Evaluation of low-cost bone and teeth processing methods for automated DNA extraction

Abstract

Bone and teeth can often be the only, or the best, biological materials available as DNA sources for the identification of decomposed or skeletal human remains. While these hard tissues are resistant to decay and therefore offer endogenous DNA a certain protection from the environment, their mineral matrices make DNA extraction challenging, a problem compounded by the low DNA content and high degradation levels usually associated with these tissues. To help release DNA from the mineral matrix, they are generally ground into a powder to increase their surface area to the extraction reagents. Furthermore, it is now common to perform a demineralization to dissolve the powder and release DNA that remains trapped. A common pulverization method consists of the use of a cryogenic grinder, which prevents heating of the sample and avoids subjecting the DNA to heat damage, but this equipment is costly. That said, little is known about the effect of lower-cost alternatives to the expensive cryogenic grinder on the quality and quantity of DNA recovered. Thus, this study aimed to evaluate inexpensive grinding methods for their potential for use in a forensic laboratory setting. The methods deemed to be viable options for this purpose were investigated to determine their effects on the quantity and quality of extracted DNA. Bone samples were also subjected to freezing prior to pulverization to assess the possibility that a lower sample temperature could limit heat damage to the DNA during processing. Pig bone and tooth powders were extracted using an original and a modified PrepFiler BTA™ Kit protocol, with the latter featuring an added demineralization step. Out of the methods explored, a coffee grinder and mortar and pestle were determined to be the most suitable for bone and teeth processing in a forensic laboratory setting. No significant differences between the two methods were found regarding the quantity and quality of DNA recovered and the rate of powder recovery, although more powder loss occurred when teeth were crushed with the mortar and pestle. Likewise, freezing samples before grinding did not have a significant impact on DNA quality and quantity. Finally, the original DNA extraction protocol performed significantly better than the modified one for the quantity of DNA recovered from both bone and teeth, while the quality was only superior for the bone.