A Direct Amplification for STR Typing from Human Bones

Abstract

Bones are the most prevalent evidence in incidences of mass disaster or long- occurred forensic cases. Individualisation and identification by conventional STR analysis is routine yet suffers from being costly and time-consuming mainly due to the DNA extraction step which also introduces risk of contamination and yield loss (up to 76%). Bone samples may need to be decalcified overnight or for a couple of days as a pre-treatment step. Past studies also found that different skeletal elements have different DNA yield and consequently different STR typing success rates. To streamline STR typing from bones and choose the best bone element for direct STR typing, this study aimed to develop a direct STR typing protocol from human bones. An optimized direct PCR protocol for STR typing from human bones was successfully developed. One hundred mg bone powder in 300 μL PBS buffer was determined to be the optimal condition. The mixture was heated at 98°C for three minutes, with the supernatant ready for subsequent DNA amplification. IDplex Plus was found to perform better than Identifiler Plus for direct STR typing (median allele recovered of 31 and median peak height of 980 RFU at optimal conditions). Fifteen of each bone elements (1st distal phalange of the hand, capitate, patella, metacarpal 4, talus and tibia) (N=105) were then subjected to direct STR typing with 92.4% of the samples resulting in high partial to full profiles. Median peak height from the profiles generated from cancellous bones were significantly better than from compact bones (p=0.033) and was also significantly different across the different bone elements (p<0.001). Bone samples of two days PMI generated significantly higher median number of allele (32+2) as compared to (28+7) for five days PMI (p<0.001). However, low success rates were obtained when applying the developed protocol on casework samples. In conclusion, the developed protocol is robust as performance was similar for all the seven bone elements investigated, as well as being rapid and easy to be applied. Further optimisation may increase success rates for casework bones and allows implementation in casework samples.