THE DEPARTMENT OF BIOTECHNOLOGY
FACULTY OF BIOLOGICAL SCIENCES
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
1.0 INTRODUCTION- - - - - - - - -
2.0 DEOXYRIBOSNUCLEIC ACID (DNA) - - - - -
2.1 DNA STRUCTURE - - - - - - -
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 - - - - -
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) -
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- - - -
6.0 SUMMARY- - - - - - - - -
6.1 CONCLUSION- - - - - - - - -
6.2 RECOMMENDATION- - - - - - - -
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).
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. 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.
DNA is a long polymer made from repeating units called nucleotides. 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). 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. 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 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. 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.
The backbone of the DNA strand is made from alternating phosphate and sugar residues. 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.
The DNA double helix is stabilized primarily by two forces: hydrogen bonds between nucleotides and base-stacking interactions among aromatic nucleobases. 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.
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. 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.
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. Another phage - Staphylococcal phage S6 - has been identified with a genome where thymine has been replaced by uracil.
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 Å wide and the other, the minor groove, is 12 Å wide. 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. 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.
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. 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.
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. 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.
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.
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. 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. These enzymes are also needed to relieve the twisting stresses introduced into DNA strands during processes such as transcription and DNA replication.
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. 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)
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. 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. A typical human cell contains about 150,000 bases that have suffered oxidative damage. 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. 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. 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.
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. As a result, DNA intercalators may be carcinogens, and in the case of thalidomide, a teratogen.
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
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).
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)
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)
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
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
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).
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).
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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