Technical note: Bone DNA extraction and purification using silica‐coated paramagnetic beads

Abstract

The goal of this study was to develop a simple method to improve DNA recovery from challenging bone samples. To this end, an optimized procedure was developed that combined the demineralization and DNA extraction into a single step, followed by DNA purification using an automated silica-coated paramagnetic bead procedure. This method replaced a previous silica-membrane-based procedure, which was able to recover sufficient DNA to obtain full autosomal and Y chromosome STR profiles from greater than 90% of the samples, including samples greater than 20 years old. The development process began with the evaluation of buffer and demineralization systems to determine the best reagent combination. During the developmental process, we observed that the addition of EDTA and DTT affected silica-based DNA purification methods by raising the pH of the digest buffer. The protocols with buffer ATL, PK, EDTA, and DTT followed by lowering the pH with sodium acetate just before purification resulted in the best yields. The method reduced the extraction volume from 10 to 1.5 ml and used commercially available reagents already being utilized in forensic DNA casework. Because of the simplicity and small volume needed for the procedure, many steps where contamination could be introduced have been eliminated or minimized. This study demonstrated a new method of recovering DNA from bone samples capable of extracting trace quantities of DNA, removing potential inhibitors, and minimizing the potential for exogenous DNA contamination.

A Concentrated Hydrochloric Acid‐based Method for Complete Recovery of DNA from Bone

“…Approximately 100 mg of bone shavings was incubated overnight at 56°C, with rotation, in 300 μL of 0.5 M EDTA, with 200 μg of proteinase K. 30 μL of 3.0 M sodium acetate, pH 5.2 was then added to the mix , and the mix was extracted with phenol:chloroform:isoamyl alcohol and chloroform and then purified by passage through a Qiagen DNeasy ® Blood and Tissue Kit silica column in accordance with the manufacturers’ instructions.…”

Nucleic Acid Sample Preparation from Teeth/Dental Remains

“…DNA binding in this process is highly specifi c and the use of guanidinium isothiocyanate (GuSCN) seems to prevent the co-extraction of potentially inhibitory compounds present in the samples [ 32 ]. Binding of DNA to silica is strongly pH dependant; pH above 7.5 can drastically reduce DNA recovery [ 33 ] so the use of compounds such as EDTA, SDS, and DTT that affect the pH needs to be done in a manner to minimize the detrimental effects. Although organic methods perform well and are less expensive, silica methods use less hazardous materials and are more amenable to automation [ 34 ].…”

Microwave-assisted digestion combined with silica-based spin column for DNA isolation from human bones

“…Additionally, silica based DNA binding procedures are known to be effective in removing PCR inhibitors such as humic acid, iron, cobalt, tannins and so forth [1,6,27,[34][35][36].…”

“…Therefore, chelating methods are routine choices in many laboratories because of the fact that DNA can be successfully isolated of from bones without co-purification of PCR inhibitors [1,[34][35]. Also silica offers many advantages such as low toxicity, biocompatibility, low cost, quick and easy, scalable, efficient removal of PCR inhibitors [27,36].…”