Genetic Extraction and Analysis from Weathered 6 Year Old Bos Taurus Bones through Bead Ruptor Homogenization

DNA analysis of human and animal skeletal remains provide vast amounts of insight for evolutionary, archeological, medical, and forensic studies. Bone structure has proven successful in housing varying amounts of genomic and mitochondrial DNA, long after the soft tissues have degraded. [1] While osseous tissues retain DNA longer than the soft tissues of the body, nuclear DNA and mitochondrial DNA found in bone are both shown to degrade exponentially at fluctuating rates depending on their post-mortem time interval and the environmental conditions which they are exposed to. [2] This degradation pattern invokes urgency for processing samples upon their retrieval, in order to mitigate as much genetic degradation as possible once in the lab. The current methodologies for the extraction of genetic material from aged bones involve multiple degradation procedure options to expose the genetic material for extraction. These methods require exposing the material to aggressive demineralizing and denaturing chemical buffers such as EDTA and DTT for prolonged periods, with some requiring up to 80 hours of incubation in these chemical baths. [5] Other methodologies rely extensively on commercially-available purification kits and multiple rounds of PCR prior to reaching a workable quantity of purified DNA. [2] Yet others, claim that multiple rounds of mechanical disruption of the sample in conjunction with demineralization treatments will yield high working quantities of extracted DNA. [5,6]

The method described herein uses a combination of chemical treatments and mechanical disruption to extract and amplify DNA from fossilized Bos taurus bone. The bone sample was obtained from the foothills of Northern Alabama as seen in Figure 1, where it sat exposed to the elements in natural conditions for approximately 6 years prior to excavation. This publication will attempt to address one of the major challenges facing post-mortem DNA extraction techniques; the limited yield of genetic recovery from aging bone, while maintaining an efficient timeline of recovery to prevent further degradation upon retrieval of the specimen. [2,3,4]

Table 2: Comparison of extracted DNA contents and the PCR products of nuclear gene GH1 and mitochondrial gene mtCOX1 in a 25 μL solution.