“migrated” across the gel,
the gel can be stained to
show the “bands” or
fragments easily
• Then comparisons can be
made
– Example: crime scene
sample to suspect
Web-Extra 9-2
• An Animated Demonstration of Gel
Electrophoresis
•
•
http://wps.prenhall.com/wps/media/objects/3801/3892550/electrophoresis.swf
Mitochondrial DNA
• Another type of DNA used for individual
characterization is mitochondrial DNA.
• Mitochondrial DNA (mDNA) is located outside the
cell’s nucleus and is inherited from the mother.
• Mitochondria are structures found in all our cells
used to provide energy that our bodies need to
function.
• A single mitochondria contains several loops of
DNA.
Mitochondrial DNA Testing
• Mitochondrial DNA typing does not approach STR
analysis in its discrimination power and thus is best
reserved for samples, such as hair, for which STR analysis
may not be possible. Advantage: We can use mDNA in
certain tissues that do not contain nuclear DNA
• Disadvantage: Forensic analysis of mDNA is more rigorous,
time consuming, and costly when compared to nuclear DNA
analysis.
• Also, all individuals of the same maternal lineage will be
indistinguishable by mDNA analysis. (possible to be either
an advantage OR a disadvantage)
• Two regions of mDNA have been found to be highly
variable and a procedure known as sequencing is used to
determine the order of base pairs.
Web-Extra 9-9
• See How We Inherit our Mitochondrial
DNA
• http://wps.prenhall.com/wps/media/objects/
3801/3892550/DNACD_mod08-1-04.swf
Combined DNA Information System
(CODIS)
• CODIS maintains a
database of DNA profiles
from
–convicted offenders
–unsolved crime scene
evidence
–profiles of missing
persons
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Probability and DNA
• http://wps.prenhall.com/wps/media/objects/
3801/3892550/DNACD_mod07-2-08.swf
Sam Sheppard
(end)
• Use the link below to investigate this
notorious murder.
• http://www.pbs.org/wgbh/nova/sheppard/
Resources
• Saferstein, Richard. Forensic Science: An Introduction. New
Jersey: Pearson Prentice Hall, 2008
• Saferstein, Richard. Forensic Science: An Introduction. 2nd
ed. New Jersey: Pearson Prentice Hall, 2011
• Saferstein, Richard. Criminalistics: An Introduction to
Forensic Science. 8th ed. Upper Saddle River, NJ; Pearson
Prentice Hall, 2004
• http://law2.umkc.edu/faculty/projects/ftrials/clinton/lewinskyd
ress.html
• http://www.pbslearningmedia.org/resource/tdc02.sci.life.gen.s
heppard/forensic-dna-analysis/
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