Forensic DNA
The Cell
The smallest unit of life
The nucleus is the “brain”
brain” of
the cell
contains all the genetic info the
cell needs to exist & to
reproduce
Chromosomes
Genes
In most types of cells,
genetic information is
organized into
structures called
chromosomes
usually X shaped
Y chromosome in
males
23 pairs in humans
Each chromosome is a single
polymeric molecule called DNA
if fully extended the molecule
would be about 1.7 meters long
unwrapping all the DNA in all your
cells
cover the distance from earth to moon
6,000 times
Each chromosome
contains hundreds to
thousands information
blocks called genes
Each gene is the
blueprint for a specific
type of protein in the body
one from mother & one
from father
Chromosomes
In most types of cells, genetic
information is organized into
structures called chromosomes
only identical twins will
have all the genes identical
Nucleotides
DNA is a polymer built from
monomers called nucleotides
DNA is found in all cells with a
nucleus (white blood cells, soft
tissue cells, bone cells, hair root
cells and spermatozoa)
Each nucleotide is consists of
deoxyribose
pentose sugar
phosphoric acid
a nitrogenous base
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Nitrogenous Bases
The phosphate
The Purines
The sugar
Nitrogenous Bases
Adenine (A)
Guanine (G)
Introduction to DNA
A nucleic acid can contain any of five
bases:
Adenine:
doubledouble-ring base (a purine)
doubledouble-ring base (a purine)
Cytosine: singlesingle-ring base (a pyrimidine)
Thymine: singlesingle-ring base (a pyrimidine)
Uracil: singlesingle-ring base (a pyrimidine)
Guanine:
The Pyrimidines
Cytosine (C)
Thymine (T)
The DNA Backbone
The monomers are
linked together by
phosphodiester
bridges (bonds)
links the 3’
3’ carbon in
the ribose of one
nucleotide to the 5’
5’
carbon in the ribose of
the adjacent
nucleotide
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The DNA Double Helix
DNA is normally a
double stranded
macromolecule
Two polynucleotide
chains are held
together by HHbonding
A always pairs with T
C always pairs with G
5’ T-T-G-A-C-T-A-T-C-C-A-G-A-T-C 3’
3’
3’ A-A-C-T-G-A-T-A-G-G-T-C-T-A-G 5’
5’
In a double helix the strands go in
opposite directions
Functions of DNA
DNA Review:
Two Functions
To
transmit information from one generation
of cells to the next
To provide the information for the synthesis of
components (proteins) necessary for cellular
function
DNA is a doubledouble-stranded molecule.
The DNA strands are made of four different building
blocks.
An individual’
individual’s DNA remains the same throughout
life.
DNA and RNA are responsible for storage and
transmission of genetic information.
In specific regions on a DNA strand each person has
a unique sequence of DNA or genetic code.
The major function of DNA is control and direction or
protein synthesis in body cells.
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Introduction to DNA
DNA
Chromosomes
Carries the information needed for making and
maintaining the different parts of an organism
Consist of DNA in the nuclei of cells coiled around
proteins (histone molecules)
Humans have 46 chromosomes, 23 from each parent
Genes
Are segments of DNA molecules that control the
production of different proteins in an organism
Vary in terms of the number and sequence of base pairs
they contain
DNA Replication
Introduction to DNA
Cell replication
Before
a cell divides, the double helix strand
begins to unwind.
Each unwinding strand serves as a template for
the formation of a new complementary strand.
Nucleotides are attracted to the exposed bases
and become hydrogenhydrogen-bonded to them: A to T,
T to A, C to G, G to C.
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.
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.
Introduction to RNA
Introduction to DNA/RNA
Ribonucleic acid (RNA)
is similar to DNA
Ribose–
–
phosphoric
acid units form backbone
Ribose
Each ribose unit is bonded to one of the four
bases
Protein synthesis: transcription
Primary structure
Protein synthesis: a series of complex steps
involving RNA
Transcription
A single strand of RNA is synthesized inside the cell
nucleus.
A segment of the DNA double helix separates into
single strands.
The exposed bases of one strand act as the template
for the synthesis of an RNA molecule.
The base sequence (messenger RNA) complements
the base sequence on the DNA strand with one
exception: RNA transcribes a uracil instead of a
thymine to adenine.
Translation
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Introduction to DNA and RNA
Protein synthesis: translation
The
code that has been copied to the new
protein is interpreted.
mRNA leaves the nucleus and takes its
chemical message to the cytoplasm of the cell,
where it binds with ribosomes.
Guided by the first codon on the mRNA strand, a
transfer RNA molecule with an anticodon that is
complementary to this codon transports a
specific amino acid to the mRNA codon
Introduction to DNA/RNA
Protein synthesis
The
actual protein synthesis occurs in the
ribosomes, which move along the mRNA one
codon at a time as the amino acid chain grows.
The mRNA is read codon by codon and the
protein is built up one amino acid at a time in the
correct sequence.
DNA—
DNA—Protein Synthesis
Introduction to DNA
The genetic code
Each
threethree-base sequence in mRNA codes
for a specific amino acid
64 threethree-base codons can be formed from the
four bases in mRNA
Recombinant DNA
Mitochondrial DNA
Mitochondria
Provide 90% of the body’
body’s energy
Contain DNA that can be used for testing purposes
Mitochondrial DNA (mtDNA)
Is circular and much smaller than nuclear DNA
Is inherited from the mother
State of Tennessee v. Ware (1996): first use of mtDNA in
court to match hair samples at the crime scene to the
suspect
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: GG-A-G codes for the amino acid
glutamine, while CC-G-T codes for alanine.
alanine.
If a nucleotide is “changed”
changed”, for example a T is
substituted for A and GG-A-G becomes GG-T-G, the
“wrong”
wrong” amino acid is placed in the protein (in this
case glutamine is replaced with valine).
valine).
As a result, the protein may not function correctly
and this is the basis for many diseases and health
issues.
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.
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DNA Fingerprinting
Cutting Variable DNA Sections
Father: DNA is
is a long,long,long long molecule that is tightly wound.
Mother: DNA is a long,long molecule that is tightly wound.
Restriction Enzymes are proteins that cut
DNA molecules at specific cut sites.
Conventional Blood Typing
Used for more than 50 years
Utilized ABO blood typing groups
Identified genetic variations in blood
proteins, tissue specific proteins and
serum protein types
Major problem is that the conventional
blood protein markers are not found in
semen.
The basic structure of everyone’
everyone’s DNA is
the same
the
difference between people is the ordering
of the base pairs
Every person can be distinguished by the
sequence of their base pairs
millions
of base pairs make this impractical
shorter method uses repeating patterns that
are present in DNA
a
Development of DNA Analysis
Techniques (1970’
(1970’s)
Restriction Fragment Length
Polymorphism
Restriction Enzymes (biological
catalysts) cut DNA whenever they
encounter a specific DNA sequence.
Gel electrophoresis separates the
fragments of DNA according to their
length.
RFLP (Restriction Fragment Length
Polymorphism)
In
1980 David Botstein and others used
RFLP to construct a human gene map.
Used genetic variations as markers
Southern Blot
Size Separation of DNA by Gel
Electrophoresis
Total DNA
Gel Electrophoresis
Restriction
Enzymes
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RFLP
A Positive RFLP Test
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.
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,
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 XX-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.
A Schematic Representation of RFLP and
Southern Blot of a SingleSingle-locus VNTR
Southern Blot
A short segment of DNA that is
complementary to a portion of the desired
DNA fragments is labeled with a
radioactive atom.
This probe binds to the fragment of
interest on the gel electrophoresis.
Visualized using XX-ray film.
Kary Mullis Invented PCR Methods
(1984)
Polymerase Chain Reaction amplifies
short specific regions of DNA
PCR is an in vitro technique that can
yield millions of copies of desired DNA
Does not use radioactivity
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PCR Testing
PCR
Polymerase chain reaction is the outgrowth of
knowledge gained from an understanding of how DNA
strands naturally replicate within a cell.
For the forensic scientist, 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, DNA polymerase and free nucleotides are added
to rebuild each of the separated strands.
Now, this process is repeated 25 to 30 times.
A Typical DNA Profile
PCR Advantages
In 1984, Alec Jeffreys developed
“DNA Fingerprinting.”
Fingerprinting.”
Was searching for disease markers
Applied the technique to personal
identification
Demonstrated that the DNA could be
retrieved from old dried blood stains
Applied the technique to highhigh-profile
forensic tests
Using shorter DNA strands -- 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.
The Pitchfork Case (1986 & 1987)
Jeffreys was consulted in the case of the
murder and rape of two British schoolgirls.
Suspect cleared from analysis of semen
samples at the scene and blood samples
from the suspect.
“Blooded”
Blooded” 4583 men
Analysis of blood sample from Colin Pitchfork
provided a match.
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VNTR’
VNTR’s
Pennsylvania v Pestinikas (1986)
First PCR case done in the United
States
DNA strands contain information which directs an organism’
organism’s
development
Also contain stretches which appear to provide no relevant genetic
genetic
information
exons
introns
repeated sequences of base pairs
Involved allegations of switching body
parts at a funeral home
Variable Number Tandem Repeats (VNTRs
(VNTRs))
can contain anywhere from 20 to 200 base pairs
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
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.
VNTRs
All humans have some VNTRs
VNTRs come from the genetic information
donated by parents
can
have VNTRs from mother, father or a
combination
will not have a VNTR that is from neither
parent
D1 = biological daughter of both parents
D2 = child of mother & former husband
S1 = couple’s biological son
S2 = adopted son
VNTR Analysis
Usually an individual will inherit a different variant of the
repeated sequence from each parent
VNTR Analysis
PCR primers bracket the locus
PCR reaction forms a nucleotide chain from the
template
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VNTR Analysis
The length of the amplified DNA & its position after
electrophoresis will depend on the number or
repeated bases in the sequence
Although some individuals have several bands in common,
the overall pattern is distinctive for each
Commercial Development of Forensic
DNA Testing in the United States:
Lifecodes Corporation—
Corporation—Founded in 1982
in Valhalla, NY as diagnostic company.
Began performing forensic DNA testing in
1987
Cellmark—
Cellmark—The US branch of Britain’
Britain’s
Imperial Chemical Industries opened in
Germantown, MD in 1987. Performed the
testing for the prosecutor in the Simpson
case.
Analysis used 3 different VNTR loci for each suspect giving
6 bands
Suspects A & C can be eliminated
B remains a suspect
Trial of Accused Rapist
Tommy Lee Andrews
November 3, 1987
First case in the U.S. to identify a criminal
by DNA
Trial held in Orlando, Florida
A scientist from Lifecodes and a biologist
from M.I.T. testified that semen left on the
victim matched Andrew’
Andrew’s DNA (1 in 10
billion)
November 6, 1987 jury returned a guilty
verdict.
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The two major private companies raced
each other to the courtroom.
In a shroud of secrecy and an environment
of expediency they disregarded the usual
methods of testing new scientific methods
Publication
and Peer Review
Standardization
DNA analysis was considered an
“infallible”
infallible” prosecution tool.
“You can’
can’t argue with science.”
science.” a juror in
Queens
“[DNA evidence] is the single greatest
advance in the search for truth since the
advent of crosscross-examination.”
examination.” Judge
Joseph Harris of Albany, NY
Replication
Evaluation
of Performance
DNA analysis was considered an
“infallible”
infallible” prosecution tool.
New York v Castro
First successful defense against DNA
analysis
Jose Castro, janitor in a nearby building,
was accused of the stabbing deaths of
Vilma Ponce and her twotwo-year old
daughter, Natasha.
“In rape cases, when the semen has
been matched with the defendant’
defendant’s and
the chance that it came from another
person is 33 billion to 1, you don’
don’t need
a jury.”
jury.” Robert Brower, defense
attorney.
New York v Castro
Lifecodes Corporation reported that the
blood found on Castro’
Castro’s watch matched
that of Vilma Ponce with a frequency of
1:189,200,000 in the Hispanic population.
Defense mounted the first successful
attempt to have DNA evidence excluded.
New York v Castro
Two defense and two prosecution
witnesses agreed that Lifecodes had failed
to use generally accepted scientific
techniques. Their data was poor and they
did not follow procedures for interpreting
the data
Castro later confessed and pled guilty to
the murders.
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The FBI and The Royal Canadian Mounted
Police Set Up DNA Laboratories
1987 FBI with NIH began collaborative
research to establish DNA identification
techniques
In late 1988 FBI set up their own
laboratory at their Pennsylvania Avenue
headquarters
RCMP also set up their own DNA
laboratory.
FBI Worked With Pioneers in the Field
GeneLex
Dr.
Raymond White of Howard Hughes
Medical Center
Lifecodes
Cellmark
The FBI and RCMP brought
standardization to the industry.
Established detailed laboratory protocols
Performed validation studies
Cut through the competitive nature that
clouded the environment of the testing
methods and tools
Defense Strategy
In 1989, National Association of Criminal
Defense Lawyers (NACDL) set up a DNA
Task Force.
Headed by Barry Scheck, a professor at
Benjamin N. Cardozo Law School and
Peter Neufeld, a private attorney in
Manhattan
Defense Strategy
Tried to reopen all the convictions
involving evidence processed by Lifecodes
Escalated the conflict surrounding DNA
testing
Launched a public relations campaign
critical of DNA typing
They used four different DNA probes
In 1990 Scheck and Neufeld Prepare
for United States vs Yee.
Three members of Hell’
Hell’s Angels
motorcycle gang of Cleveland Ohio were
accused of killing David Hartlaub thinking
he was a member of a rival gang
The victim was shot 14 times with a MAC
10 machine gun.
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Blood Evidence on the Scene
Most was determined to be that of the
victim
Some belonged to one of the defendants
Theory—
Theory—a ricocheting bullet had hit one of
the suspects.
Prosecution Expert Witnesses
Thomas Caskey,
Caskey, Baylor College of
Medicine
Kenneth Kidd, Yale geneticist
Bruce Budowle,
Budowle, FBI DNA scientist
Defense Expert Witnesses
Richard Lewontin,
Lewontin, geneticist from Harvard
Daniel Hartl,
Hartl, geneticist at Washington
University
Eric Lander, mathematician and geneticist
at M.I.T.
Defense Argued:
FBI’
FBI’s published articles on matching
criteria were “ambiguous”
ambiguous” and
“inconsistent.”
inconsistent.”
Prosecution Countered:
In spite of disputes over match criteria, the
multimulti-probe match produced a highly
likelihood that the specimens came from
two different people.
United States vs Yee
DNA matches were admitted as
evidence at the trial and at the appeal.
The documents from the FBI would later
find their way into the courtroom.
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The National Academy of Science’
Science’s
National Research Council (NCR)
Report
NCR Report—
Report—DNA Technology in
Forensic Science (1992) Criticized:
A federal study intended to be the
definitive study on the problems of forensic
DNA
The most contended issue-How to
issue--How
calculate statistical probability
Ceiling principle
No population geneticists or statisticians
on the panel
Inconsistencies
Did not address paternity cases or PCR
The frequencies
of subsub-groups could differ
widely from that in the larger population
NCR Report—
Report—DNA Technology in
Forensic Science
The report was to be the final word in
clarifying the role of DNA forensics.
This document is often cited by defense
lawyers in arguing of the exclusion of DNA
evidence.
The Media and DNA Forensics
First the Media proclaimed that the “new
technology was a miracle.”
miracle.”
It recorded it’
it’s “glowing victories.”
victories.”
Followed by giving opponents to DNA
analysis a venue for airing objections and
charges (many of which were unfounded
and unchallenged)
Nature Published Articles Exploring
Forensic DNA
The Media and DNA Forensics
The Washington Post headline read,
“Panel Backs DNA Tests as Crime
Evidence.”
Evidence.”
Alec Jeffreys announced his methods in
Nature.
“DNA fingerprinting dispute laid to rest”
rest”
Budowle and Lander Oct. 27, 1994
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1994
“Lander and Budowle declare that after
400 technical papers, 100 scientific
conferences, three sets of DNA analysis
guidelines, 150 court cases, and an
exhaustive threethree-year study by the
National Research Council—
Council—’The DNA
fingerprinting wars are over.’”
over.’”
National Research Council reconvened to
try to rectify the deficiencies of their first
report.
The trial of the century convened: “Dollars
v DNA”
DNA” or California v OJ Simpson.
Summary
Forensic DNA testing was developed
rapidly by shortshort-sighted commercial
interests
Standards were not developed as quickly
as necessary
Prosecution oversold DNA evidence
Expert witnesses had a vested interest in
fueling the controversy.
Summary
Sensational and inaccurate media
coverage spread misinformation about
DNA.
The judicial system is sometimes
indifferent to choices that could expedite
justice.
Where is Forensic DNA Analysis
Today?
The Media and DNA Forensics
The DNA Technology in Forensic Science
report strongly endorsed the continued
use of DNA typing in the courts.
The New York Times front page article
headline read “U.S. Panel Seeking
Restriction on Use of DNA in Courts.”
Courts.”
Forensic labs have implemented a
process of peer review, selfself-regulation and
accreditation
DNA analysis has freed over 156 convicts.
The Innocence Project at Yeshiva
University is run by Barry Sheck and Peter
Neufeld.
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Short Tandem Repeats
Where is Forensic DNA Analysis
Today?
Paternity testing.
Historical case of Thomas Jefferson as the
likely father of children of slave, Sally
Hemmings.
Hemmings.
Resolved questions surrounding the
disputed descendent of the Czar Nicholas
II
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 limitedlimited-samplesamplesize problem often associated with crimecrime-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.
This gives rise to the concept of multiplexing.
Using the technology of PCR, one can
simultaneously extract and amplify a
combination of different STRs.
STRs.
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.
Y- STR
Another tool available in the arsenal of the DNA
analyst is the ability to type STRs located on the Y
chromosome, which is male specific.
More than 20 different YY-STR markers have been
identified.
Y-STRs will prove useful when multiple males are
involved in a sexual assault.
A YY-STR analysis will have only one band or peak,
rather than the conventional STR which is derived
from two chromosomes and has two bands or peaks.
The YY-STR is therefore less complicated in
appearance and interpretation.
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Mitochondrial DNA
Mitochondrial DNA Testing
Another type of DNA used for individual
characterization is mitochondrial DNA.
Mitochondrial DNA (mDNA
(mDNA)) is located
outside the cell’
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.
CODIS
Perhaps the most significant tool 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 convicted
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.
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.
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 DNADNA-destroying
bacteria and fungi.
Packaging Biological Evidence
Each stained article should be packaged
separately in a paper bag or in a wellwell-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).
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