Abstract
DNA analysis is very important in forensics as it is a method to discover a victim or perpetuator of a crime. The study done was to extract DNA using a buccal swab and analyse it using a capillary gel electrophoresis which was then compared to determine the perpetuator of a crime. The DNA was extracted, quantity of DNA determined using a nanodrop and then a capillary gel electrophoresis was done. The DNA collected was of low quantity being 0.0686 ug/ul. The capillary gel electrophoresis results presented as an electrophoretogram showed that the sample collected from the coffee cup matched the DNA of the individual tested.
Introduction
DNA profiling also called DNA fingerprinting is a process of determining an individual's DNA characteristics. DNA profiling is a technique used in forensics to investigate victims or perpetrators of a crime by comparing their DNA to the DNA evidence found in a crime scene (Murphy, 2018). A buccal swab is a method of retrieving DNA samples from an individual in a cheap, reliable and non-invasive way (McMichael et al., 2009). It collects a sample of DNA in the form of saliva from the inside of the cheek in order to form a basis of comparison. Before the obtained sample can be used it needs to be purified and DNA needs to be extracted from the cells. It can be easily done using a Buccalyse DNA release kit also known as Isohelix BEK 50 kit, also prepares the sample for PCR (Scientific, 2019). The DNA sample is then analysed for sequences called short tandem repeats (STR) which are unique for every individual. They are repeated units of bases and has multiple alleles defined by the number of repeat units called flanking regions (HMM202 practical manual, 2019). It is done using a capillary gel electrophoresis which provides a rapid, high resolution separation of the amplified DNA (Mansfield et al., 1998). In this practical a sample of DNA was collected using a the Isohelix kit mentioned and prepared for STR analysis using capillary gel electrophoresis. Due to time constraints an already prepared electrophoretogram was given to analyse. The purpose of this experiment is to be able to extract DNA and analyse it as potential suspects of a crime.
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Method
The first step was to obtain a buccal swab. Drinking caffeine had to be avoided prior to the experiment and mouth had to be rinsed to avoid any contamination. A tube was labelled with individuals initials and 200ul of Buccalyse was added. The swab was rubbed on the inner cheeks around 10 times to ensure a good sample, and then was placed in the tube guaranteeing most of the liquid was captured. The tube was then incubated at 70 degrees for 15 minutes and 95 degrees for 2 minutes. The tube was vortexed in between each incubation. The next step involved the nanodrop. From task 1, 2ul of the isolated genomic DNA was obtained and the concentration detected.
This then followed an agarose gel electrophoresis at 120V for 30 minutes. A sample of 10ul of DNA was obtained and aliquot into wells. The final step was DNA profiling done by a capillary gel electrophoresis with the results given by the staff (HMM202 practical manual, 2019).
Discussion
The results in figure A mean that the DNA sample collected from the buccal swab was successful as there was a black band present behind the well. The DNA was condensed, and the weight of the DNA was too heavy for it to migrate through the matrix thus producing that black colour behind the wells (Chauhan, 2018). The smearing present represents that the DNA is contaminated with RNA (Chauhan, 2018). Compared to other samples in the gel electrophoresis there were high quality DNA samples present like in lane 6, 7, 11 and 12. This indicates that the sample collected in lane 14 was of low quality which could be due to insufficient swabbing of the inner mouth, which in turn did not collect enough DNA sample. The nanodrop results obtained also showed that the sample of DNA collected was contaminated with protein, phenol or other contaminants, thus yielding a low quantity of DNA (Scientific, 2019). The highest ratio for A260/280 obtained by the nanodrop was 1.03 however still lower than the preferred range. To ensure less contamination enzymes can be used like proteinase K or RNase and then incubated overnight (Bradburn, 2018). It is important to reduce contamination as it interferes with the results and could cause an individual to become a suspect that has no relation to a case.
Every individual will have differing numbers of bases and repeated sequences in an STR region thus the graphs formed in an electropherogram based on those alleles would be unique to every individual (Encyclopedia Britannica, 2018). The aim of the experiment was achieved as DNA was successfully extracted however was highly contaminated. In future if the experiment was to be conducted the DNA sample should be purified to eliminate any contaminants. Not only is capillary gel electrophoresis used for DNA profiling it also helps in clinical and health studies. It is used to determine endogenous and exogenous compounds in body fluids and tissue extracts (Thormann et al., 1994). It also used in open-tubular column systems, identify separation patterns of serum proteins and even used to separate haemoglobin variants (Chen, Liu and Sternberg, 1991).
RefePrences
- Dr Liza Raymond and Dr Richard Williams (2019), Molecular Diagnostics HMM202 Practical Manual, pp.23-27.
- Mansfield, E., Robertson, J., Vainer, M., Isenberg, A., Frazier, R., Ferguson, K., Chow, S., Harris, D., Barker, D., Gill, P., Budowle, B. and McCord, B. (1998). Analysis of multiplexed short tandem repeat (STR) systems using capillary array electrophoresis. Electrophoresis, 19(1), pp.101-107
- McMichael, G., Gibson, C., O'Callanghan, M., Goldwater, P., Dekker, G., Haan, E. and MacLennan,, A. (2009). DNA from Buccal Swabs Suitable for High-Throughput SNP Multiplex Analysis, 20(5), pp.232–235.
- Murphy, E. (2018). Forensic DNA Typing. Annual Review of Criminology, [online] 1(1), pp.497-515. Available at: https://www.annualreviews.org/doi/10.1146/annurev-criminol-032317-092127[Accessed 11 Sep. 2019]
- Scientific, B. (2019). Buccalyse Rapid Buccal Cell DNA Isolation Kit 25 extractions. [online] Bocascientific.com. Available at: https://www.bocascientific.com/simhelix-buccalyse-rapid-buccal-cell-dna-isolation-kit-50-extractions-with-sk1-swabs-p-4452.html [Accessed 11 Sep.2019]
- Scientific, T. (2019). Assessment of Nucleic Acid Purity. [ebook] Available at: https://s3-us-west-2.amazonaws.com/oww-files-public/d/d7/T042- NanoDrop-Spectrophotometers-Nucleic-Acid-Purity-Ratios.pdf [Accessed 14 Sep. 2019]
- Chauhan, T. (2018). A complete guide for analysing and interpreting gel electrophoresis results. [online] Genetic Education. Available at: http://geneticeducation.co.in/a-complete-guide-for-analysing-and-interpreting-gel-electrophoresis-results/ [Accessed 14 Sep. 2019].
- Bradburn, S. (2018). The Nanodrop Results Explained - Top Tip Bio. [online] Top Tip Bio. Available at: https://toptipbio.com/the-nanodrop-results-explained/ [Accessed 16 Sep. 2019].
- Encyclopedia Britannica. (2018). DNA fingerprinting | Definition, Examples, & Facts. [online] Available at: https://www.britannica.com/science/DNA- fingerprinting [Accessed 16 Sep. 2019]
- Thormann, W., Molteni, S., Caslavska, J. and Schmutz, A. (1994). Clinical and forensic applications of capillary electrophoresis. Electrophoresis, 15(1), pp.3-12.
- Chen, F., Liu, C. and Sternberg, J. (1991). Capillary electrophoresis--a new clinical tool. The American Association for Clinical Chemistry, 37(1), pp.1-2.
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