The following article will focus on PCR –
its unquestionable role in modern medicine; its prime role in crime scene
investigation where everything One needs are pieces of evidence deeply rooted in
the background of hard science – the science which is not easily put under
question or doubt.
Reverse transcription of
polyadenylated RNA (the vast majority of cellular mRNA) with the subsequent
amplification of the copied DNA via the polymerase chain reaction is an
important technique in assaying so-called “fusion” transcripts. Fusion
transcripts occur in rare instances in which chromosomal translocation fuses
transcribed regions of two genes. These may result in fusion proteins in which
coding information from one gene is fused to the coding information of another
gene, producing a hybrid protein. These proteins may retain functionality, as
in the case of the bcr/abl fusion protein, but the regulation of these proteins’
functions may be abnormal. Chronic myelogenous leukemia (CML) is typified by
the presence of the Philadelphia
Forensic serology is based on findings from empirical and theoretical
studies in many disciplines of biology. Progress involves applying technologies and concepts from immunology and blood group
serology to forensic serology. Molecular biology and
population genetics have played developmental and supportive roles in new advances
utilizing DNA technology.
Example Case 001
Three armed men break into a home.
There are four people, two males and two females at home. All are related to
each other. Both females are sexually assaulted and the males are beaten with a
pistol. One male who lives in the home forcibly obtains a weapon from an
assailant and fires. The robbers flee the scene, and two are later apprehended
at another location. The evidence collected from the crime scene consists of:
(1) blood stains on the carpet, (2) a leather handbag, (3) several articles of
clothing, and (4) cigarette butts and a baseball cap left by an assailant. Both
female victims are wearing blue jeans at the time of the attack. Item (5): the
women are taken to the sexual assault treatment center and suspect semen
samples are collected on swabs from the vaginal vault and perineal area. Item
(6): there are also six blood stains on the clothing of the two suspects in
custody. Item (7): underwear from both suspects contain what appear to be body fluid
stains outside and inside the garment. Item (8): the two suspects are taken to
the hospital, where penile swabs are collected and placed into evidence. Item
(9): there also are hairs on all of the clothing of both victims and
defendants.
“The power of DNA lies in its level of variation among individuals and
greater potential to identify them”
Most samples that come into a crime laboratory have dried and degraded,
a fact that both aids and hinders the serologist. The stains on the blue jeans and on the leather handbag present a
special problem. Special procedures must be used, as the chemicals in leather
and blue jeans may interfere with testing for some genetic markers. The swabs
collected at the sexual assault treatment center are immediately placed into a
paper envelope so they can air-dry. The only liquid samples collected at the
center are vials of blood, preserved to minimize cell membrane breakage and
protect the integrity of the DNA molecules. As an example there is an analysis of semen samples from cases involving a sexual
assault submitted. Semen is processed by RFLP/VNTR analysis, as in the case of a blood
stain. In this example, the amount of semen is graded by use of a presumptive
test for the enzyme acid phosphatase, which is found in large quantities
inhuman semen. It could also be graded by the amount of sperm cells detected.
If the sample is too small or not amenable to RFLP, a DNA/PCR analysis is
performed. If RFLP/VNTR analysis gives clear results, the DNA profile from the
suspect and or evidence may be entered into the database.
Blood is not the only biological fluid or tissue shed or left at a crime
scene. Body fluids represent a significant contribution to crime scene
material. For instance seminal fluid is often part of the evidence in sexual
assaults. Skin and other tissue are left many times at hit-and-run scenes.
Epithelial cells are deposited on licked stamps, cigarette butts, envelopes,
and chewing gum. Fetal material such as cord, cord blood, and fetal tissue
originates from an aborted fetus. With the use of PCR and mitochondrial DNA
analysis, dental pulp and bone marrow can now yield substantial genetic
information to the investigator. The PCR process is a very powerful technique
which allows the analyst to copy or amplify the amount of DNA present in a
small or degraded sample. Mitochondrial DNA (mtDNA) is located to some extent
in the hair shaft itself. Early
results show that use of PCR/DNA analysis is easily accomplished in tissue
attached to fingernail scraping and broken fingernails from a crime scene.
There are three methods of detecting items of serological evidence:
-
visual,
-
microscopic,
-
chemical.
Body fluid stains are either visible to the eye or invisible because of
the quantity of stain or because they are hidden in another body fluid or on
material which precludes visual detection. For example, a semen or saliva stain
may be masked by a large quantity of blood on a garment. Some stains, such as
semen, have a characteristic texture or color which ranges from milky white to
light tan when blood cells are present.
There are eight categories of genetic markers which a forensic
serologist uses routinely:
-
conventional markers — cellular antigens,
-
serum proteins,
-
cellular antigens
-
DNA-based markers — RFLPs (restriction fragment length
polymorphism),
-
STRs (short tandem repeats),
-
AmpFlps (amplified fragment length polymorphism),
-
ASO (allele specific oligo),
-
mtDNA
(mitochondrial DNA) analysis
There are eight categories of tests in the forensic serologist’s arsenal. The
forensic serologist’s arsenal includes tests based on antigenic and enzyme
assays, as well as DNA tests. Antigenic assays couple antibodies which
recognize specific forms of the antigen; the familiar ABO blood groups are
antigen/antibody assays. Other antigenic tests are listed in the CELLULAR
ANTIGENS category. Another set of tests focuses on proteins found in serum or
in cells. Some of these use antibodies to recognize specific forms of the
protein, while other assays depend on enzymatic activity to visualize the
different forms. These are categorized as serum or cellular tests rather than
as antigen or enzymatic tests, because both types of assays are used in
detecting serum and cellular protein differences. Three of the classes of DNA
tests identify length variation at specific human genes. RFLP (restriction
fragment length polymorphisms) or, more specifically, VNTR (variable number
tandem repeat polymorphisms) are the original DNA tests. STR (simple tandem
repeat polymorphisms) and AmpFLPs (amplified fragment length polymorphisms) are
tests which can be run with very small samples, because they depend on
amplification of DNA. The DNA in ASO (allele specific oligonucleotide) tests is
also amplified, but the specificity of these assays is not dependent on length
variation; it is dependent on nucleotide sequence differences or short
deletions or insertions. Finally, forensic tests may be based on actual
sequence data. Mitochondrial DNA (mtDNA) is often used for sequencing, because
it is a relatively small and abundant molecule with variable regions. In modern forensic science and investigative technique an autoradiogram
or autorad is a photographic image of the pattern of DNA fragments, arranged in
vertical columns, produced by known and unknown samples. Larger DNA fragments
are at the top of the autorad, because, by convention, the origin or well into
which DNA samples were placed for separation by electrophoresis is placed at
the top. Since the agarose gel containing the DNA fragments has a sieving
effect, the larger pieces do not move as far as the smaller pieces; they stay
close to the origin and are at the top of the autorad.
Example.0.1
Since the DNA molecule is housed within the nucleus of all types of
cells in the human body, except mature red blood cells, scientists can rely on
different tissue sources for collection. Nucleic acid can be extracted from
bone, hair, or dried skin; these are the sources for most DNA isolation from
older material such as mummies. If we are dealing with a maternity or paternity
case in which the putative parent is alive, blood will be the tissue chosen for
DNA isolation because it is relatively noninvasive and allows almost painless
access. Buccal (mouth) swabbing of epithelial cells contained in the cheek area
is another tissue of choice. DNA is a very long polymer. Human cells contain 46
nuclear DNA molecules; each is composed of millions of nucleotide pairs. The
long DNA chains are susceptible to degradation. There are factors acknowledged
to contribute to DNA breakdown. The most ideal and perfect in structure are
tissue samples that are dried as soon as possible for DNA profiling. The reason
is simple: many of the degradative processes require the DNA to be hydrated
(surrounded by water); if the DNA is not in solution, it will retain its
integrity longer. When samples arrive in the lab, it must be decided whether
the DNA isolation will begin immediately. If circumstances do not permit DNA
purification, dried samples are stored frozen in a dry environment while wet
samples should be just frozen. PCR
(polymerase chain reaction) is a process in which small fragments of DNA
(usually less than 2 to 3 thousand bases long) are repeatedly copied using a
semiconservative mechanism. The PCR process consists of three major steps:
denaturing, annealing, and extension. This process is repeated from 20 to 30
times. Starting with a single copy of a specific nucleotide sequence flanked by
primer nucleotide sequences, it is possible to produce millions or even
billions of copies of this specific sequence. The number of copies produced
depends on the number of replication cycles.
Example.0.2.
Example.0.3.
Primer binding is called annealing (55 to 72°C ). Next, a thermostable
DNA polymerase is added to the reaction which adds the bases A, T, G, or C to
the single-strand template, thus making a new, complementary single strand. The
primer initiates the extension (72 to 75°C ) process. Since primersequences are
present in vast excess, the process may be repeated through 2 to 30 cycles creating
millions of copies — an exponential chain reaction. Where we started with one
double-stranded DNA sequence or two single denatured strands, we now have four
single strands. The next cycle will produce eight (4 × 2) double strands.
Example.0.4.
The polymerase chain reaction multiplies copies of specific DNA segments
or genes were originally produced by cloning, cutting out the segment wanted
and inserting it into a host cell which would, as it reproduced, make copies of
the inserted gene along with its own DNA. Now DNA may be copied enzymatically
using a temperature-insensitive DNA copying enzyme or polymerase. Starting with
double-stranded DNA, the specific site to be amplified is targeted by using
primers which flank the target site and act as anchors for the synthesis of a
new DNA strand. (A) The first step of a cycle involves melting the DNA to
expose the nucleotides of each strand allowing the primers to bind. (B) Next
the temperature is lowered and the polymerase enzyme facilitates the synthesis
of two new DNA strands using the old strands as templates. (C) These two
double-stranded DNA molecules are melted or denatured in cycle 2 to begin the
process anew. It is called a chain reaction, because at each cycle the number
of previously existing DNA molecules is doubled. Theoretically it is possible to multiply
single strands of DNA to essentially millions of copies of that single
sequence, PCR is extremely sensitive, and from 1 to 5 ng of DNA can be
successfully typed using the process. The amplification, the DNA sample is
separated on a gel, usually polyacrylamide, and the amplified fragments are
visualized with silver stain or fluorescent dyes. As in VNTR analysis, size
ladders are included on the analytical gels. In AmpFLP analysis, however,
alleles are treated as discrete units which allows visual comparison of alleles
with the ladder alleles, unlike VNTR analysis which requires computer sizing.
Human identification depends on two things:
- characteristics which vary
among individuals,
- knowledge of character
percentages or frequencies.
An identification system using measurements of a person’s head size,
right ear size, left foot size, color of the iris of the left eye, and hair
color, among other characteristics, was introduced to forensics by the French
anthropologist Alphonse Bertillon in the late 1800s.
Fingerprint identification depends on similar exclusion and inclusion
logic; if a print is an arch, then all loop and whorl prints (those which are
not arch) are excluded. Both physical description and fingerprinting are based
on character variability and frequency information. Fingerprinting has the
advantage that prints may be left at a crime scene, but it shares the
disadvantage that explicit frequency calculations are based on the assumption
of character independence which is, of course, statistically tested in forensic
labs. Blood group and serum protein characteristics share the variability and
frequency knowledge requirements seen in the use of physical description and
fingerprinting. Individual evidence is at the opposite pole from class evidence.
Individual evidence itself is in such a rare class, or its individual
characteristics are so uncommon, as to make it unique. A good example of this
is a fracture pattern of a broken piece of glass. Every piece of glass that is
broken produces a unique and individual fracture pattern. If you were to break
a glass an infinite number of times, a particular fracture pattern would never
be reproduced. A serologist uses a
simple statistical test to measure the value of genetic markers for
individualization. This is the power of discrimination (PD) test. A serologist
has a 54% chance of discriminating between two stains of different individuals.
It really does not matter how you want to think about it; both numbers guide
the serologist toward the choice of a genetic marker. The goals of the forensic scientist in regard
to error are to minimize its occurrence and to limit its effects when it does
occur. In comparisons of evidence characteristics, and suspect characteristics
the general question is “Do they or do not they match?”. Are both samples,
evidentiary and suspect, likely to have come from the same person, the suspect,
or the victim? Errors may occur at this stage of the analysis either by
declaring a match when, in fact, the evidentiary and suspect DNA is different.
In other words, the evidentiary DNA did not come from the suspect, but the
examiner has declared a match suggesting that the origin of both samples could
have been the same individual. Alternatively, a nonmatch may be declared in
which the DNA characteristics are said not to match when they actually have
come from the same individual
Type I/Type II Errors
1. Null Hypothesis:
Evidentiary phenotype = suspect phenotype. The data prediction is that
the bands are visually in the same position and within the match-window after
sizing of the DNA fragments.
2. Alternative Hypothesis:
Evidentiary phenotype/suspect phenotype. The data prediction is that the
bands do not match visually. The presumption of innocence is not violated by
formulating the null hypothesis in this way, and we may attach an error
probability to the test of type I, i.e., false exclusion. In this case, we
accept the hypothesis match, because the bands are the same, suggesting that
the samples came from the suspect.
The investigative methods underline the importance of science and DNA which is so small that You can barely see it with an electron microscope. Nonetheless people learnt how to figure it out, read it, copy it, cut into pieces, sort it, put it together in new combinations only to prove its undeniable role in crime solving process. No matter how horrid the crime is, no matter what the suspect or the victim says, ultimately in this particular matter all lies lead to the truth and this truth is PCR, it scarcely discriminates the judgement and the final outcome of the case.
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Forensic Science.2006
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Techniques of Crime Scene Investigation.2012
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„A Short History of the Polymerase Chain Reaction". PCR Protocols. Methods in Molecular
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Molecular Cloning: A Laboratory Manual (3rd ed.). Cold
Spring Harbor ,N.Y. : Cold Spring Harbor
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"Antibodies as Thermolabile Switches: High Temperature Triggering
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Chemistry. 2013
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Acknowledgements:
The Police Department;
https://www.politie.nl/mijnbuurt/politiebureaus/05/burgwallen.html and a Chief Inspector – Mr. Erik Akerboom ©
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