Human skeletal remains: development of DNA extraction and typing methods

In forensic cases human remains are often the only biological material available for identification of missing persons or unknown remains found in different circumstances such as mass disasters, wars or socio-political events and to solve paternity issues. DNA extracted from bones is often present in low copy number (LCN) and in various states of degradation due to chemical and physical damages produced by intrinsic and extrinsic bone characteristics. Efficient DNA extraction procedures, as well as accurate DNA amplification, are critical steps involved in the process of successful DNA analysis of skeletal samples. Unfortunately, at present there is not an infallible method to recover DNA from very degraded samples due to variations in DNA yield from larger bone fragments that may be attributed to heterogeneity within a bones. In this study different types of human bones ranging in age from few months to 90 years post mortem, found in various states of preservation and conserved in different places, were analyzed. We developed a modified silica based spin columns protocol, consisting in an initial separation of DNA from proteins and waste material, by using phenol-chloroform to better purify samples. Moreover, as the recovery of information from these degraded samples is enhanced by the use of smaller PCR products (Mini Short Tandem Repeats) rather than conventional STRs, eight STR markers included in available commercial multiplex PCR kits, were redesigned by moving forward and reverse primers in close proximity to the STR repeat region. Two PCR quadruplexes were assembled to obtain PCR products less than 130 bp in size. Our modified protocol was successfully employed to extract DNA from long bones of different ages and preservation state. Importantly the use of phenol chloroform consistently increased the amount of DNA that could be extracted from long bones, because it allowed to clean samples preventing that waste material interferes with columns or magnetic beads. Environmental conditions under which remains were exposed, had stronger influence on the state of DNA quality than the age of skeletal remains. Moreover the use of miniSTRs has proposed here could be used in addition to commercial kits, to increase as much as possible the number of markers analyzed. Using amplification commercial kits and the two new mini-STR quadruplex systems we always obtained genetic profiles of at least 12 STR from DNA typing of femur samples. The improvement of DNA extraction methods and the inclusion of robust and powerful miniSTR loci in addition to the commercial available kits, are effective solutions for forensic practices of degraded DNA samples because ensure that difficult casework samples with low amounts of degraded DNA can be fully typed.