Dna Profiling Techniques in Forensic Science

Literature Review and Project Proposal
AS6P01

Title: DNA Profiling Techniques in Forensic Science

Abstract

Since 1985, DNA profiling in forensic science has become very important in this virtual era of technology and in the world of science that solves both major and minor crimes. Small traces of DNA are considered in all circumstances from how the DNA was collected to fully obtaining the profile in its significant form. Traces of sweat, blood and semen are the most common type’s evidence found at crime scenes. There are several different methods for creating a DNA profile such as STR (Short Tandem Repeat), PCR (Polymerase Chain Reaction), Y chromosome analysis, Restriction Fragment Length Polymorphism (RFLP) and Mitochondrial DNA (MtDNA) analysis. All these types of methods are able to extract DNA from a chosen sample taken from a crime scene. DNA profiling is the information of how a sample is processed and analysed and a DNA profile must be created by collecting and analysing VNTR’s (Variable Number Tandem Repeats), these are unique sequences on the loci which is an area on chromosomes. Most DNA sequences in different people look too similar to tell apart whereas VNTR result in bands that are unique enough for identification of individuals.

Introduction
DNA (deoxyribonucleic acid) is the hereditary material in almost all living organisms. In 1953 researchers J. Watson and F. Crick saw the structure of DNA. DNA consists of two long strands that are built up chain like, each consisting four nucleotide subunits, attached to a sugar phosphate backbone. Adenine (A), guanine (G), cytosine (C) and thymine (T) are bases that are arranged pairwise in the middle of the DNA stand. The nucleotides are covalently linked together, from which the bases A and T, G and C bind by a hydrogen bond (Bray et al 2010: 173). Figure 1 shows the order of the bases, which determine the biological information available for building, and maintaining an organism, the sugar phosphate group molecules form the vertical side piece and the base pairs form a ring shape to create a spiral called a double helix. The two backbone chains run in opposite directions, this is specific for base to base bonding which allows this genetic information contained in DNA to be copied accurately from one generation of cells to the next.

Figure 1 – DNA
By Jaspreet (Bray et al 2012) There are 23 pairs of chromosomes in humans inherited from our parents, with each parent contributing one half of each pair. Chromosomes are made up of DNA, 22 pairs are autosomes and the last pair is a sex chromosome fig 2 shows this. Autosomes are chromosomes that are not sex chromosomes; they are individual which means that each person has a DNA profile as unique as a fingerprint. No two DNA profiles or fingerprints can be the same due to the combination of marker sizes found in each person makes up his/her unique genetic profile. When determining the relationship between two individuals, their genetic profiles are compared to see if they share the same inheritance patterns at a conclusive rate. Chromosomes are located in the nucleus of each cell and consist of long DNA strands where they are tightly packed and coiled around specific proteins called histones, which are looped and fixed to specific regions of the chromosome. There are 5 different kinds of histones (H1, H2A H2B, H3 and H4); they all bind to DNA to form chromatin in the nucleus during cell division where the chromatin condenses into visible structures that are the chromosomes itself. The DNA wrapped around each histone core is 200bp (base pair) long. Histones can be purified from DNA as H2A and H2B stick together as do H3 and H4 therefore making 8 proteins in each histone core with DNA wrapped is called a nucleosome which is 10nm (nano meter) fibre thickness, H1 is not part of the histone core as it binds to the nucleosome to give an even more structure to the chromatin and waits for the next interaction to take place. Fig 2 – 23 pairs of Chromosomes

DNA is specific to its kind where identical twins are formed when a fertilised egg splits into two and develop into a full embryo. Identical twins will have the same genes making them alike in everything controlled by the genes itself. Although they have different phenotypes the DNA itself is expressed in different ways too. If identical twins are raised in different environments and stimuli they can develop some changes such as a genotype for a particular weight and height but this can only happen if they have enough food or a genotype for a potential IQ but again this will rely on the education and the right kind of stimulus received for that individual. Variation within humans result in the genes inherited from your parents and the environment you live in. (David Wright, 2000: 162)
Main Body
The techniques for identification of DNA profiling only need just a small amount of DNA but in some methods a larger amount is needed. DNA profiling is carried out when human biological fluid or human tissue is found at crime scenes and is used for evidence to link or exclude a possible suspect from the scene. DNA profiling can only be used if there is enough DNA within the sample taken and is only useful for comparing the samples. Samples taken can be compared to a national database of DNA profiling where there are over 700,000 samples in the UK national database alone. When there is a match, the two samples may have originated from the same person but before DNA can be profiled, it must be extracted from the sample in any of the following analysis depending on what the sample is and how much of it you have.
One of the first techniques that were adapted for forensic DNA profiling was Restriction Fragment Length Polymorphism (RFLP) where this kind of analysis determines variation in the length of a single DNA fragment. If two samples originate from different sources, RFLP can differentiate them using fewer loci than other systems. RFLP can determine whether a single sample contains DNA from more than one person but this can only happen if there is a large amount of greater quality DNA, this technique is stated to be ‘laborious and difficult to automate’ (Rudin and Inman. 2002: 41) (2). Fig 3 shows the RFLP process.

Fig 3 – RFLP by Jaspreet (Botstein 2012)
Polymerase Chain Reaction (PCR) is a process where a specific region of DNA is replicated over and over again to make copies of a particular sequence. Fig 4 shows the process which involves the heating and cooling of samples in a thermal cycle pattern of three steps denaturation, annealing and elongation. The strands get separated and bind to primers (fig 5), which are pieces of DNA so that they attach to the DNA at each end of the region for it to be copied. PCR is very sensitive and effective in many ways in the use of forensic science. PCR only needs a small amount of blood compared to RFLP needs about a quarter.

Fig 4 – PCR by Jaspreet (Prof Santiago 2012)
The primers used in PCR define the region of the genome that will be analysed. Primers are short pieces of DNA that anneal to the template molecule at either end of the specific region Fig 5 illustrates this. For a forensic PCR analysis the primers must bind to the regions of the DNA sequence to effectively amplify the human DNA while at the same time taking precautions not binding DNA to any other species. (Goodwin et al, 2007)

Fig 5 – Primers binding to the DNA strand which is known as the annealing stage. By Jaspreet (Davidson 2012)
Short Tandem Repeat (STR) technology is a forensic analysis that evaluates specific regions, loci that are found on a DNA strand. STR is when the repeat units are shorter and each loci can be used simultaneously of two to six bases long. STR regions are analysed for forensic testing between one DNA profiles to another. The purpose of having a core set of STR loci (13 set loci) is to ensure that all forensic laboratories can get the DNA from databases and share valuable forensic information. STR’s does have some limitations on sensitivity where it will work on degraded DNA samples such as damaged body tissue or bone destroyed by fire but sometimes there just isn’t enough sample to be tested giving no results at all for example aged bone. To test even smaller sample of DNA currently mtDNA is the choice of technique. (Gill et al, 2001)

Mitochondria produce 90% of a cell's energy, and contain their own genomes in the form of a double-stranded circular molecule known as mitochondrial DNA (mtDNA). MtDNA is important for finding missing person’s investigations, mass disasters, and other forensic investigations. MtDNA is valuable for determining DNA recovered from damaged, degraded, or very small biological samples that techniques such as STR cannot extract with small amounts of DNA. MtDNA is a small circular genome located in the mitochondria, which are located outside of a cell's nucleus. Fig 6 illustrates this.
There are two properties of mtDNA: high copy number and maternal inheritance. Samples such as hair, bone and teeth can be used to examine common ancestry between individuals. Maternal inheritance such as grandmother, mother and daughter all have statistically the same mtDNA sequence within them as they have been passed on from generation to generation. This can help to find unidentified remains for analysis and comparison of the mtDNA profile to any maternal relative. High copy number is valuable for when the amount of material within the cell is very small for analysis. (Goodwin et al, 2007)

Fig 6 – MtDNA by Jaspreet (Ferullo 2012)

Y-chromosome analysis targets only the male population of biological samples as they are passed down from father to son unchanged, except when mutations occur. They can also be used to trace family members amongst males only. A reference Y-chromosome profile has to be compared with an unknown sample match for significance, to confirm that the match actually exists. The Y chromosome DNA testing is important in situations where a small amount of male DNA may be recovered in the presence of excess female DNA, such as in sexual assault evidence. Y chromosome analysis can also benefit missing person’s investigations as it extends the range of potential reference samples. Since fathers pass their Y chromosome onto their sons unchanged all males in a paternal lineage will possess a common Y chromosome haplotype.
Conclusion
DNA profiling technology is constantly evolving where techniques such as PCR, STR and mtDNA and new loci are being discovered and are being used widely around the world to solve crimes globally. From research and experience we know that the technique RFLP requires too much DNA for the process to take place, it also takes longer too this is the reason why forensic scientist do not use this anymore. However, PCR can be used to amplify very small amounts of DNA, usually in 2-3 hours, to the levels required for RFLP analysis. Therefore, more samples can be analyzed in a shorter time. The ability to analyse such small amounts of samples of evidence taken from crime scenes increases the automation and promises faster and more effective results for forensic evidence in court. DNA degradation can be easily identified on an electropherogram where the process can reduce the height of some alleles, making them too low to be recognised from the data. When there are too many samples of DNA degraded it is classed as no results being obtained and can complicate the interpretation of the samples if two or more are similar to each other. Degradation is more likely to occur during the technique of STR as the amplification of specific regions on the DNA strand will not be successful.

References
Books
1. John M. Butler, 2005, Forensic DNA Typing – Biology, Technology and Genetics of STR Marker. Elsevier Academic Press (USA). 2nd Edition, page 42 and 63. 2. Norah Rudin and Keith Inman, An introduction to Forensic Analysis, 2nd Edition, CRC Press LLC 2002, page 41, 58 3. Alberts Bray et al, 2010, Essential Cell Biology. Garland Science, Taylor & Francis Group LLC. 3rd Edition, page 173 4. William Goodwin et al, 2007. An Introduction to Forensic Genetics. John Wiley & Sons Ltd. Page 41, 71, 127-132 5. David Wright, 2000. Human Physiology and Health. Heinemann Educational Publishers. Page 22, 162.
Websites
6. http://www.nij.gov/topics/forensics/evidence/dna/basics/analyzing.htm#mitochondrial - accessed 21st November 2012 at 11.30am 7. http://www.iitk.ac.in/infocell/Archive/dirnov3/science.html - accessed November 20th, 2012 8. Ferullo, Daniel. 7.342 Powerhouse Rules: The Role of Mitochondria in Human Diseases,Spring 2011. (Massachusetts Institute of Technology: MIT OpenCourseWare), http://ocw.mit.edu (Accessed 29 Nov, 2012). License: Creative Commons BY-NC-SA
Journals
9. Gill, P., Sparkes, R. and Tully, G. (2001). DNA Profiling in Forensic Science. John Wiley & Sons Ltd. (1), 1-6.
(http://onlinelibrary.wiley.com/doi/10.1038/npg.els.0001001/pdf) - accessed 19th November 2012 10. Renata Jacewicz, Krzysztof Lewandowski, Joanna Rupa-Matysek, Maciej Jedrzejczyk, Mieczysław Komarnicki and Jarosław Berent. 2012. Genetic investigation of biological materials from patients after stem cell transplantation based on autosomal as well as Y-chromosomal markers. Int J Legel Med. (1), 1-4. 11. Andreas Meyerhans, Jean-Pierre Vartanian and Simon Wain-Hobson. (1991). Strand Specific PCR amplification of Low Copy Number DNA. Nucleic Acids Research. 20 (3), 521-523.