Application Of Real Time PCR In Forensic Science

The Use of PCR in DNA Profiling

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Forensic science is the study of evidences through different scientific methodology in order to identify the criminal or the process of the crime and food analysis (Reischcl, Witter, Coceril, 2012). Polymerase Chain Reaction is used for amplification of a single DNA fragment into multiple DNA copies (Dietmaier, Witter and Sivasubramanian, 2013). Real time PCR technique is the polymerase chain reaction method  the monitoring of the whole reaction process is done in the real time format (Giampaoli et al, 2012). Criminal acts such as murder, rape, sexual assaults are testified through different methods of forensic science. DNA profiling which includes techniques such as DNA typing, DNA testing and DNA fingerprinting is one of the most used techniques used by forensic scientists (Caniglia et al, 2010). To carry out such techniques, PCR is used as a tool. Nowadays, Real Time PCR is used for more specific identification of the evidences (Johnson, Wilson-Wilde and Linacre, 2014). The real-time fluorescence based quantitative PCR is one of the significant benchmark technology used in forensic science application. Another aspect where PCR is used based on mitochondrial genes is used for its matrilineal inheritance (McLaughlin, Doty and Lednav, 2014).

As discussed in a research paper by Sinha and his coworkers, Retro transposable elements which is comprised of two regions known as LINEs (Long interspersed nuclear element) and SINEs (short interspersed nuclear element) can be used as marker for human identification and bio-ancestry testing. As Retro transposable elements does not cause shutter artifacts due to slippage at the time of PCR, the interpretation of the result becomes much easier (Sinha et al, 2015).

In another research paper by Dawnay and his colleagues carried out real time PCR using HyBeacon probe technology which enabled them species detection. They used this method for rapid onsite non-human forensic testing. In cases of illegal animal killing, species identification is one of the major issues. In this method they have used a single HyBeacon probe and melt curve analysis for rapid screening. Melt curve detection process helps to detect species specific SNP sites present on the COI genes. This helps the forensic scientists to specifically identify a particular species of animal from unknown DNA sample (Dawnay, et al, 2016).

The aim of the research carried out by Sudhir Sinha and his coworkers was to develop a novel and sensitive DNA marker which can be applied in forensic science for the identification of human DNA with an unknown DNA sample (Sinha et al, 2015).

Real Time PCR Technique

The aim of the research carried out by Nick Dawnay and his coworkers is to develop a technique which can be used for non-expert genetic species identification among different species. Another aspect of the aim was to design the application of this method for on-human forensic authentication (Dawnay, et al, 2016).

In order to achieve the mentioned aim, Sinha and his colleague used novel primer design methodology. In this method they have used real time PCR technique for the novel primer design. Methylene specific polymerase chain reaction or MSP is used. MSP method removed the intra-spacing between the locus competition, which are found in case of heterozygote DNA. The samples were analyzed by Hardy-Weinberg expectation to demonstrate the linkage and disequilibrium between the sequences. Using a capillary electrophoresis technique the allelic nature of the alu typing system was also carried out. After that, the splicing reduces the amplicon size of loci region and in two allelic states of INNULs. In the second part they used real time qPCR. qPCR is used for the development of quantitative and qualitative assay.

In case of development of PCR technique using HyBeacon probe technology. At first Nick and his colleagues used the Sample authentication-DNA sequencing. They extracted DNA samples from 42 different fish species. After that thee HyBeacon assay design was done. They downloaded the sequence data of Atlantic cod from BO LD and NCBI. Databas2.0 It is seen that Hybeaconprobes are generally 20 to 30 base pair long having short COI homology regions . The process also included pautive species specific SNP sites. Using this this technology they carried out the identification of key target species using Mega 6.0. After this multiple sequences were aligned using Clustal. After the design was done, they Develop the HyBeacon assay. This is the most crucial part, as the result of the entire research was dependent on this method. For the development of the specific cod assay, data was gathered from the genomic DNA. They categorized the experiment sensitivity and specificity studies were made.

Sinha and his co workers observed that Retro transposable elements are in generally novel markers. They have a property of high copy number. They can be used for the most reproducible and sensitive DNA quantification. In the result Sinha observed that, Innotyper*21 amplification of A DNA sample collected from 2cm long rootless hair shaft. The result collected showed that there was a significant amount of DNA degradation and low level quantity was found. The sample which was yielded had the characteristics of a full genotype consistent with the buccal swab sample from the donor. As a result the Innotyper Retro transposable elements can be combined with ancestry informative ALU RE markers. These combination can be made small samples as less than 100 base pair. As a result the forensic samples will provide additional information. The detection of  an ancestry gene within the sub-continental and continental population will diversify to detect human population in different forensic cases.  The other major aspect of this method is that it can detect sample in highly degrade and low level conditions as well.  The development aspect of small amplicon and the multiplex primer kit will help the scientists to prepare next generation sequencing libraries as well. These libraries will also help in forensic and bio-ancestral identification from unknown DNA sample.

PCR and Mitochondrial Genes

Nick and his coworkers found in their research that the detection capability of this process was about 7.5 pg of DNA. This is a high sensitivity detection phenomenon as compared to other c0onventional methods (De Bruyne et al, 2011). The result obtained showed that none of the non-target species in the tests showed repeated amplification or melt curve detection in the same regions (Winder et al, 2011). One of the reasons behind this finding is the lack of homology found in the primers and the test specimen (Filonzi et al, 2010). A diagnostic peak of Haddock was amplified to produce a melt peak. The result collected showed that T_m was 45 degrees Celsius and it was not miscalled. ANOVA analysis showed that there was no significant difference between the delta RFU  for each melt curve. The results seen in ParaDNA software measured all three melt targets. As a result, it can be said the, using this technique more than one species can be detected in one sample at the same time. The ability of detecting numerous different species composition in a single sample may allow identification of separate form of the species (Cawthorn, Steinman and Witthuhn, 2012). Another aspect of this method is that it can be used to identify different sort of animal species in food departments as well.

Application of these two techniques will allow a whole net domain in forensic sciences (Nielsen et al, 2012). Using detection technique through the retro transposable elements will allow a diverse detection method. Population or a single individual can be identified with the help of low amount of samples such as body fluids, blood, hair or other DNA extractable samples. In case of forensic application, criminals can be easily identified and brought to justice. In many cases, forensic cannot detect DNA samples as the sample is of low amount or in degrading condition (Hennessy et al, 2014). This technique will allow the scientists to test sample which are not in good condition. In other hand, the technique of HyBeacon will allow the forensic scientist to test samples in illegal animal poacher or other assault (Grubaugh et al, 2013). The most important aspect of this method is that, more than one species can be detected through the analysis of one single sample (Dawnay et al, 2014). This increases the sphere of detection, the method is also very much time consuming and easy to develop (Ball et al, 2015). This technology is ideal for non-expert users as well. As the method is very much easier to adopt and there is no use of hazardous element, the set up for such experiments can be established in current laboratories as well. Traditional detection methods often take a numerous number of days, but in this method, the results will be collected very much sooner. It can be concluded from the analysis of these two method that if these techniques are used in current forensic studies, the identification sphere will increase as well as it will increase the specificity of the identification (Tamura et al, 2013).          

Research Methodologies

References

Ball, G., Dawnay, N., Stafford-Allen, B., Panasiuk, M., Rendell, P., Blackman, S., Duxbury, N. and Wells, S., 2015. Concordance study between the ParaDNA® Intelligence Test, a Rapid DNA profiling assay, and a conventional STR typing kit (AmpFlSTR® SGM Plus®). Forensic Science International: Genetics, 16, pp.48-51.

Caniglia, R., Fabbri, E., Greco, C., Galaverni, M. and Randi, E., 2010. Forensic DNA against wildlife poaching: identification of a serial wolf killing in Italy. Forensic Science International: Genetics, 4(5), pp.334-338.

Cawthorn, D., Steinman, H.A. and Witthuhn, R.C., 2012. DNA barcoding reveals a high incidence of fish misrepresentation and substitution on the South African market.

Dawnay, N., Hughes, R., Court, D.S. and Duxbury, N., 2016. Species detection using HyBeacon® probe technology: Working towards rapid onsite testing in non-human forensic and food authentication applications. Forensic Science International: Genetics, 20, pp.103-111.

Dawnay, N., Stafford-Allen, B., Moore, D., Blackman, S., Rendell, P., Hanson, E.K., Ballantyne, J., Kallifatidis, B., Mendel, J., Mills, D.K. and Nagy, R., 2014. Developmental Validation of the ParaDNA® Screening System-A presumptive test for the detection of DNA on forensic evidence items. Forensic Science International: Genetics, 11, pp.73-79.

De Bruyne, K., Slabbinck, B., Waegeman, W., Vauterin, P., De Baets, B. and Vandamme, P., 2011. Bacterial species identification from MALDI-TOF mass spectra through data analysis and machine learning. Systematic and applied microbiology, 34(1), pp.20-29.

Dietmaier, W., Wittwer, C. and Sivasubramanian, N. eds., 2013. Rapid Cycle Real-Time PCR—Methods and Applications: Genetics and Oncology. Springer Science & Business Media.

Filonzi, L., Chiesa, S., Vaghi, M. and Marzano, F.N., 2010. Molecular barcoding reveals mislabelling of commercial fish products in Italy. Food Research International, 43(5), pp.1383-1388.

Giampaoli, S., Berti, A., Valeriani, F., Gianfranceschi, G., Piccolella, A., Buggiotti, L., Rapone, C., Valentini, A., Ripani, L. and Spica, V.R., 2012. Molecular identification of vaginal fluid by microbial signature. Forensic Science International: Genetics, 6(5), pp.559-564.

Grubaugh, N.D., Petz, L.N., Melanson, V.R., McMenamy, S.S., Turell, M.J., Long, L.S., Pisarcik, S.E., Kengluecha, A., Jaichapor, B., O’Guinn, M.L. and Lee, J.S., 2013. Evaluation of a field-portable DNA microarray platform and nucleic acid amplification strategies for the detection of arboviruses, arthropods, and bloodmeals. The American journal of tropical medicine and hygiene, 88(2), pp.245-253.

Hennessy, L.K., Mehendale, N., Chear, K., Jovanovich, S., Williams, S., Park, C. and Gangano, S., 2014. Developmental validation of the GlobalFiler® express kit, a 24-marker STR assay, on the RapidHIT® System. Forensic Science International: Genetics, 13, pp.247-258.

Johnson, R.N., Wilson-Wilde, L. and Linacre, A., 2014. Current and future directions of DNA in wildlife forensic science. Forensic Science International: Genetics, 10, pp.1-11.

McLaughlin, G., Doty, K.C. and Lednev, I.K., 2014. Discrimination of human and animal blood traces via Raman spectroscopy. Forensic science international, 238, pp.91-95.

Nielsen, E.E., Cariani, A., Mac Aoidh, E., Maes, G.E., Milano, I., Ogden, R., Taylor, M., Hemmer-Hansen, J., Babbucci, M., Bargelloni, L. and Bekkevold, D., 2012. Gene-associated markers provide tools for tackling illegal fishing and false eco-certification. Nature Communications, 3, p.851.

Reischl, U., Wittwer, C. and Cockerill, F. eds., 2012. Rapid Cycle Real-Time PCR—Methods and Applications: Microbiology and Food Analysis. Springer Science & Business Media.

Sinha, S., Murphy, G., Brown, H., Montgomery, A., Carrol, M. and Tabak, J., 2015. Retrotransposable elements: Novel and sensitive DNA markers and their application in human identity. Forensic Science International: Genetics Supplement Series, 5, pp.e627-e629.

Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular biology and evolution, 30(12), pp.2725-2729.

Winder, L., Phillips, C., Richards, N., Ochoaâ€ÂCorona, F., Hardwick, S., Vink, C.J. and Goldson, S., 2011. Evaluation of DNA melting analysis as a tool for species identification. Methods in Ecology and Evolution, 2(3), pp.312-320.