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Chapter 9
DNA: THE INDISPENSIBLE
FORENSIC SCIENCE TOOL
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-1
Introduction
• The gene is the
fundamental unit of
heredity.
• Each gene is actually
composed of a DNA
sequence that specifically
code for a protein.
• DNA is a polymer made
by linking a series of
repeating units called
nucleotides (monomers).
• Each nucleotide is
composed of a sugar, a
phosphate group, and a
nitrogen base.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-2
The Bases
• Four types of bases used
in DNA: adenine (A),
guanine (G), cytosine
(C), and thymine (T).
• DNA’s structure is a
double-stranded helix as
discovered by Rosalind
Franklin, Crick and
Watson.
• Chargaff discovered that
A always pairs with T
and C always pairs with
G – AKA base pairing.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-3
DNA at Work
• DNA codes for proteins (
also polymers), which
are formed by amino
acids (monomers) in a
long chain.
• The sequence of amino
acids determines the
shape and function of
the protein.
• A codon (three
nucleotides) codes for a
particular amino acid.
G-A-G codes for the
amino acid
glutamine, while CFORENSIC SCIENCE
An Introduction
G-T codes for
By Richard Saferstein
•
G-A-G codes for
glutamine, C-G-T codes
for alanine.
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-4
Nucleotide Errors
• If a nucleotide is
changed or omitted, a T
is substituted for A then
G-A-G becomes G-T-G,
and a different amino
acid is placed in that
position 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.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-5
Intracellular DNA Replication
• DNA duplicates itself in
the nucleus prior to cell
division.
• DNA replication begins
with the unwinding of
the DNA strands of the
double helix.
• Many enzymes are
involved in unwinding
the DNA (helicases), and
assembling the new DNA
strands (polymerases).
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-6
Extracellular DNA Replication
• Polymerase chain
reaction (PCR) is a
technique for replicating
a small DNA sample
found at a crime scene.
• The ability to multiply
small bits of DNA means
that a single sample is no
longer a limitation in
analyzing crime scene
DNA.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-7
PCR
• PCR uses the enzyme
taq-polymerase
(taken from
bacteria), and
Adenine, Thymine,
Cytosine and
Guanine to copy a
segment of DNA.
• Very little DNA is
needed (0.2 ul)
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
• All the ingredients
are placed into the
thermal-cycler.
• Thermal-cycler heats
the DNA sequence to
unravel the strand.
• The A, T, C and G
will pair with the
sample in the correct
order, the copy will
be released when the
cycle cools
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-8
Recombinant DNA
• Recombinant DNA relies on the ability of proteins,
known as restriction enzymes to cut through both
strands in DNA sequence.
• Restriction enzymes can be thought of as highly
specialized scissors that cut a DNA molecule when
recognizing 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 is to
insert the DNA fragment into another DNA strand
(usually in bacteria).
• The bacteria divide rapidly passing the newly-altered
DNA on to all new bacteria.
• Method used to produce insulin, penicillin,
botox.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-9
Recombinant DNA
Recombinant Cloning:
Plasmid insertion of cut
DNA sequence.
New piece of DNA is
incorporated into the
bacterial DNA.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-10
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 are found throughout our genome
between the coding areas of DNA.
• What is important to understand is that all humans
have the same repeat regions, but there is tremendous
variation in the number of repeats and each person
has a unique number in each region of their genome.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-11
DNA Typing
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-12
RFLP
• Long repeating DNA sequences are called
restriction fragment length polymorphisms
(RFLP’s) and form the basis for the first DNAtyping procedure.
• The sequence consists of a range of 15-40 basepairs in length and can be repeated 20-1000 times
in each region.
• The key to understanding DNA typing is that
total number of repeats of the specific region is
inherited from each parent to form a unique
combination for each child.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-13
RFLP Analysis
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-14
Electrophoresis
• A technique analogous to TLC is
electrophoresis.
– Here, materials are forced to move
across a gel-coated plate under the
influence of an electrical potential.
• In this manner, substances such as
DNA can be separated and
characterized.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-15
Agarose Gel Electrophoresis
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-16
DNA Typing
• 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
through the electrical field 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 (or bioluminescent) probes containing a
base sequence complementary to the RFLPs being
identified (a process called hybridization).
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-17
DNA Typing
• Next, the nylon sheet is placed against X-ray film and
exposed for several days.
• When the film is processed, dark bands will 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.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-18
PCR Testing
• Polymerase chain reaction is based on the way DNA
strands naturally replicate within a cell.
• In forensics, PCR offers a distinct advantage in that it can
amplify minute quantities of DNA many millions of times.
• First, the DNA is heated to separate it.
• Second, primers (short strands of DNA used to target
specific regions of DNA for replication) are added, which
hybridize with the strands.
• Third, DNAtaq- polymerase and free nucleotides
(A,T,C,G) are added to the mixture to rebuild each of the
separated strands.
• The heating-cooling cycles are repeated 25 to 30 times.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-19
PCR Technology
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-20
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.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-21
PCR Advantages
• First advantage in moving to shorter DNA
strands is that they are more stable and less
subject to degradation (breakdown) due to
adverse environmental conditions.
• Second advantage is long RFLP strands tend to
break apart under the adverse conditions
(which are all too common at crime scenes).
• Third advantage is PCR can amplify minute
(tiny) quantities of DNA, producing an
unlimited supply of reliable crime scene
evidence.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-22
Short Tandem Repeats
• The CURRENT method of DNA typing, short
tandem repeat (STR) analysis is the most
successful and widely used DNA procedure.
• STRs are locations on the chromosome that
contain short sequences that repeat themselves
inside the DNA code.
• They are valuable markers for identification
because they are located throughout the human
genome.
• CODIS requires 13 standard STR sites for
submission.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-23
STR Advantages
• STRs normally consist of repeating sequences of
3 to 7 bases in length. The entire strand of an
STR is 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.
• Due to shortened sequences, STRs are easily
amplified by PCR.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-24
The Power of STR
• Hundreds of STRs are found in human genes.
• The more STRs that can be matched to a
suspect, the more individual the evidence.
• Now forensic labs wil perform multiplexing
DNA testing, which will identify more
correlation between the suspect and the crime.
• PCR allows the scientist to simultaneously
extract and amplify a combination of different
STRs.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-25
Standardizing STR Testing
• Currently, U.S. crime laboratories have
standardized on 13 STRs for entry into a
national database (CODIS).
• The 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.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-26
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.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-27
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.
• 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.
• 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.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-28
CODIS
• Perhaps the most significant development to
arise from DNA typing is the ability to compare
DNA types recovered from crime scene
evidence to those of convicted sex offenders and
other criminals.
• CODIS (Combined DNA Index System) is a
computer software program developed by the
FBI that maintains local, state, and national
databases of DNA profiles from convicted
offenders, unsolved crime scene evidence, and
profiles of missing persons.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-29
Packaging Biological Evidence
• Before the collection of biological evidence
begins, it is important that it be photographed
and recorded on sketches.
• Wearing disposable latex gloves while handling
the evidence is required.
• Clothing from victim and suspect with blood
evidence must be collected.
• The packaging of biological evidence in plastic
or airtight containers must be avoided because
the accumulation of residual moisture could
contribute to the growth of DNA-destroying
bacteria and fungi.
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-30
Packaging Biological Evidence
• Each stained article should be packaged separately in a
paper bag or in a well-ventilated box.
• Dried blood is best removed from a surface by using a
sterile cotton swab lightly moistened with distilled
water that is air dried before being placed in a swab
box, then a paper or manila envelope.
• All biological evidence should be refrigerated or stored
in a cool location until delivery to the laboratory.
• Standard/reference DNA specimens must also be
collected, such as blood or the buccal swab (swabbing
the mouth and cheek).
FORENSIC SCIENCE
An Introduction
By Richard Saferstein
PRENTICE HALL
©2008 Pearson Education, Inc.
Upper Saddle River, NJ 07458
9-31