THE DEPARTMENT OF BIOTECHNOLOGY
FACULTY OF
BIOLOGICAL SCIENCES
ABSTRACT
Forensic science is a broad spectrum of
science and technology which is used to investigate situations after fact and
to establish what occurred based on collected evidence. This is especially
important in law enforcement where forensic is done in relation to criminal or
civil law. Forensic science deals more in crime detection. Crime can be said to
be a harmful act or omission against the public which the state is trying to
prevent and when convicted is punishable by law. The application of
biotechnology to crime detection lies on the discovery of DNA typing or genetic
finger printing which confirms that every person has a unique DNA pair
material. DNA profiling uses various repeat sequences which includes Variable
Number Tandem Repeat (VNTR) and Short Tandem Repeats (STR).DNA finger printing
helps forensic science in the identification of an individual through the
process of DNA profile and this uses techniques like southern blotting and
polymerase chain reaction. The different techniques of DNA finger printing
share the same steps, that is, isolation and extraction of different DNA
samples, cutting the DNA with a restriction enzyme, separating it with the use
of a gel electrophoresis, transferring and binding the DNA and developing an
X-ray film to make it visible.
TABLE OF
CONTENTS
TITLE
PAGE- - - - - - - - - 1
CERTIFICATION - - - - - - - - 2
DEDICATION
- - - - - - - - - 3
ACKNOWLEDGMENTS- - - - - - - 4
ABSTRACT
- - - - - - - - - 5
TABLE
OF CONTENT - - - - - - - 6
CHAPTER ONE
1.0
INTRODUCTION- - - - - - - - -
CHAPTER
TWO
2.0 DEOXYRIBOSNUCLEIC ACID (DNA) - - - - -
2.1 DNA STRUCTURE - - - - - - -
CHAPTER THREE
3.0 DNA FINGERPRINTING - - - - - - -
3.1 DNA
FINGERPRINTING TECHNIQUES - - - - -
3.1.0
SOURCES
OF DNA SAMPLE - - - - - -
3.1.1
ISOLATION OF DNA SAMPLE - - - - - -
3.1.2 CLEAVAGE OF DNA FRAGMENT- - - - -
3.1.3 SEPERATION USING GEL ELECTROPHORESIS
TECHNIQUE
3.1.4 SOUTHERN
BLOTTING TECHNIQUE - - - - -
CHAPTER
FOUR
4.0 MODIFICATION IN DNA FINGERPRINTING
TECHNIQUES -
4.1 POLYMERASE CHAIN
REACTION
4.2 VARIABLE NUMBER TANDEM REPEATS (VNTR) - -
4.3 SHORT TANDEM REPEAT (STR)- - - - - -
4.4 RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)
4.5 AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP) -
CHAPTER FIVE
5.0 DNA DATABASE- - - - - - - - -
5.1 THE TWO CODIS INDEXES- - - - - - -
5.2 APPLICATION OF DNA FINGERPRINTING- - - -
5.3 DNA STORAGE- - - - - - - - -
5.4
COMPARISON OF DNA SAMPLES FOR FORENSIC ANALYSIS-
5.5 PROBABILITIES OF A MATCH- - - - - -
5.6 LIMITATIONS IN DNA FINGERPRINTING IN FORENSIC
ANALYSIS
5.6.0 CONTAMINATION IN DNA SAMPLE- - - - -
5.6.1 DEGRADATION OF DNA SAMPLE- - - - -
5.6.2 MISSING OF DNA
PROFILE
5.7 PREVENTION OF
DNA CONTAMINATION- - - -
CHAPTER
SIX
6.0
SUMMARY- - - - - - - - -
6.1
CONCLUSION- - - - - - - - -
6.2 RECOMMENDATION- - - - - - - -
CHAPTER
ONE
1.0 INTRODUCTION
Forensic is the usage of science and
technology to aid in criminal investigation. Crime is a harmful act or omission
against the public which the state wishes to prevent and which upon conviction
is punishable by fine, imprisonment, and or death. The application of biochemical
approach to crime detection is based on the discovery of the
DNA profiling which lies on the
fact that the DNA of each
person is unique and has a unique
sequence of base pairs which allow a person
to be identified by examining their genetic materials . DNA profiling (also called DNA
testing, DNA typing, or genetic fingerprinting) is a technique
employed by forensic scientists
to assist in the identification of individuals by their respective DNA profiles. DNA profiles are encrypted sets of numbers
that reflect a person's DNA makeup, which can also be used as the person's
identifier. It is used in, for parental
testing and criminal investigation (Cummins, 1941).
Although
99.9% of human DNA sequences are the same in every person, little of the DNA fragment is enough to distinguish
one individual from another, unless they are monozygotic twins. DNA profiling
uses repeat sequences that are highly variable, called variable number
tandem repeats (VNTRs), Particularly short tandem
repeats (STRs).
VNTR loci
are very similar between closely related humans, but so variable that unrelated
individuals are extremely unlikely to have the same VNTRs (Joseph and David,
2001).
The
DNA profiling technique was first reported in 1984 by Sir Alec
Jeffreys at the University of Leicester in England, and is now the basis of several national DNA databases.(Amy, 2008) Unlike a convention fingerprint that
occurs only on the fingertips and can be altered by surgery or mutation, a DNA
fingerprint is the same for every tissue, and organ of a person. It cannot be
altered by any known treatment. The
finger print itself is a distinct pattern that resembles a barcode. It can be
displayed directly on a gel or on a nitrocellulose sheet visualized by x-ray film. (Amy, 2008).
Henry Erlich (1987) developed a method
of DNA fingerprinting so sensitive that it could be used to identify an individual
from an extremely small sample of hair, blood, semen, or skin. Erlich’s
techniques used Jeffrey’s traditional method and combined it with a new
technique called polymerase chain reaction (PCR). Using his new method, Erlich was able to
duplicate and heat separate the DNA fragments from a single human hair root
many times. The amplified DNA was then used to obtain a DNA fingerprint. The
different DNA techniques employed in “fingerprinting” all share common steps in
procedure and analysis that include
(1)
Isolation
and extraction of the DNA from the unidentified sources of biological remnants
such as blood.
(2)
Cutting
the DNA into fragments with a restriction enzyme.
(3)
Separating
it into bands through electrophoresis, transferring and then binding the
emerging DNA band pattern with radiation.
(4)
Applying
and developing x-ray film to make the DNA band pattern visible for comparison
with known DNA samples from an individual or source.
Genetic
finger printing has already proved to be a very useful tool. Initially, it was
used to be exclusively in forensic (criminal) science and law. This technique
has helped to link suspect to crimes where a single drop of blood was the only
evidence. When there is a need to know who an individual’s biological mother or
father is, genetic fingerprinting can provide the answer by matching DNA
elements between parents and child. DNA fingerprinting must be done with great
care, since any contamination of blood samples by mixing or touching them with
ungloved hands can produce false results, with serious consequences in legal
proceedings such as a trial. If carefully done, however, genetic fingerprinting
can provide accurate identification of an individual (Erlich, et al., 1987).
CHAPTER TWO
2.0 DEOXYRIBOSNUCLEIC ACID (DNA)
Deoxyribonucleic acid (DNA) is a molecule that encodes the genetic instructions used in the development and functioning of all
known living organisms
and many viruses. DNA is a nucleic
acid; alongside proteins and carbohydrates, nucleic acids compose the three major macromolecules essential for all known forms of life. Most DNA molecules consist of two biopolymer strands coiled around each other to form a double helix. The two DNA strands are known as polynucleotides since they are composed of simpler
units called nucleotides. Each nucleotide is composed of a nitrogen-containing nucleobase—either
guanine (G), adenine
(A), thymine (T), or cytosine (C)—as well as a monosaccharide sugar called deoxyribose and a phosphate
group. The nucleotides are joined to one
another in a chain by covalent
bonds between the sugar of one nucleotide
and the phosphate of the next, resulting in an alternating sugar-phosphate
backbone. According to base
pairing rules (A with T and C with G), hydrogen
bonds bind the nitrogenous bases of the
two separate polynucleotide strands to make double-stranded DNA.
DNA is well-suited for biological information storage. The DNA backbone is resistant to cleavage, and
both strands of the double-stranded structure store the same biological
information. Biological information is replicated as the two strands are
separated. A significant portion of DNA (more than 98% for humans) is non-coding, meaning that these sections do not serve a function of
encoding proteins.
The two strands of DNA run in
opposite directions to each other and are therefore anti-parallel. Attached to each sugar is one of four types of nucleobases
(informally, bases). It is the sequence
of these four nucleobases along the backbone that encodes biological
information. Under the genetic
code, RNA strands are translated to specify the sequence of amino
acids within proteins. These RNA strands
are initially created using DNA strands as a template in a process called transcription.
Within cells, DNA is organized into
long structures called chromosomes.
During cell division
these chromosomes are duplicated in the process of DNA
replication, providing each cell its own
complete set of chromosomes. Eukaryotic
organisms (animals, plants,
fungi, and protists)
store most of their DNA inside the cell
nucleus and some of their DNA in organelles, such as mitochondria or chloroplasts.[1] In contrast, prokaryotes (bacteria
and archaea) store their DNA only in the cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organize DNA. These compact structures guide
the interactions between DNA and other proteins, helping control which parts of
the DNA are transcribed.
Scientists use DNA as a molecular
tool to explore physical laws and theories, such as the ergodic
theorem and the theory of elasticity.
The unique material properties of DNA have made it an attractive molecule for
material scientists and engineers interested in micro- and nano-fabrication.
Among notable advances in this field are DNA
origami and DNA-based hybrid materials.[2]
Properties
DNA is a long polymer made from repeating units called nucleotides.[3][4][5] DNA was first identified and isolated by Friedrich Miescher
and the double helix structure of DNA was first discovered by James
Watson and Francis
Crick. The structure of DNA of all
species comprises two helical chains each coiled round the same axis, and each
with a pitch of 34 ångströms (3.4 nanometres) and a radius of 10 ångströms (1.0 nanometres).[6] According to another study, when measured in a particular
solution, the DNA chain measured 22 to 26 ångströms wide (2.2 to 2.6 nanometres), and one nucleotide unit measured 3.3 Å
(0.33 nm) long.[7] Although each individual repeating unit is very small, DNA
polymers can be very large molecules containing millions of nucleotides. For
instance, the largest human chromosome, chromosome number 1,
consists of approximately 220 million base
pairs[8] and is 85 nm long.
In living organisms DNA does not
usually exist as a single molecule, but instead as a pair of molecules that are
held tightly together.[9][10] These two long strands entwine like vines, in the shape of
a double helix.
The nucleotide repeats contain both the segment of the backbone of the
molecule, which holds the chain together, and a nucleobase, which interacts
with the other DNA strand in the helix. A nucleobase linked to a sugar is
called a nucleoside
and a base linked to a sugar and one or more phosphate groups is called a nucleotide. A polymer comprising multiple linked nucleotides (as in
DNA) is called a polynucleotide.[11]
The backbone of the DNA strand is
made from alternating phosphate
and sugar
residues.[12] The sugar in DNA is 2-deoxyribose, which is a pentose (five-carbon)
sugar. The sugars are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. These asymmetric bonds mean a strand of DNA has a direction. In a double helix the
direction of the nucleotides in one strand is opposite to their direction in
the other strand: the strands are antiparallel. The asymmetric ends of
DNA strands are called the 5′ (five prime) and 3′ (three prime) ends, with the 5′ end having a
terminal phosphate group and the 3′ end a terminal hydroxyl group. One major
difference between DNA and RNA
is the sugar, with the 2-deoxyribose in DNA being replaced by the alternative
pentose sugar ribose
in RNA.[10]
The DNA double helix is stabilized
primarily by two forces: hydrogen
bonds between nucleotides and base-stacking
interactions among aromatic
nucleobases.[14] In the aqueous environment of the cell, the conjugated π
bonds of nucleotide bases align
perpendicular to the axis of the DNA molecule, minimizing their interaction
with the solvation shell
and therefore, the Gibbs
free energy. The four bases found in DNA are adenine (abbreviated A), cytosine (C), guanine
(G) and thymine (T). These four bases are attached to the sugar/phosphate
to form the complete nucleotide, as shown for adenosine monophosphate.
Nucleobase
classification
The nucleobases are classified into
two types: the purines,
A and G, being fused five- and six-membered heterocyclic compounds, and the pyrimidines, the six-membered rings C and T.[10] A fifth pyrimidine nucleobase, uracil (U), usually takes the place of thymine in RNA and differs
from thymine by lacking a methyl
group on its ring. In addition to RNA and
DNA a large number of artificial nucleic acid analogues have also been created to study the properties of nucleic
acids, or for use in biotechnology.[15]
Uracil is not usually found in DNA,
occurring only as a breakdown product of cytosine. However, in a number of
bacteriophages – Bacillus subtilis bacteriophages PBS1 and PBS2 and
Yersinia bacteriophage piR1-37 – thymine has been replaced by
uracil.[16] Another phage - Staphylococcal phage S6 - has been
identified with a genome where thymine has been replaced by uracil.[17]
Grooves
Twin helical strands form the DNA
backbone. Another double helix may be found tracing the spaces, or grooves,
between the strands. These voids are adjacent to the base pairs and may provide
a binding site.
As the strands are not symmetrically located with respect to each other, the
grooves are unequally sized. One groove, the major groove, is 22 Ã… wide
and the other, the minor groove, is 12 Ã… wide.[26] The narrowness of the minor groove means that the edges of
the bases are more accessible in the major groove. As a result, proteins like transcription factors that can bind to specific sequences in double-stranded DNA
usually make contacts to the sides of the bases exposed in the major groove.[27] This situation varies in unusual conformations of DNA
within the cell (see below), but the major and minor grooves are always
named to reflect the differences in size that would be seen if the DNA is
twisted back into the ordinary B form.
Base
pairing
In a DNA double helix, each type of
nucleobase on one strand bonds with just one type of nucleobase on the other
strand. This is called complementary base
pairing. Here, purines form hydrogen
bonds to pyrimidines, with adenine
bonding only to thymine in two hydrogen bonds, and cytosine bonding only to
guanine in three hydrogen bonds. This arrangement of two nucleotides binding
together across the double helix is called a base pair. As hydrogen bonds are
not covalent,
they can be broken and rejoined relatively easily. The two strands of DNA in a
double helix can therefore be pulled apart like a zipper, either by a
mechanical force or high temperature.[28] As a result of this complementarity, all the information in
the double-stranded sequence of a DNA helix is duplicated on each strand, which
is vital in DNA replication. Indeed, this reversible and specific interaction
between complementary base pairs is critical for all the functions of DNA in
living organisms.[4]
The two types of base pairs form
different numbers of hydrogen bonds, AT forming two hydrogen bonds, and GC
forming three hydrogen bonds. DNA with high GC-content is more stable than DNA with low GC-content. Most DNA
molecules are actually two polymer strands, bound together in a helical fashion
by noncovalent bonds; this double stranded structure (dsDNA) is
maintained largely by the intrastrand base stacking interactions, which are
strongest for G,C stacks. The two strands can come apart – a process known
as melting – to form two single-stranded DNA molecules (ssDNA)
molecules. Melting occurs at high temperature, low salt and high pH (low pH
also melts DNA, but since DNA is unstable due to acid depurination, low pH is
rarely used).
The stability of the dsDNA form
depends not only on the GC-content (% G,C basepairs) but also on sequence
(since stacking is sequence specific) and also length (longer molecules are more
stable). The stability can be measured in various ways; a common way is the
"melting temperature", which is the temperature at which 50% of the
ds molecules are converted to ss molecules; melting temperature is dependent on
ionic strength and the concentration of DNA. As a result, it is both the
percentage of GC base pairs and the overall length of a DNA double helix that
determines the strength of the association between the two strands of DNA. Long
DNA helices with a high GC-content have stronger-interacting strands, while
short helices with high AT content have weaker-interacting strands.[29] In biology, parts of the DNA double helix that need to
separate easily, such as the TATAAT Pribnow
box in some promoters,
tend to have a high AT content, making the strands easier to pull apart.[30]
In the laboratory, the strength of
this interaction can be measured by finding the temperature necessary to break
the hydrogen bonds, their melting
temperature (also called Tm
value). When all the base pairs in a DNA double helix melt, the strands
separate and exist in solution as two entirely independent molecules. These
single-stranded DNA molecules (ssDNA) have no single common shape, but
some conformations are more stable than others.[31]
Super coiling
DNA can be twisted like a rope in a
process called DNA supercoiling.
With DNA in its "relaxed" state, a strand usually circles the axis of
the double helix once every 10.4 base pairs, but if the DNA is twisted the
strands become more tightly or more loosely wound.[38] If the DNA is twisted in the direction of the helix, this
is positive supercoiling, and the bases are held more tightly together. If they
are twisted in the opposite direction, this is negative supercoiling, and the
bases come apart more easily. In nature, most DNA has slight negative
supercoiling that is introduced by enzymes called topoisomerases.[39] These enzymes are also needed to relieve the twisting
stresses introduced into DNA strands during processes such as transcription and DNA
replication.[40]
DNA usually occurs as linear chromosomes in eukaryotes,
and circular chromosomes in prokaryotes. The set of chromosomes in a cell makes up its genome; the human
genome has approximately 3 billion base
pairs of DNA arranged into 46 chromosomes.[86] The information carried by DNA is held in the sequence of pieces of DNA called genes. Transmission
of genetic information in genes is achieved via complementary base pairing. For
example, in transcription, when a cell uses the information in a gene, the DNA
sequence is copied into a complementary RNA sequence through the attraction
between the DNA and the correct RNA nucleotides. Usually, this RNA copy is then
used to make a matching protein
sequence in a process called translation,
which depends on the same interaction between RNA nucleotides. In alternative
fashion, a cell may simply copy its genetic information in a process called DNA
replication. The details of these functions are covered in other articles; here
we focus on the interactions between DNA and other molecules that mediate the
function of the genome.
2.1 DNA STRUCTURE
The
naturally occurring double helix structure of DNA
Figure
1: DNA structure (Waston J.D. and Crick F.H.C,1953)
Deoxyribonucleic
acid (DNA) is the genetic material of cell defined as a polynucleotide having a
specific sequence of deoxyribonucleotide units covalently joined through 31,
51 – phosphodiester bonds; which serve as the carrier of genetic
information. DNA is composed of nucleotide subunits which consists of
deoxyribose (a sugar), a phosphate group and a nitrogenous bases. The
nitrogenous bases are thymine (T) guanine (G) adenine (A) and cytosine (C)
these subunits bonds to each other covalently to form strands of DNA. Hydrogen
bonds links nitrogenous bases together. The bases are specific in bonding in
that adenine (A) bonds to thymine (T) and Guanine (G) only bonds to cytosine
(C). These bonds form the naturally occurring double helix structure as shown above (David and Michael, 2005)
DAMAGE
DNA can be damaged by many sorts of mutagens, which change the DNA sequence. Mutagens include oxidizing
agents, alkylating
agents and also high-energy electromagnetic radiation such as ultraviolet light and X-rays. The type of DNA damage produced depends on the type of mutagen.
For example, UV light can damage DNA by producing thymine
dimers, which are cross-links between
pyrimidine bases.[73] On the other hand, oxidants such as free radicals
or hydrogen peroxide
produce multiple forms of damage, including base modifications, particularly of
guanosine, and double-strand breaks.[74] A typical human cell contains about 150,000 bases that have
suffered oxidative damage.[75] Of these oxidative lesions, the most dangerous are
double-strand breaks, as these are difficult to repair and can produce point
mutations, insertions and deletions
from the DNA sequence, as well as chromosomal translocations.[76] These mutations can cause cancer. Because of inherent limitations in the DNA repair
mechanisms, if humans lived long enough, they would all eventually develop
cancer.[77][78] DNA damages that are naturally occurring, due to normal cellular processes that produce reactive
oxygen species, the hydrolytic activities of cellular water, etc., also occur
frequently. Although most of these damages are repaired, in any cell some DNA
damage may remain despite the action of repair processes. These remaining DNA
damages accumulate with age in mammalian postmitotic tissues. This accumulation
appears to be an important underlying cause of aging.[79][80][81]
Many mutagens fit into the space
between two adjacent base pairs, this is called intercalation. Most intercalators are aromatic and planar molecules; examples include ethidium
bromide, acridines, daunomycin,
and doxorubicin.
For an intercalator to fit between base pairs, the bases must separate, distorting
the DNA strands by unwinding of the double helix. This inhibits both
transcription and DNA replication, causing toxicity and mutations.[82] As a result, DNA intercalators may be carcinogens, and in the case of thalidomide, a teratogen.[83]
CHAPTER THREE
3.0 DNA FINGERPRINTING
DNA
fingerprinting is a relatively new techniques that has revolutionized forensic
science. It is a technique employed by forensic scientists
to assist in the identification of individuals by their respective DNA profiles. DNA profiles are encrypted sets of numbers
that reflect a person's DNA makeup, which can also be used as the person's
identifier. A
DNA fingerprinting is a unique barcode pattern of information derived from
assaying DNA. Each person has a unique sequence of DNA base pairs which allows
a person to be identified by examining their genetic material. The fingerprint
itself is a distinct pattern that resembles a barcode. It can be displayed
directly on a gel or on a nitrocellulose sheet visualized by x – ray film
(Schwartz and Vissing, 2002).
3.1
DNA FINGERPRINTING TECHNIQUES
DNA fingerprinting technique was first
discovered in England in 1985 and is based on the fact that the DNA of each
person is unique. In 1992, DNA fingerprinting was approved by the U.S National
Research Council as a reliable identification technique. Now, DNA
fingerprinting can be used in court cases as evidence. Techniques like Southern
Blotting and polymerase chain reaction can be used to create DNA fingerprinting
(Barttle and Stirling, 2003).
Techniques
in DNA Fingerprinting involves:
1.
Sources
of DNA sample
2.
Isolation of DNA sample
3.
Cleavage
of DNA fragment
4.
Separation
using Gel Electrophoresis technique.
5.
Southern
Blotting technique
3.1.0 SOURCES OF DNA
Source of DNA sample must be known. DNA
can be recovered from the cells or tissues of the body, this source could be a
single strand of hair, semen, blood, saliva, cheek cell, etc. During criminal
investigations the hair, semen, or blood of the suspect is collected for
further analysis.
3.1.1 ISOLATION OF DNA
The
next step is to extract the DNA sample from its source. The extraction process
is devised in a way to break down the cell membrane and release the DNA to its
outer environment. Detergents are used for this purpose. They tend to break
open the cell membrane by forming micelles with protein and lipid entities of
the membrane (Bhattacharya, 2011).
3.1.2 CLEAVEAGE
OF DNA FRAGMENT
Once the DNA is extracted, it is subjected to
digestion by Restriction Endonucleases. These are enzymes that cut a DNA
fragment at specific sites which it recognizes (Iborra et al., 2004). It means
that each restriction enzyme (R.E.) recognizes a specific DNA sequence and cuts
at a specific site. Remember that the cleaving is a double strand cut,
producing DNA fragmentsof varied lengths. These fragments are also called RFLP
(Restricted Fragment Length Polymorphism). Many of these fragments will contain
the Variable Number Tandem Repeats (VNTRs) (chenqet al., 2004).
3.1.3 SEPARATION USING GEL ELECTROPHORESIS
TECHNIQUE.
DNA
fragments are then separated by difference in their length using Gel
Electrophoresis technique. This technique employs electric current to move the
DNA fragments over a gel-based matrix. The DNA molecule is negatively charged
(due to a phosphate group) and hence will move towards the positive anode in
the set up. The gel-based matrix is
usually made of agarose which provides tiny pores in them through which the DNA
molecules can travel. The gel may be treated with an acid like Hcl to break the
DNA into smaller sizes. This process is
known as depurination, so as to allow efficient transfer from gel to
membrane. (Iborra et al 2004). The DNA samples are loaded at one end of the gel and
moves to the other when electric current is applied. The larger fragments
travel slowly through the gel. Note: To determine DNA Fragment of larger than
15kp use DNA Sizes marker or ladder.
However,
the smaller fragments travel quickly and reach further away from the sample
loading point. The different-sized pieces of DNA will therefore, be separated
by size, with the smaller pieces towards the bottom and the larger pieces
towards the top (Christoffersen, 2011).
3.1.4 SOUTHERN BLOTTING TECHNIQUE FOR THE DETECTION
OF A SPECIFIC DNA SEQUENCE
Southern
blotting is a technique used for analyzing the related genes in a DNA
restriction fragment. A southern blotting technique is a method used in
molecular biology for detection of a specific DNA sequence in DNA sample.
(Iborra et al 2004)
STEPS INVOLVED IN SOUTHERN BLOTTING ANALYSIS
a.
Sample
preparation
b.
Gel
electrophoresis
c.
Transfer of DNA
fragments from gel to membrane
d.
Blocking
e.
Detection
f.
Result
A.
SAMPLE
PREPARATION
The genomic DNA is first extracted from the
sample using a suitable procedure or by using DNA extraction kit, then, one or
more restriction endonucleases are used to cut the high molecular DNA at a
specific site into smaller fragments. Restriction enzymes or endonucleases are
a class of enzymes that cut DNA molecules of specific site generating DNA
fragments (Iborra et al, 2004)
B.
GEL
ELECTROPHORESIS
The
DNA fragments are placed in an agarose gel electrophoresis to separate them by
sizes, If compared with a DNA marker and the size are larger than 15kb, the gel
may be treated with an acid like HCl to break the DNA into smaller sizes. This process is known as depurination.
(Iborra et al 2004). This is to allow
efficient transfer from gel to membrane. Note : To determine DNA Fragment of
larger than 15kp use DNA Sizes marker or ladder.
C.
TRANSFER OF DNA
FRAGMENTS FROM GEL TO MEMBRANE
The
gel containing separated DNA fragment is placed into a transfer buffer in an
alkaline buffer solution. The DNA gel is incubated with an alkaline solution
containing NaOH to denature the double stranded DNA.This separate double
stranded DNA into a single stands of DNA for hybridization to the probe.This
improves the binding of the negatively charge DNA to a positively charged
membrane and destroys any residual RNA that may still be present in the DNA
preparation. A sheet of nitrocellulose or nylon membrane is placed on top of
the gel or below, pressure is applied evenly to the gel either by using suction
or by placing a stack of paper towels and a weight on top of the membrane and
gel to ensure good and even contact between gel and membrane. Buffer transfer
by capillary actions from a region of high water potential to a region of low
water potential then moves the DNA fragment from the gel into the membrane. Ion
exchange interactions bind the DNA to the negative charged DNA and positively
charged membrane, the membrane is then removed and baked in a vacuum or oven at
80oC for 2 hours to permanently attach the transferred DNA fragment
into the membrane. The reason for transferring the DNA fragment to a solid support
using a nitrocellulose membrane is to make the DNA accessible to sequence
specific labels (probes). When DNA fragment are in the gel they are sort of
buried. But transferring them to the nitrocellulose membrane exposes them for
detections by probes. The relative position of the DNA fragment is preserved
during their transfer to the filter membrane. (Iborra et al 2004).
D.
BLOCKING
The
membrane is treated with e.g. sodium dodecyl sulphate (SDS) to block the
membrane surface not converted by the target DNA to reduce non-specific binding
of the probe, this allows the probe to bind specifically to the target
sequence. (Iborra et al 2004)
E.
DETECTION
The
membrane is exposed to a hybridization probe. A hybridization probe is a single
stranded DNA fragment with a specific sequence whose complementary sequence in
a sample DNA is labeled so that it can be easily detected by radioactivity,
tagging with a fluorescent or chromogenic dye from the membrane. The pattern of
hybridization is visualized on X-ray film by autoradiography in the case of a
radioactive or fluorescent probe or by development of color on the membrane if
a chromogenic detection is used. (Iborra et
al 2004)
F.
RESULTS
Hybridization
of the probe to a specific DNA fragment on the filter membrane indicates that
this fragment contain DNA sequence it is complimentary to the probe.(Iborra et al 2004).
|
DNA
FINGERPRINTING TECHNIQUE
|
CHAPTER FOUR
4.0 MODIFICATION IN DNA FINGERPRINTING
TECHNIQUES
These
modifications include:
4.1 POLYMERASE CHAIN REACTION (PCR)
The process, the polymerase chain reaction (PCR), mimics the biological process of DNA
replication, but confines it to specific DNA
sequences of interest. With the invention of the PCR technique, DNA profiling
took huge strides forward in both discriminating power and the ability to
recover information from very small (or degraded) starting samples.
PCR
greatly amplifies the amounts of a specific region of DNA. In the PCR process,
the DNA sample is denatured into the separate individual polynucleotide strands through heating. Two oligonucleotide DNA primers are used to hybridize to two corresponding nearby sites on
opposite DNA strands in such a fashion that the normal enzymatic extension of
the active terminal of each primer (that is, the 3’ end) leads toward the other
primer. PCR uses replication enzymes that are tolerant of high temperatures,
such as the thermostable Taq
polymerase. In this fashion, two new copies of
the sequence of interest are generated. Repeated denaturation, hybridization,
and extension in this fashion produce an exponentially growing number of copies
of the DNA of interest. Instruments that perform thermal cycling are now
readily available from commercial sources. This process can produce a
million-fold or greater amplification of the desired region in 2 hours or less.
Two primers that are complementary to the 3' (three prime) ends of each of the sense
and anti-sense strand of the DNA target. Primers are
short artificial strands of a DNA sequence which bind to the beginning and end
of the target sequence and allow replication to occur. The primers are designed
to anneal to specific STR loci (Lawyer et
al., 1993)
PRINCIPLE AND
PROCEDURES OF PCR INVOLVES;
Figure 2: polymerase chain reaction
(Rychlick et al., 1990)
Polymerase Chain Reaction is an in vitro
technique based on the principle of DNA polymerization reaction. It relies on
thermal cycling consisting of repeated cycles of heating and cooling of the
reaction for DNA melting and enzymatic replication of the DNA using
thermostable DNA polymerase, primer sequence (complementary to target region)
and dNTPs. It thus can amplify a specific sequence of DNA by as many as one
billion times. Most PCR methods can amplify DNA fragments of up to 10 kilo base
pairs (kb), although some techniques allow for amplification of fragments up to
40 kb in size (Lawyer et al., 1993).
The PCR is commonly carried out in a
reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a
thermal cycler. The thermal cycler heats and cools the reactiontubes to achieve
the temperatures required at each step of the reaction. Many modern thermal
cyclers make use of the Peltier effect, which permits both heating and cooling
of the block holding the PCR tubes simply by reversing the electric current.
Thin-walled reaction tubes permit favorable thermal conductivity to allow for
rapid thermal equilibration. Most thermal cyclers have heated lids to prevent
condensation at the top of the reaction tube. Older thermocyclers lacking a
heated lid require a layer of oil on top of the reaction mixture or a ball of
wax inside the tube.
Typically, PCR consists of a series of
20-40 repeated temperature changes, called cycles, with each cycle commonly
consisting of 2-3 discrete temperature steps, usually three (Fig. 2). The
cycling is often preceded by a single temperature step (called hold) at a high
temperature (>90°C), and followed by one hold at the end for final product
extension or brief storage. The temperatures used and the length of time they
are applied in each cycle depend on a variety of parameters. These include the
enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in
the reaction, and the melting temperature (Tm) of the primers.
Steps Involved
In PCR Process Includes;
1.
Initialization
step
2.
Denaturation
of template DNA to make single strands (usually at 950C)
3.
Primer
annealing to STRs (usually 330C)
4.
DNA
elongation (usually at 720C)
Usually the 3 steps are repeated 35-50
times to amplify the STR DNAs (Barttle andStirling, 2003)
1.
Initialization
step:
This step consists of heating the reaction to a temperature of 94–96 °C (or 98
°C if extremely thermostable polymerases are used), which is held for 1–9
minutes. It is only required for DNA polymerases that require heat activation
by hot-start PCR.
2.
Denaturation step: This step is
the first regular cycling event and consists of heating the reaction to 94–98
°C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting
the hydrogen bonds between complementary bases, yielding single-stranded DNA
molecules.
3.
Annealing step: The reaction
temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the
primers to the single-stranded DNA template. Typically the annealing
temperature is about 3-5 degrees Celsius below the Tm of the primers used.
Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very
closely matches the template sequence. The polymerase binds to the
primer-template hybrid and begins DNA formation. The mixture is now cooled to a
temperature of 50–65 degreecentigrade for 20-40 seconds which helps in
annealing of the primers to the single-stranded DNA template. Stable DNA-DNA
hydrogen bonds are only formed when the primer sequence very closely matches
the template sequence that permits annealing of the primer to the complementary
sequences in the DNA. As a rule, these sequences are located at the 3′-end of
the two strands of the segment to be amplified.The duration of annealing step
is usually 1 min during the first as well as the subsequent cycles of PCR.
Since the primer concentration is kept very high relative to that of the
template DNA, primer-template hybrid formation is greatly favored over
re-annealing of the template strands.
4.
Extension/Elongation
step:
The temperature at this step depends on the DNA, commonly a temperature of 72°C
is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA
strand complementary to the DNA template strand by adding Deoxynucleotide
Triphotsphates (dNTPs) that are complementary to the template in 5' to 3'
direction, condensing the 5'-phosphate group of the Deoxynucleotide
Triphotsphates (dNTPs) with the 3'-hydroxyl group at the end of the nascent
(extending) DNA strand. The extension time depends both on the DNA polymerase
used and on the length of the DNA fragment to be amplified. As a rule-of-thumb,
at its optimum temperature, the DNA polymerase will polymerize a thousand bases
per minute. Under optimum conditions, i.e., if there are no limitations due to
limiting substrates or reagents, at each extension step, the amount of DNA
target is doubled, leading to exponential (geometric) amplification of the
specific DNA fragment.
5.
Final
elongation:
This single step is occasionally performed at a temperature of 70–74 °C for
5–15 minutes after the last PCR cycle to ensure that any remaining
single-stranded DNA is fully extended.
6.
Final hold: This step at
4–15 °C for an indefinite time may be employed for short-term storage of the
reaction.
STAGES IN PCR
PROCESS
The
PCR process can be divided into three stages:
Exponential
amplification:
At every cycle, the amount of product is doubled (assuming 100% reaction
efficiency). The reaction is very sensitive: only minute quantities of DNA need
to be present.
As
a result of each cycle, the number of copies of the desired segment becomes
twice the number present at the end of the previous cycle. The more times the
three PCR cycles are repeated the more DNA you can obtain. This is because
every cycle of a PCR reaction theoretically doubles the amount of target
copies, so we expect a geometric amplification. In other words PCR is an
exponential process.
Leveling off
stage:
The reaction slows as the DNA polymerase loses activity and as consumption of
reagents such as Deoxynucleotide Triphotsphates (dNTPs) and primers causes them
to become limiting.
Plateau: No more productsaccumulate
due to exhaustion of reagents and enzyme. The term “plateau effect” is used to
describe the attenuation in the exponential rate of product accumulation that
occurs during late PCR cycles. The plateau effect is affected by: Utilization
of substrates (dNTPs or primers), stability of reactants (dNTPs or enzyme), end-product
inhibition (pyrophosphate, duplex DNA), competition for reactants by
nonspecific products or primer-dimer, reannealing of specific product at
concentrations above 1E8 M (may decrease the extension rate or processivity of
Taq DNA polymerase or cause branch-migration of products strands and
displacement of primers. Incomplete denaturation /strand separation of product
at high concentration.
STR DNA
FINGERPRINTING PROCEDURE
Running
STR DNA Fingerprinting involves the following
1.
Isolation
of DNA from the rest of the cellular material. This is done chemically and by
applying a high pressure.
2.
STR
bands do not need to be cut by restriction nucleases to be sized on a gel. They
are already short.
3.
These
STR are amplified and can be directly seen on a gel.
4.
The
amplified short STR fragments are then sorted by gel electrophoresis.
5. The DNA is added to a gel which then
undergoes an electrical charge; the top of the gel receives a negative charge,
while the bottom of the gel receives a positive charge. Due to DNA’s negative
phosphate groups, the DNA will migrate towards the bottom. Once the
electrophoresis is completed the smallest pieces of DNA will be at the bottom
of the gel whereas the largest pieces of DNA will be at the top. The gel is
then stained with methylene blue which allows analysis of the fingerprints
(Rubin, 2003).
4.2 VARIABLE NUMBER TANDEM REPEAT (VNTR)
ANALYSIS
In VNTR analysis, the DNA sample is cut
with restriction enzymes and then transferred to nitrocellulose membrane. They
are then hybridized to a specific VNTR Probe. These probes are radioactive DNA
sequences that are designed to complement specific VNTR sequences of
nucleotides. The probe is introduced to strands of DNA on the membrane that
have been denatured and thus, are single stranded. The membrane-probe mixture
is then shaken which allows the probes to bind to their complementally strands.
The membrane sheet can then be exposed to x-ray film to examine where the probe
bound. They result in a bar-code liked produced. (Chenget al.1994).
However, it is the chromosomal “flotsam
and jetsam” or “junk DNA” that is of a special interest to the forensic
scientist. Specific base-pair sequences
in the genes are known to perform a certain function, while the non-coding DNA
contain repeated base –pair sequences arranged in tandem, which having no known
function are inherited as functional
genes are also inherited. (Coding DNA also contain such repeat but less than
the non-coding DNA).
These tandem repeats make up a molecular
–DNA Fingerprinting that is believed to be unique for each individual (with
possible exception of identical twins) because the number of repeated sequences
that varies from one person to another. These non-coding base-pair repeated
sequences bear the complicated name Variable Number Tandem Repeats (Diamond,
2011).
4.3 SHORT TANDEM REPEAT (STR)
The Short Tandem Repeat (STR)
methodology for extracting DNA is the system most wide used form of DNA
fingerprinting. This system is based on the features of PCR, as it utilizes
specific areas that have short sequential repeat DNA. This is similar to VNTRS,
except that the repeated units are much shorter. These fragments choose for
forensic use or crime detection generally have unit of 3-4 base pairs, which
may be repeated in the DNA molecules for a dozen times. The smaller base pair
unit gives only small amounts of degraded DNA that may be sufficient for forensic
use (Dolan, 2011).
The big advantage in this method is that
the DNA comparison can match the possibilities into an almost endless range.
The problem of short-DNA segment can be increased in size for analytical
convenience and efficiency. This is done through the polymerase chain reaction
techniques.
4.4 RESTRICTION FRAGMENTS LENGTH POLYMORPHYSM
Restriction fragment length polymorphism
(RFLP) analysis the length of the strand of DNA molecules with repeating base
pair pattern. The term polymorphism refer to the fact that genes and non-coding
DNA sequences, can exit in more than one form on separate chromosome i.e.
sequences will differ in one or more nucleotide pairs (Erlich et al., 2008)
Chromosome
exits in pairs; one pair is inherited from each parent. Where a specific gene
or non-coding DNA sequences, is identical on each of the pair of chromosomes
(homozygous condition) where the genes or sequences differ in some way. Within
each DNA strand are numbers of genes that determine the particular character of
an individual. While about 5% of the gene compositions on DNA contain this type
of genetic information, the other 95% are the non-coding genes containing
identifiable repetitive sequences of base pairs. Which are called Variable
Number Tandem Repeat (Heldeman, 2011)
To
extract a DNA Fingerprint, DNA sample is
extracted an sorted via agarose Gel electrophoresis follow by a southern
blotting techniques and the DNA is
analyzed via a radioactive probe .The restriction fragment length polymorphism
analysis is used to detect the repeated sequences by determining a specific
pattern of the VNTR, which becomes the person DNA Fingerprint.
Bacteria produce extremely useful
proteins called restriction enzymes that are used to cleave foreign DNA that
enters the bacterial cell. These enzymes cut DNA at specific nucleotide sequences.
These enzymes can be used to detect DNA polymorphisms. A polymorphism is a
change in a nucleotide sequence at a specific site in two individuals. This
polymorphism can create a new restriction site or destroy an old one which can
be detected by restriction digestion.RFLP are changes in the lengths or
patterns of DNA fragment cut with specific restriction enzymes that result from
the presence of different DNA recognition sites. Thus RFLP analysis techniques
can be used to run DNA fingerprint to identify someone genetically.The loci on
human chromosomes contain a variable number of tandem repeats (VNTR): repeating
segments of DNA base pairs. These VNTRs occur in introns (the DNA between
coding DNA within a gene), or also in the “Junk DNA” between genes, and differ
in length between individual (Cheng, et
al.,2004).
4.5 AMPLIFIED FRAGMENT LENGTH POLYMORPHISM
(AFLP)
The AFLP technique is a powerful DNA
fingerprinting technology applicable to any organism without the need for prior
sequence knowledge. The AFLP technique
is based on the selective PCR amplification of restriction fragments from a total
digest of genomic DNA. The technique involves three steps: (i) restriction of
the DNA and ligation of oligonucleotide adapters, (ii) selective amplification
of sets of restriction fragments, and (iii) gel analysis of the amplified
fragments.
PCR amplification of restriction fragments is
achieved by using the adapter and restriction site sequence as target sites for
primer annealing. The selective amplification is achieved by the use of AFLP primers
that extend into the restriction fragments, amplifying only those fragments in
which the primer extensions match the nucleotides flanking the restriction
sites. Each of primer will yield unique fingerprints.
Visualization of AFLP fingerprints after gel
electrophoresis of AFLP products is described using either a conventional autoradiography
platform or an automated LI-COR system. The number of fragments that can be
analyzed simultaneously, however, is dependent on the resolution of the
detection system. Typically 50-100 restriction fragments are amplified and
detected on denaturing polyacrylamide gels.
The AFLP technique provides a novel and very
powerful DNA fingerprinting technique for DNAs of any origin or complexity
CHAPTER FIVE
5.0 DNA DATA BASE
A DNA DATABASE is a collection of DNA
profiles on a computer used to compare a single DNA fingerprint against a large
number of DNA samples. This chapter will discuss the background, methods, uses
and problems associated with DNA databases (Khan et al., 2002).
Combined
DNA index system (CODIS), the world’s largest DNA database of the FBI
laboratory CODIS is the U.S National DNA Database that connects federal state
and local law enforcement agencies.CODIS began in 1990 as a pilot program
serving 14 state and local laboratories CODIS National DNA INDEX system became
national in 1994 when the DNA identification Act was passed in 2001. However,
it did not begin national operation until 1998. (Suter, 2010).
5.1 THE TWO CODIS INDEXES
i.
The
forensic indexes which contain DNA profiles from crime scene evidence.
ii.
Offender
index which contains DNA profiles of individuals convicted of violent crimes
and sex offenses.
Forensic scientists in U.S analysis 13
core loci which have been carefully selected over the years to provide reliable
data with excellent probabilities. The core loci contain no known medical
information, but instead analyze highly unique sites in the junk regions
between genes.Currently, whose DNA gets collected varies from state to state. For
example, Massachusetts in 1997 approved legislation requiring all convicted
felons to submit their DNA to CODIS. However, in 1998 an appeal by the American
Civil Liberties Union (ACLU) was passed in a Boston court stating it was a clear
violation of human rights. Virginia law authorizes the collection of a DNA
sample from people accused of a violent crime upon their arrest, and a number
of officials around the U.S have proposed collecting DNA from people arrested
for any crime serious enough to require processing at a station house (Kondo,et al 1992).
5.2 APPLICATION OF DNA FINGERPRINTING
DNA fingerprinting has a wide variety of
application. The usage of DNA evidence in the courtroom has saved some innocent
persons while sending the quality to a lifetime of imprisonment. There is no
doubt that DNA evidence is one of the most powerful tools in forensic science.
Many cases have been solved because of this useful tool. Child support payments
have been made, fatherhood has been conformed, criminal offenders have been
brought to justice and countless other successes have occurred due to DNA
fingerprinting. The most prevalent one is its usage as evidence in court cases.
DNA fingerprinting is an aspect of a science known as forensics. Forensic is
the usage of science and technology to aid in criminal investigation.
Therefore, DNA can be used as evidence for criminal investigations Another use of DNA fingerprinting is in
paternity testing for the case of woman who is pregnant but is unsure of whom
the father is. For such cases, DNA fingerprinting can be used to discover the
father of the child. DNA fingerprinting can also be used to identify body
remains, for instance, the DNA fingerprinting of the remains of soldiers who
died in war can be extracted genetically and compare against family members DNA
fingerprinting for identification. An additional use of DNA fingerprinting
exists in molecular archaeology which allows ancient genetic remains to be
analyzed to determine species, blood lines, or gender of ancient species. In
this the differences between an ancient and modern species can be revealed by
comparing the modern and the ancient genetic material which will give a much
better view of the past. (Vos et al.,2005).
5.3 DNA STORAGE
Storage of blood requires low
temperature and dark places that are not humid. Low temperature protects DNA
from degradation and allow those samples that are to be used many years after
to be preserved. Purified DNA in aqueous solutions can be stored for up to 3
years if stored at 40C and -700C for storage time period
longer than 3 years, for longer time storage blood requires temperature of 800C
to ensure the DNA remains intact(Bieber and Frederick 2006).
5.4 COMPARISON OF DNA SAMPLES FOR FORENSIC
ANALYSIS
A statistical evaluation enables the
forensic pathologist to compare the suspect’s DNA with a DNA recovered from a
crime scene and to state with high degree of certainty (usually 99%) when
comparison is done and compatibility is known. When the suspect is not
identified DNA sample are analyzed and compare with the DNA Profile at the
database. Databases of DNA fingerprints are only available for known offenders
(Jeffreyset al., 2000).
Comparison of
DNA bands samples of suspects(Jeffreys et al., 2000).
In
the example shown above, DNA collected at the scene of a crime is compared with
DNA samples collected from 4 possible suspects. The DNA has been cut up into
smaller pieces which are separated on a gel. The fragments from suspect 3 match
those left at the scene of the crime, betraying the guilty party.
5.5 PROBABILITIES OF A MATCH
The usefulness and accuracy of the
database grows proportionally to the number of profiles stored in database.
Database is used to determine specific allele frequencies at forensic loci. For
instance locus-A might be determined to have a frequency of 0.1 and locus-B a
frequency of 0.2 in a database
population, if both loci are analyzed for a given DNA sample, the
probability of a similar match occurring randomly would be 0.1 x 0.2. = 0.02.
So about 2% of the general population is expected to have a similar profile.
DNA as determined from a database of a million entries is far more accurate
than those determined from only 100 entries. Thus, when using the 13 CODIS core
loci, one can typically achieve a frequency on the order of one – in-a-billion
to one-in-a-trillion that the profiles will match random non related subsets
.(Kondo et al., 2002).
5.6 LIMITATION OF DNA FINGERPRINTING IN
FORENSIC ANALYSIS
There are
limitations which include:
5.6.0 Contamination in DNA Sample
Contamination implies the accidental transfer
of DNA. There are three potential sources of contamination when performing PCR:
sample contamination with genomic DNA from the environment, contamination from
samples during preparation, and contamination of a sample with amplified DNA
from a previous PCR reaction (Lygoet al. 1994). It is often said that
the most critical source of PCR contamination is DNA from previous PCRs. Again,
a PCR produces many DNA copies of the target DNA sequences. Due to shear
number, these copies (called amplicons) are a hazard for future PCRs… However,
a more dangerous source of contamination is what is called genomic DNA. This is
DNA that hasn't yet been amplified. Genomic DNA doesn't have the high
concentration of the target DNA copies but is a hazard because genomic DNA
could produce an entirely false DNA profile (Mullis,2000).
The first source of contamination is largely
dependent on sample collection at the crime scene and the care taken there by
the evidence collection team. Environment contamination can be monitored only
in a limited sense by ‘substrate controls’ (Gill 1997). The latter two sources
of contamination can be controlled and even eliminated by using appropriate
laboratory procedures and designated work areas.
5.6.1 DNA Degradation
DNA Degradation is the breakdown of DNA
into smaller units DNA degradation can be caused by a number of factors like
extreme heat, UV rays moisture and humidity. Transporting and storage of DNA
evidence therefore requires much care and must be kept under some conditions
like DNA sample must be kept dry, out of the sun, at low temperature, If these
needs are not met the DNA will stand degraded.(Bieber and Frederick 2006).
5.6.2 Missing of DNA Profile
Missing of DNA profile due to lack
of ability to produce test due to limited DNA sample at crime scene (Park,
2004).
5.7 PREVENTION OF DNA CONTAMINATION
Contamination of DNA can have horrible
consequences on DNA analysis; it can lead to evidence being discarded in a
court case, or incorrect results. Contamination occurs when outside material is
introduced to the sample, for example another person’s DNA or a chemical that
was spit (Rychlik et al., 2000).
The
following are some guidelines to prevent these errors:
1.
Avoid
excessive exposure to heat or humidity. Then it should be refrigerated or
freeze if possible.
2.
Never
handle evidence with bare hands; never allow two items of evidence to come into
contact with each other.
3.
Air-dry
evidence completely before packaging.
4.
Package
each item separately.
5.
Ship
evidence with dry ice or leak-proof ice packet sample must remain dry, Wear
gloves, change them often
6.
Use
disposable instruments or clean them thoroughly before and after handling each
sample.
7.
Avoid
touching the area where you believe DNA may exist.
8.
Avoid
talking, sneezing and coughing over evidence.
9.
Avoid
touching your face, nose, and mouth when collecting and packaging evidence.
10.
Put
evidence, into new paper bags or envelopes, not into plastic bags. Do not use
staples.
Evidence collection and transportation
are not the only places where mistakes can be made. Laboratory Technicians must
use great care and follow strict guideline in order to process the DNA material
correctly so it can be submitted as valid evidence. Although many precautions
are taken, human error still exists (Vos et
al., 2005)
1.
The
National Research Council (NRC) recommends that evidence be divided into
multiple samples; this supplies forensic scientists with back up samples in
case contamination or other mistakes subsequently occur.
2.
The
NRC also recommend analysis be conducted by unbiased laboratory technicians
that have a low error rate to assure accurate and fair results. It is important
to have all the right equipment to carry out the task and also know different
collection methods for specific types of evidence. For instance
(a) Large blood stains should be collected with
sterile gauze or cotton cloth allowed to air dry, and then refrigerated or
frozen immediately.
(b) In the case of dried blood stains,
collection depends on the materials to which the blood has adhered to. If the
blood has adhered to clothing or a small object, it should be wrapped in clean
paper and sent to the laboratory. (c)
If it is on a larger solid object, such
as a table, the stain should be scraped with a clean knife onto clean paper,
and then placed into a collection envelope and shipped to the laboratory.
(d) For seminal stains on clothing or other
cloth materials, allow the evidence to air-dry, then wrap the evidence in paper
bags and ship it to the laboratory as soon as possible.
(e) For sex offenses cases the victim should be
examined by a physician as quickly as possible. If hair is found it should be
collected with tweezers, and sent to the laboratory in a labeled envelope.
It
is important to treat all evidence with great care caution. This will protect
the evidence from being contaminated by your skin cells, hair, sweat or any
other contaminant that may be transference from you to the evidence through direct
or even close contact. (Bieber and Frederich 2006).
CHAPTER SIX
6.0 SUMMARY
6.1 CONCLUSION
The use of DNA Fingerprinting without
doubt represents one of the most significant advancement in forensic science, since
criminals can be easily identified by DNA Fingerprinting analysis.
DNA fingerprinting has become a strong
staple in our judicial system, but to ensure that the evidence is considered
valid a lot of care and effort must go into evidence collection and handling.
From the crime scene to the lab, all of the members who work on a case must use
extreme care and follow guidelines diligently so that the evidence can be submitted and help the court
system come to correct conclusion (Suter, 2010).
6.2 RECOMMENDATION
Nigeria
government should buy the idea of DNA fingerprinting analysis in handling criminal
issues because most of Nigerians are convicted of one crime or the other in
which they have not committed.
The
populations of young people in our cells and prisons today are greatly
increasing. If DNA fingerprinting
technique can be employed in criminal investigation a greater number of wrong
convicted criminals will gain freedom, our congested cells and prisons will
reduce thereby increases our productivity as a nation.
Issues of unknown gunmen (criminals)
militating against our country can be addressed if there is proper and
efficient DNA fingerprinting analysis of every suspects arrested and if found
guilty, prosecuted according to the law.
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