Human DNA Identification and sex determination from bloodstains using duplex PCR analysis
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Published:
Dec 29, 2017
Keywords:
Duplex PCR Bloodstains Cytochrome b SRY
Main Article Content
Abstract
This study aimed to identify DNA of human origin and determine human sex from bloodstains using duplex PCR analysis. Two pairs of primers amplifying the Amelogenin gene, AMEL(1) and AMEL(2) and a primer pair targeting the Sex Determining Region Y gene [SRY] were compared at the preliminary stage to measure the accuracy of sex determination. The human specific region of the Cytochrome b (Cyt b) gene, sized 412 bp, successfully targeted only human-origin DNA, while other types of animal DNA (dog, cat, cow, chicken and pig) did not produce any PCR product. The SRY primer showed 100% accuracy for male identification with a 197 bp amplicon. Therefore, the SRY primer pair was chosen for sex identification. The optimal annealing temperature for duplex PCR analysis was 55 °C, producing the most distinctive PCR products, determining the male gender with 197 bp and 412 bp bands and female with only 412 bp. For sensitivity, the smallest blood sample that could be detected was 20 ml. For specificity, both human origin and sex determination could be detected in all ratios of mixed human and dog bloods, including 100 fold less human blood to animal blood. Determination of male blood in mixed male and female blood samples was also investigated. The results showed that the presence male blood could be determined in mixed female blood sample at ratios of less than 10 fold. This study demonstrated that duplex PCR analysis of Cyt b and SRY is a reliable tool to investigate human DNA and sex of questioned bloodstains.
Article Details
How to Cite
Amornpan, P., Petthai, A., Monthatong, M., & Lomthaisong, K. (2017). Human DNA Identification and sex determination from bloodstains using duplex PCR analysis. Asia-Pacific Journal of Science and Technology, 22(4), APST–22. https://doi.org/10.14456/apst.2017.34
Section
Research Articles
References
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[26] Suwa, N., Ikegaya, H., Takasaka, T., Nishigaki, K., Sakurada, K., 2012. Human blood identification using the genome profiling method. Legal Medicine 14, 121-125.
[27] Kanthaswamy, S., Premasuthan, A., Ng, J., Satkoski, J., Goyal, V., 2012. Quantitative real-time PCR (qPCR) assay for human-dog-cat species identification and nuclear DNA quantification. Forensic Science International: Genetics. 6, 290-295.
[28] Hu, Z., Wang, Z., Zhang, S., Bian, Y., Li, C., 2015. Species identification through pyrosequencing 12S rRNA gene. Forensic Science International: Genetics 5, e561-e563.
[2] Ono, T., Miyaishi, S., Yamamoto, Y., Yoshitome, K., Ishikawa, T., Ishizu, H., 2001. Human identification from forensic materials by amplification of a human-specific sequence in the myoglobin gene. Acta Medica Okayama 55, 175-184.
[3] Parson, W., Pegoraro, K., Niedertatter, H., Foger, M., Steinlechner, M., 2000. Species identification by means of the cytochrome b gene. International Journal of Legal Medicine 114, 23-28.
[4] Sullivan, K.M., Hopgood, R., Gill, P., 1992. Identification of human remains by amplification and automated sequencing of mitochondrial DNA. International Journal of Legal Medicine 105, 83-86.
[5] Kreike, J., Lehner, A., 1995. Sex determination and DNA competition in the analysis of forensic mixed stains by PCR. International Journal of Legal Medicine 107, 235-238.
[6] Faerman, M., Filon, D., Kahila, G., Greenblatt, C.L., Smith, P., Oppenheim, A., 1995. Sex identification of archeological human remains based on amplification of the X and Y amelogenin alleles. Gene 167, 327-332.
[7] Aasen, E., Medrano, J.F., 1990. Amplification of the ZFY and ZFX genes for sex determination in humans, cattle, sheep and goats. Nature Biotechnology 8, 1279-1281.
[8] Steinlechner, M., Berger, B., Niederstatter, H., Parson, W., 2002. Rare failures in the amelogenin sex test. International Journal of Legal Medicine 116, 117-120.
[9] Michael, M., Brauner, P., 2004. Erroneous gender identification by amelogenin sex test. Journal of Forensic Sciences 49, 258-259.
[10] Drobnic, K., 2006. A new primer set in a SRY gene for sex identification. International Congress Series 1288, 268-270.
[11] Tyler, M.G., Kirby, L.T., Wood, S., Vernon, S., Ferris, J.A., 1986. Human bloodstains identification and sex identification in dried bloodstains using recombinant DNA techniques. Forensic Science International 31, 267-272.
[12] Kornkaew, A., 2012. Application of multiplex PCR for bloodstains analysis for sex and human blood identification [Thesis]. Khon Kaen: Graduate school, Khon Kaen University. (Thai.)
[13] Tozzo, P., Giuliodori, A., Scorato, S., Ponzano, E., Rodrigues, D., Caenazzo, L., 2013. Deletion of amelogenin Y-locus in forensics: Literature revision and description of a novel method for sex confirmation. Journal of Forensic and Legal Medicine 20, 387-391.
[14] Nakahori, Y., Takenaka, O., Nakagome, Y., 1991. A human X-Y homologous region encode “amelogenin”. Genomics 9, 264-269.
[15] Matsuda, H., Seo, Y., Kakizaki, E., Kozawa, S., Muraoka, E., Yukawa, N., 2005. Identification of DNA of human origin based on amplification of human-specific mitochondrial cytochrome b region. Forensic Science International 152, 109-114.
[16] Chang, Y.M., Perumal, R., Keat, P.Y., Yong, R.Y., Kuehn, D.L., Burgoyne, L., 2007. A distinct Y-STR haplotype for amelogenin negative males characterized by a large Y(p)11.2 (DYS458-MSY1-AMEL-Y) deletion. Forensic Science International 166, 115-120.
[17] Turrina, S., Filippini, G., Voglino, G., De Leo, D., 2010. Two additional reports of deletion on the short arm of the Y chromosome. Forensic Science International: Genetics 5, 242-246.
[18] Quincey, D., Carle, G., Alunni, V., Quatrehomme, G., 2013. Difficulties of sex determination from forensic bone degraded DNA: A comparison of three methods. Science & Justice 53, 253-260.
[19] Drobnic, K., 2006. A new primer set in a SRY gene for sex identification. International Congress Series 1288, 268-270.
[20] Inturri, S., Robino, C., Gino, S., Caratti, S., Torre, C., 2009. Integration of the AmpFISTR Identifiler PCR amplification kit with SRY-specific primers for gender identification. Forensic Science International: Genetics Supplement Series 2, 36-37.
[21] Zehethofer, K., 2011. A molecular analysis of three amelogenin negative males in two routine paternity tests. Forensic Science International: Genetics 5, 550-551.
[22] Asamura, H., Sakai, H., Kobayashi, K., Ota, M., Fukushima, H., 2006. MiniX-STR multiplex system population study in Japan and application to degraded DNA analysis. International Journal of Legal Medicine 120, 174-181.
[23] Alonso, A., Martin, P., Albarran, C., Garcia, P., Garcia, O., de Simon, L.F., Garcia-Hirschfeld, J., Sancho, M., de la Rua, C., Fernandez-Piqueras, J., 2004. Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies. Forensic Science International 139, 141-149.
[24] Andreasson, H., Nilsson, M., Budowle, B., Lundberg, H., Allen, M., 2006. Nuclear and mitochondrial DNA quantification of various forensic materials. Forensic Science International 164, 56-64.
[25] Tobe, S.S., Linacre, A.M.T., 2008. A technique for the quantification of human and non-human mammalian mitochondrial DNA copy number in forensic and other mixtures. Forensic Science International: Genetics 2, 249-256.
[26] Suwa, N., Ikegaya, H., Takasaka, T., Nishigaki, K., Sakurada, K., 2012. Human blood identification using the genome profiling method. Legal Medicine 14, 121-125.
[27] Kanthaswamy, S., Premasuthan, A., Ng, J., Satkoski, J., Goyal, V., 2012. Quantitative real-time PCR (qPCR) assay for human-dog-cat species identification and nuclear DNA quantification. Forensic Science International: Genetics. 6, 290-295.
[28] Hu, Z., Wang, Z., Zhang, S., Bian, Y., Li, C., 2015. Species identification through pyrosequencing 12S rRNA gene. Forensic Science International: Genetics 5, e561-e563.