Improved DNA recovery and STR profile development from weathered Bos taurus bones using demineralized bone slices.

Abstract

Current processing techniques for harvesting DNA from osseous elements are destructive, and success rates vary widely. When skeletal elements are homogenized into a fine powder, endogenous DNA may be subjected to fragmentation, and the likelihood of introducing co-purified inhibitory substances to the sample increases. While a limited number of articles in the relevant literature have challenged the status quo of pulverization, powdering hard tissue samples before DNA isolation continues to be standard practice in the forensic and ancient DNA communities. In this work, we have developed and optimized an alternative front-end processing method for demineralizing and slicing cortical bone using aged and weathered bovine skeletal samples as a model for degraded human bone. Additionally, this study evaluated the enzymatic digestion of demineralized bone slices using collagenase I as a powder-free alternative for cell isolation. The efficacy of pulverization, demineralized slices, and collagenase-digested demineralized slices was assessed via DNA quantitation and STR profile data. The combined treatment incorporating collagenase digestion of demineralized slices did not improve the recovery of endogenous DNA yields or STR profile development; however, profiles developed from demineralized slices retained a significantly greater percentage of expected alleles and higher peak heights than samples processed with conventional powdering and organic chemistry extraction. By eliminating pulverization of bone and employing modified silica-based extraction chemistry, the susceptibility to inhibitory and competitive effects from native and foreign components often found in degraded skeletal remains can be minimized when utilizing total demineralization of small fragments with subsequent slicing before lysis.