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Forensic DNA Analysis | Basis of DNA profiling

Forensic DNA analysis was started back in the middle of the 1980’s. (Jeffreys, Wilson et al. 1985) It all started with the ABO blood group system and later was expanded to body fluids and forensic analysis. In the 1970s, polymorphism in DNA sequence was detected and shifted the paradigm towards itself, these sequences vary from person to person and they were helpful in finding out the difference between victim and the guilty.(Council 1992)DNA analysis thus played an immensely important role in our society regarding the justice system by assisting the sentence of guilty and discharge of the innocent ones. Tremendous progress has been made in developing efficient and robust DNA analysis protocols.

By re-combining and re-associating the remains from the crime scene, we can devise results by various tests and DNA technologies.(Jeffreys, Wilson et al. 1985). DNA profiling has the number of applications in; finding an unknown parent (father) of a child, Identification of victims after a natural disaster, creating a family pedigree, finding missing person either alive or dead.(Butler 2009)

New methodologies are being introduced day by day with increased sensitiveness and they are well informed.(Gill, Jeffreys et al. 1985).DNA profiling of body fluids has been used in forensic analysis for more than the past 50 years (Gaensslen 1983). The term ‘DNA fingerprinting’ was coined in 1985 by Jeffery and his co-workers. In forensic samples, DNA is present in a sufficient amount and that amount varies in different biological samples.

1.1. DNA ANALYSIS:

In (Coyle 2012), it was mentioned that the DNA is found in each and every cell of a body, it is the hereditary material that carries information in the form of genes from parents to offsprings. It a double helical structure with grooves in it. Through studies, we found that 99% of the DNA sequence is the same in every human body while only 1% is unique because of polymorphism. Forensic analysts are interested in only this unique sequence of DNA. The probability of individuals having the same DNA is one in 594 trillion people. Forensic DNA analysis involves the following major steps:

Sample Collection:

DNA from the crime scene is collected, it can be in the form of fingerprints, body fluid, hair, blood or etc. The sample was then stored and transported safely to the laboratories where further testing was being done.

DNA Extraction:

We ought to isolate DNA from the samples being collected from the crime scene, isolation of DNA can be done either manually or using DNA isolation Kits.

Amplification:

The sample thus being collected from the crime scene won’t be enough for analysis, to make it detectable amplification of DNA was done using PCR.

DNA Quantification:

DNA fragments are then separated based on their size and quantified using a spectrophotometer, their absorbance was measured and the amount of it present in the sample was then calculated.

Profile Matching:

 DNA was then compared with different DNA profiles present in the Forensic Database, or compared directly with that of the suspect’s DNA collected to prove whether the suspect is guilty or innocent.(Romeika and Yan 2013)

DNA technology plays an immensely important role in forensic biology. After 1980’s forensic scientists came to know about the potential of DNA typing being done on the biological samples collected from the crime scene. It has revolutionized the justice community of our society by pinpointing the criminal, finding missing persons, paternity testing and identifying dead bodies of people after some natural mass disasters. Gill, Jeffreys et al. (1985) Gill, Jeffreys et al. (1985) Gill, Jeffreys et al. (1985) Gill, Jeffreys et al. (1985)  Gill, et al. 6 Gill, Jeffreys et al. (1985) Gill, Jeffreys and Werrett Gill, Jeffreys [3] Gill, Jeffreys et al. (1985) Gill, Jeffreys et al. (1985)  (Gill, Jeffreys et al. 1985). With the advent of new robust technologies, better and efficient results can be obtained using small degraded DNA samples collected from the crime scene.(Clayton, Whitaker et al. 1995).

According to (Chakraborty and Kidd 1991), polymorphism is present throughout the human genome (Wyman and White 1980). DNA profiling was a major tool in forensic biology because, with this, we can differentiate between individuals of the same species. In earlier times, the variable number of tandem repeats VNTR loci were used. This repeating unit can be as small as dinucleotide consisting of only two nucleotides and as large as consisting of 30 nucleotides. These nucleotides differ in the length of fragments they form, giving rise to variation because the number of these repetitive sequence varies from one person to another (Nakamura, Carlson et al. 1988).

After completing the DNA profile according to the mentioned guidelines, three possibilities can be there;(Chakraborty and Kidd 1991; Wilson, Stoneking et al. 1993)

  1. The first possibility includes, DNA profiling may give insufficient result either because of the degraded sample or very low amount of the sample available. Or maybe it’s because of improper handling.(Wambaugh 1995)
  2. If the results after DNA fingerprinting do not match with that of suspect’s, it means that the evidence fails to justify the criminal intention of the suspect and he should be set free. (Forman et al. 1991)
  3. Third and the last possibility is if the DNA match is found then the legal procedure starts from here. That match is either because of mishandling or is it true? Is the person really a criminal?

In (Hochmeister 1995), it is stated that in the past, justice in criminal courts was not this much easy because of undersupplying of information. However, after DNA profiling has been recognized as a powerful tool in forensics, it became easier to distinguish guilty and the person with a lack of criminal intent. It all starts with the examination of the body fluids and the pattern of stains found at the crime scene. (Brinkmann et al.)

1.2. GENETIC BASIS OF DNA TYPING:

(Council 1992) stated that, DNA typing is based on the structure and polymorphic character of DNA determined after Mendel’s discovery in the 1980s according to which DNA is transferred from parents to offsprings and carries genetic information with it in the form of genes, alleles in the form of pairs are present that determines the trait being inherited. These genes reside on a structure called a chromosome, thread-like structure which is present in the nucleus., traits of an individual represent its phenotype whereas, its total genetic make-up (pairs of alleles) is referred to as its genotype. These traits differ from one individual to another, thus giving rise to polymorphism, (Roychoudhury and Nei 1988) variation in the DNA sequence. With the advent of DNA technology, we can directly study these variations and this forms the basis of Forensic analysis.(Council 1992)

1.3. STRUCTURE AND VARIATION IN DNA:

According to  (Council 1992) there are 22 pairs of autosomes and one pair of sex chromosomes (two X in female or an X and Y in a male) in the human body. These chromosomes are made up of DNA ( a double-helical structure), which is made up of four nucleotides ( Adenine, Guanine, Cytosine, Thymine ), bonded by a hydrogen bond. Complementary base pairing occurs between them (A-T, G-C). These nucleotides are arranged in the form of spiral thus giving rise to double-helical DNA structure.

Structure of double-helical DNA in the Chromosome. The line shown in the chromosome is further expanded to show the detailed structure of DNA.(Council 1992)Structure of double-helical DNA in the Chromosome. The line shown in the chromosome is further expanded to show the detailed structure of DNA.(Council 1992)
fig. 01: Structure of double-helical DNA in the Chromosome. The line shown in the chromosome is further expanded to show the detailed structure of DNA.

With the advancement in DNA technology, we found out variations in the genetic make-up of an individual either because of insertion, mutation, deletion or difference in single- nucleotide. In the junk part of DNA out of 300 to 1000 nucleotides, one nucleotide varies among two persons.(Cooper, Smith et al. 1985).This one nucleotide difference can alter the restriction enzyme recognition site, and thus DNA with new restriction sites will be obtained. Apart from this, some parts of DNA contain a repetitive sequence, also known as tandem repeats. These tandem repeats can be found both either in the coding region or in the non-coding region of the DNA, but their probability in the coding region is very low.(Council 1992)

The major two forms of variations are VNTR (Variable number of tandem repeats) and RFLP (Restriction fragment length polymorphism). In VNTR, length of tandem repeats differs they can be in clusters of two, four, six etc., whereas in RFLP, restriction site of the enzymes differs because of the variation in the DNA sequence. VNTR’s have large number of repeating units (thousands of base pairs long) (Shetty and Bhagat 2014).

1.4. BASIS OF DNA TYPING:

DNA fingerprinting includes a comparison between the sample of DNA collected from the crime scene and that of the suspect (Sample collected from the blood of the suspect). Major tools required for DNA typing includes Probes, Electrophoresis, PCR etc. Each of these methods has its own limitations and benefits.

RESTRICTION FRAGMENT LENGTH POLYMORPHISM:

According to (Southern 1975),  when DNA is fragmented under controlled conditions, nick is produced in the double-stranded DNA helix at various positions, and thus giving rise to restriction fragments, RFs (from hundreds to thousands of base pairs long). Each of these RF has its own specific length and sequence different from the rest of the fragments. To study these fragments, they are run on agarose/polyacrylamide gel electrophoretically and are separated on the basis of size. Their travelling speed depends on their size, smaller fragments will move faster as compared to the larger ones when the electric field is applied. The gel is then transferred to the nylon membrane for further analysis.

A probe (It is actually a single-stranded DNA tagged with fluorescent, radioactive groups for detection of its complementary sequence) is then added that binds itself with the complementary sequence. The membrane (nylon or PVC membrane) is then placed in the probe containing a bath, this will lead to the hybridization of the probe with the targeted restriction fragments. After a few hours, the non-specifically bound probe was then washed off and then it is visualized under UV or by autoradiography technique. Major steps involved were; Digestion of DNA, Electrophoresis (PAGE/SDS), Southern Blotting, Hybridization with the probe.

 Restriction Fragment Length Polymorphism. This figure shows how DNA is extracted from blood and is cleaved by restriction enzymes that lead to the formation of restriction fragments, which is then observed by electrophoresis and then southern blotting is being performed. (Council 1992) Restriction Fragment Length Polymorphism. This figure shows how DNA is extracted from blood and is cleaved by restriction enzymes that lead to the formation of restriction fragments, which is then observed by electrophoresis and then southern blotting is being performed. (Council 1992)
fig.02: Restriction Fragment Length Polymorphism. This figure shows how DNA is extracted from blood and is cleaved by restriction enzymes that lead to the formation of restriction fragments, which is then observed by electrophoresis and then southern blotting is being performed.

POLYMERASE CHAIN REACTION:

(Metzker and Caskey 2009) mentioned in their work that polymerase chain reaction was developed in 1980 by Kary Mullis. Its Principle is based on the usage of DNA polymerase enzyme’s replication ability which can add nucleotides at the 3’OH group of the strand in the presence of primers. Using PCR, a short fragment of DNA can be amplified and millions of copies can be made, makes it easier for us to analyze the sample. It is an in-vitro amplification method being used in molecular biology. PCR can be used for various purposes which includes; identification, genetics and in diagnostics. PCR cycle is an in-vitro amplification technique, which amplifies our required DNA sample for further analysis. PCR cycle consists of three steps;

CLICK HERE TO READ POLYMERASE CHAIN REACTION IN DETAIL.

Denaturation:

          The double-stranded helical structure of DNA is unwound by keeping the temperature at 94°C. This weakens the hydrogen bonds between strands and they get dissociated.

Annealing:

         Due to lowering of temperature at this step, primers bind themselves with their complementary DNA sequence (our targeted DNA sequence)

Extension:

           At this step, the temperature is kept at 74°C, best working temperature for DNA polymerase enzymes which will add nucleotide bases, which are the major ‘building blocks’ to the DNA strand in 5’ to 3’ direction.

Polymerase Chain Reaction PCR, it consists of 3 steps; Denaturation, Annealing and Elongation. The cycle repeats itself until the product gets amplified.  Source: NCBI - NIH, Principle of PCRPolymerase Chain Reaction PCR, it consists of 3 steps; Denaturation, Annealing and Elongation. The cycle repeats itself until the product gets amplified.  Source: NCBI - NIH, Principle of PCR
fig. 03: Polymerase Chain Reaction PCR, it consists of 3 steps; Denaturation, Annealing and Elongation. The cycle repeats itself until the product gets amplified.

The PCR cycle is repeated for 25-30 times, thus producing billions of copies of our targeted DNA that can be used for analysis in future. Using PCR both genetic typing and the amplification of the targeted sequence can be done.(Council 1992)

References:

Butler, J. M. (2009). Fundamentals of forensic DNA typing, Academic Press.

Chakraborty, R. and K. K. Kidd (1991). “The utility of DNA typing in forensic work.” Science 254(5039): 1735-1739.

Clayton, T., J. Whitaker, et al. (1995). “Identification of bodies from the scene of a mass disaster using DNA amplification of short tandem repeat (STR) loci.” Forensic Science International 76(1): 7-15.

Cooper, D. N., B. A. Smith, et al. (1985). “An estimate of unique DNA sequence heterozygosity in the human genome.” Human genetics 69(3): 201-205.

Council, N. R. (1992). DNA technology in forensic science, National Academies Press.

Coyle, H. M. (2012). “The importance of scientific evaluation of biological evidence—Data from eight years of case review.” Science and Justice 52(4): 268-270.

Gaensslen, R. E. (1983). Sourcebook in forensic serology, immunology, and biochemistry, US Department of Justice, National Institute of Justice.

Gill, P., A. J. Jeffreys, et al. (1985). “Forensic application of DNA ‘fingerprints’.” Nature 318(6046): 577.

Hochmeister, M. N. (1995). “DNA technology in forensic applications.” Molecular aspects of medicine 16(4): 315-437.

Jeffreys, A. J., V. Wilson, et al. (1985). “Hypervariable ‘minisatellite’regions in human DNA.” Nature 314(6006): 67.

Jeffreys, A. J., V. Wilson, et al. (1985). “Individual-specific ‘fingerprints’ of human DNA.” Nature 316(6023): 76.

Metzker, M. L. and C. T. Caskey (2009). “Polymerase chain reaction (PCR).” eLS.

Nakamura, Y., M. Carlson, et al. (1988). “New approach for isolation of VNTR markers.” American journal of human genetics 43(6): 854.

Romeika, J. and F. Yan (2013). “Recent advances in forensic DNA analysis.” J Forensic Res 12: 001.

Roychoudhury, A. K. and M. Nei (1988). Human polymorphic genes: world distribution, Oxford University Press, USA.

Shetty, C. K. and V. Bhagat (2014). “Amazing Advances in Forensic DNA Analysis–past, present and the future.” International Journal of Scientific & Engineering Research 5(6): 1418-1422.

Southern, E. M. (1975). “Detection of specific sequences among DNA fragments separated by gel electrophoresis.” J mol biol 98(3): 503-517.

Wambaugh, J. (1995). The blooding, Bantam.

Wilson, M. R., M. Stoneking, et al. (1993). “Guidelines for the use of mitochondrial DNA sequencing in forensic science.” Crime Lab Digest 20(4): 68-77.

Wyman, A. R. and R. White (1980). “A highly polymorphic locus in human DNA.” Proceedings of the National Academy of Sciences 77(11): 6754-6758.

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