A New and Efficient Method for DNA Extraction from Human Skeletal Remains Usable in DNA Typing

Document Type: Original Article

Authors

1 Human Genetics Research Center, Baqiyatallah University of Medical Sciences Tehran, Iran

2 Young Researchers and Elite Club, Islamic Azad University, Tehran, Iran

Abstract

In historical cases, mass disasters, missing person’s identification and ancient DNA investigations, bone and teeth samples are often the best and the only biological material available for DNA typing. This is because of the physical and chemical nature of the protein-mineral matrix of bone which is relatively resistant to the adverse environmental effects and biological attacks. Most bone extraction protocols used in the forensic laboratories involve an incubation period of bone powder in extraction buffer for proteinase digestion, followed by the collection of the supernatant, and the elimination of large quantities of undigested bone powder. Here we demonstrate an extremely effective protocol for recovery of DNA. This is performed in a method that retains and concentrates all the reagent volume for complete DNA recovery. For our study, we selected challenging bone samples of skeleton remains of the martyred individuals in Iraq’s imposed war on I.R. Iran (1980-1988). The bones that were extracted with our new protocol showed that this new protocol significantly enhances the quantity of DNA that can be used for amplification from degraded skeletal remains. At the same time we tested in parallel the samples by using of QIAamp DNA Investigator Kit and attained the best results by using new protocol. In fact, our new DNA extraction method is based on previous standard methods such as Chelex and salting out. We have used this technique to successfully recover authentic DNA Typing from extremely challenging samples that failed repeatedly using the standard protocols. However, the amount of recovered DNA was very small but it was possible to extract genomic DNA from these challenging bone samples. The results indicated that our procedure for DNA extraction although yielded little amount of genomic DNA; however, it was pure DNA that can be used for further analysis such as PCR amplification and DNA profiling. Since the new procedure is fast and needed less time than previously standard procedures, we have named it FDEB (Fast DNA Extraction of Bone).

Keywords


  1. Ariffin, S.H., Wahab, R.M., Zamrod, Z., Sahar, S., Razak., M.F., Ariffin, E.J., Senafi, S., Molecular archeology of ancient bone from 400 year old shipwreck. Asia Pacific J Mol Biol Biotechnol, 2007, Vol. 15(1), pp. 27-31.
  2. Putkonen, M.T., Palo, J.U., Cano, J.M., Hedman, M., Sajantila, A., Factors affecting the STR amplification success in poorly preserved bone samples. Invest Genet, 2010, Vol. (1), pp. 9-15.
  3. Loreille, O.M., Diegoli, T.M., Irwin, J.A., Coble, M.D., Parsons, T.J., High efficiency DNA extraction from bone by total demineralization. Forensic Sci Int Genet, 2007, Vol. 1(2), pp. 191-195.
  4. O'Rourke, D.H., Hayes, M.G., Carlyle, S.W., Ancient DNA studies in physical anthropology. Annu Rev Anthropol, 2000, Vol. 29(1), pp. 217-242.
  5. Katzmarzyk, A.R., Parsons, T.J., Success rates of nuclear short tandem repeat typing from different skeletal elements. Croat Med J, 2007, Vol. 48, pp. 486-93.
  6. Marjanović, D., Durmić-Pašić, A., Bakal, N., Haverić, S., Kalamujić, B., Kovačević, L., Ramić, J., Pojskić, N., Škaro, V., Projić, P., Bajrović, K., DNA identification of skeletal remains from World War II mass graves uncovered in Slovenia. Croat Med J, 2007, Vol. 48(4), pp. 513-519.
  7. Vanek, D., Saskova, L., Koch, H., Kinship and Y-chromosome analysis of 7th century human remains: novel DNA extraction and typing procedure for ancient material. Croati Med J, 2009, Vol. 50(3), pp. 286-295.
  8. Kalmár, T., Bachrati, C.Z., Marcsik, A., Raskó, I., A simple and efficient method for PCR amplifiable DNA extraction from ancient bones. Nucleic Acids Res, 2000, Vol. 28(12), pp. 67-71.
  9. Loreille, O.M., Parr, R.L., McGregor, K.A., Fitzpatrick, C.M., Lyon, C., Yang, D.Y., Speller, C.F., Grimm, M.R., Grimm, M.J., Irwin, J,A,, Robinson, E,M., Integrated DNA and fingerprint analyses in the identification of 60‐year old mummified human remains discovered in an alaskan glacier. J Forensic Sci, 2010, Vol. 55(3), pp. 813-818.
  10. Kemp, B.M., Smith, D.G., Use of bleach to eliminate contaminating DNA from the surface of bones and teeth. Forensic Sci Int, 2005, Vol. 154(1), pp. 53-61.
  11. Bogdanowicz, W., Allen, M., Branicki, W., Lembring, M., Gajewska, M., Kupiec, T., Genetic identification of putative remains of the famous astronomer Nicolaus Copernicus. Proc Natl Acad Sci USA, 2009, Vol.106 (30), pp. 12279-12282.
  12. Nelson, K., Melton, T., Forensic mitochondrial DNA analysis of 116 casework skeletal samples. J Forensic Sci, 2007, Vol. 52 (3), pp. 557-561.