Comparison across grinding and broaching methods of DNA extraction from dental pulp
Corresponding Author(s) : Leena Kumari
International Journal of Allied Medical Sciences and Clinical Research,
Vol. 8 No. 3 (2020): 2020 Volume - 8 Issue-3
Abstract
Various methods are available for obtaining yield of High Molecular Weight DNA for subsequent Multiplex PCR from teeth sample. However, a proper validation and comparison of these methods for obtaining maximum output with quantifiable output not clearly established. The primary objective of this study was to measure the effectiveness across the grinding and broaching methods of DNA extraction in freshly extracted tooth pulp, its quality check using Real Time-Polymerase Chain Reaction (RT-PCR) and subsequent multiplex STR typing.
Method
A total of 40 freshly extracted normal teeth were randomly collected. Isolation and extraction of DNA was done by organic extraction method. Precipitation of samples was done using 100% chilled ethyl alcohol followed by concentration and washing of DNA via column- based technique using DNA binding buffer and DNA wash buffer. Agarose gel electrophoresis was done to roughly estimate the DNA content while exact quantity of DNA was estimated by RT-PCR technique.
Result
Independent sample t test analysis revealed that the mean quantity of DNA (in ?g/l) was significantly higher in broaching method (M=29.91, SD=0.65) than grinding (M=9.71, SD=0.45), t (38) =114.19, p<0.000. Similarly, quality of DNA was analyzed using smear quality and it was found that the quality of DNA for broaching (M=4.55, SD=0.51) was significantly higher that the grinding method (M=2.55, SD =0.6), t (38) =11.3, p<0.000. As far as multiplex STR typing was concerned, a more clear, sharp and balanced genotype plot was obtained from the DNA obtained via broaching method in comparison to grinding method.
Conclusion
The significant quantitative and qualitative loss of DNA was observed in tooth samples processed via grinding method compared to broaching method which was further supported by the fact that DNA profiles generated from the DNA extracted using broaching method provided adequate resolution of the autosomal markers and sex identification marker (amelogenin marker) in the present study which is valuable for human identification and the gender identification finally leading to individualization.
Keywords
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- . Alia-garcía, E., Parra-pecharromán, D., Sanchez-díaz, A., Mendez, S., Royuela, A., Gil-alberdi, L., Lopez-palafox, J. & Del campo, R.. Forensic identification in teeth with caries. Forensic science international, 257, 2015, 236-241.
- . Burger, J., Hummel, S., Herrmann, B. & Henke, W. DNA preservation: a microsatellite‐dna study on ancient skeletal remains. Electrophoresis: an international journal, 20, 1999, 1722-1728.
- . Datta, P. & Datta, S. S. Role of deoxyribonucleic acid technology in forensic dentistry. Journal of forensic dental sciences, 4, 2012, 42.
- . Drancourt, M., Aboudharam, G., Signoli, M., Dutour, O. & Raoult, D. Detection of 400-year-old yersinia pestis dna in human dental pulp: an approach to the diagnosis of ancient septicemia. Proceedings of the national academy of sciences, 95, 1998, 12637-12640.
- . Ginther, C., Issel-tarver, & King, M.-C. Identifying individuals by sequencing mitochondrial dna from teeth. nature genetics, 2, 1992, 135-138.
- . Gunn, A., Essential forensic biology, John wiley & sons., 2019.
- . Hanni, C., Laudet, V., Sakka, M., Begue, A. & Stehelin, D. Amplification of mitochondrial dna fragments from ancient human teeth and bones. comptes rendus de l'academie des sciences. serie iii, Sciences de la vie, 310, 1990, 365-370.
- . Hervella, M., Iniguez, M. G., Izagirre, N., Anta, A. & De‐la‐rua, C, Nondestructive methods for recovery of biological material from human teeth for dna extraction. journal of forensic sciences, 60, 2015, 136-141.
- . Higgins, D. & Austin, J. J. teeth as a source of dna for forensic identification of human remains: a review. science & justice, 53, 2013, 433-441.
- . Manjunath, B., Chandrashekar, B., Mahesh, M. & Rani, R. V. DNA profiling and forensic dentistry–a review of the recent concepts and trends. journal of forensic and legal medicine, 18, 2011, 191-197.
- . Nelson, S. J. Wheeler's dental anatomy, physiology and occlusion-e-book, Elsevier health sciences., 2014.
- . Salman, M., Baumann, M., Hellmich, M., Roggendorf, M. & Termaat, S. sem evaluation of root canal debridement with sonicare canalbrush irrigation. international endodontic journal, 43, 2010, 363-369.
- . Sivagami, A., Rao, A. R. & Varshney, U. A simple and cost-effective method for preparing dna from the hard tooth tissue, and its use in polymerase chain reaction amplification of amelogenin gene segment for sex determination in an indian population. forensic science international, 110, 2000, 107-115.
- . Sweet, D. & Hildebrand, D. Recovery of dna from human teeth by cryogenic grinding. Journal of forensic science, 43, 1998, 1199-1202.
References
. Alia-garcía, E., Parra-pecharromán, D., Sanchez-díaz, A., Mendez, S., Royuela, A., Gil-alberdi, L., Lopez-palafox, J. & Del campo, R.. Forensic identification in teeth with caries. Forensic science international, 257, 2015, 236-241.
. Burger, J., Hummel, S., Herrmann, B. & Henke, W. DNA preservation: a microsatellite‐dna study on ancient skeletal remains. Electrophoresis: an international journal, 20, 1999, 1722-1728.
. Datta, P. & Datta, S. S. Role of deoxyribonucleic acid technology in forensic dentistry. Journal of forensic dental sciences, 4, 2012, 42.
. Drancourt, M., Aboudharam, G., Signoli, M., Dutour, O. & Raoult, D. Detection of 400-year-old yersinia pestis dna in human dental pulp: an approach to the diagnosis of ancient septicemia. Proceedings of the national academy of sciences, 95, 1998, 12637-12640.
. Ginther, C., Issel-tarver, & King, M.-C. Identifying individuals by sequencing mitochondrial dna from teeth. nature genetics, 2, 1992, 135-138.
. Gunn, A., Essential forensic biology, John wiley & sons., 2019.
. Hanni, C., Laudet, V., Sakka, M., Begue, A. & Stehelin, D. Amplification of mitochondrial dna fragments from ancient human teeth and bones. comptes rendus de l'academie des sciences. serie iii, Sciences de la vie, 310, 1990, 365-370.
. Hervella, M., Iniguez, M. G., Izagirre, N., Anta, A. & De‐la‐rua, C, Nondestructive methods for recovery of biological material from human teeth for dna extraction. journal of forensic sciences, 60, 2015, 136-141.
. Higgins, D. & Austin, J. J. teeth as a source of dna for forensic identification of human remains: a review. science & justice, 53, 2013, 433-441.
. Manjunath, B., Chandrashekar, B., Mahesh, M. & Rani, R. V. DNA profiling and forensic dentistry–a review of the recent concepts and trends. journal of forensic and legal medicine, 18, 2011, 191-197.
. Nelson, S. J. Wheeler's dental anatomy, physiology and occlusion-e-book, Elsevier health sciences., 2014.
. Salman, M., Baumann, M., Hellmich, M., Roggendorf, M. & Termaat, S. sem evaluation of root canal debridement with sonicare canalbrush irrigation. international endodontic journal, 43, 2010, 363-369.
. Sivagami, A., Rao, A. R. & Varshney, U. A simple and cost-effective method for preparing dna from the hard tooth tissue, and its use in polymerase chain reaction amplification of amelogenin gene segment for sex determination in an indian population. forensic science international, 110, 2000, 107-115.
. Sweet, D. & Hildebrand, D. Recovery of dna from human teeth by cryogenic grinding. Journal of forensic science, 43, 1998, 1199-1202.