Human skeletal remains: development of DNA extraction and typing assays

Extraction and successful PCR amplification of DNA from human remains in historical and forensic cases has great importance, but is particularly difficult because the methods employed at present are not always satisfactory. Several of them are in fact complicated and time consuming and none methods has reached on acceptance level such that they are routinely used on a widespread basis. Bone extraction protocols currently employed in forensic laboratories tend to be limited because they often fail to give reproducible results, e.g. in cases where bones are exposed to environmental conditions for a long time. 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. Different kind of bone, femur, homerus, tibia, jaw, rib, belonging to different cadavers were analyzed. We established a semi-standardized protocol for DNA extraction from bones, verifying which kind of bone yields the best quality of DNA and evaluating different characteristics such as preservation, place of conservation and age of skeletal remains. A comparison in terms of quality of electrophoretic products, was performed from human skeletal remains considering five different DNA extraction methods and starting from a low amount of bone powder (50 -100 mg). In addition to the traditional phenol–chloroform organic method for DNA extraction, four commercial kits were evaluated: QIAamp® DNA Mini kit (Qiagen, Hilden, Germany), QIAamp® DNA Investigator kit (Qiagen, Hilden, Germany), DNA IQ™ System (Promega, Milan, Italy) and PrepFiler™ Forensic DNA Extraction Kit (Applied Biosystems, Foster City, CA). Experiments were also performed with a modified protocols developed by Forensic Genetic Laboratory of University of Verona, consisting in an initial separation of DNA from proteins and waste material, by using phenol-chloroform to better purify samples. The new step was introduced after incubation in lysis buffer of different kit solutions. The phenol-chloroform step allowed to clean samples avoiding that the waste material would interfere with columns or magnetic beads. Moreover as recovery of information from degraded samples is enhanced by the use of smaller PCR products called miniSTR, primer pairs of eight STR markers, included in available commercially multiplex PCR kits, were redesigned by moving forward and reverse primers in close proximity to the STR repeat region. They were assembled in two PCR-multiplexes to obtain PCR products less than 130 bp in size. The choice of this size was determined by the observation that in degraded samples amelogenin is usually the only marker amplified because it is the lowest in size (106-112 bp). In addition to the new miniSTRs multiplexes all samples were amplify also with two kits widespread in forensic use such as AmpFlSTR® Identifiler™ PCR Amplification Kit (Applied Biosystems), AmpFlSTR® MiniFiler™ PCR Amplification Kit (Applied Biosystems) and PowerPlex® ESI 17 System (Promega). The improvement of DNA extraction methods and increasing number of miniSTRs in addition to the commercial available kits, may be effective solutions for forensic practices of degraded DNA samples. The application of the DNA extraction protocol based on the use phenol-chloroform for bones exposed to critical environmental conditions for long periods and for low amounts of bone gave good results. 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.