Application of the intermittent microwave irradiation to accelerate the decalcification of bone specimens in DNA extraction

2005 Fiscal Year Final Research Report Summary

• Project/Area Number: 16590553
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Legal medicine
• Research Institution: National Research Institute of Police Science
• Principal Investigator: IMAIZUMI Kazuhiko National Research Institute of Police Science, First forensic science division, Senior researcher, 法科学第一部, 主任研究官 (00356148)
• Project Period (FY): 2004 – 2005
• Keywords: forensic science / personal identification / skeletal remains / DNA extraction / microwave / decalcification / bone

Research Abstract

A decalcification is an essential process in bone DNA extraction since it makes the bone specimens soluble at the proteinase K digestion. This takes long periods of time so that some techniques improving its speed would bring a high throughput of the caseworks. In this study, we examined the accelerating effects of the microwave (MW) on the decalcification. The bovine compact bone shaped into a cubic, 2.5 mm each side was decalcified with 0.5 M EDTA solution with or without an intermittent MW irradiation under temperature control, 37℃, 56℃ and 80℃. The DNA was extracted from each sample by proteinase K digestion followed by phenol-chloroform extraction. The volume of the bone blocks before decalcification and after enzymatic digestion were measured by micro X-ray CT imaging system, then the progress of the decalcification was assessed from the value of solubility calculated. The amount of the amplifiable DNA was also quantified by the competitive PCR targeting 419 bp of bovine mitochondrial DNA control region to assess the quality of the DNA obtained by each treatment. In non-MW irradiation specimens, temperature-dependent acceleration was clearly observed between 37℃ and 56℃. However, this effect significantly decreased at 80℃. No clear acceleration of the decalcification due to the MW irradiation was observed even in the specimens treated for 24 hours. The best result was obtained from the sample with non-MW, at 56℃. The amplifiable DNA dropped in amount in proportion to the MW irradiation periods, indicating the MW irradiation would degrade the DNA with regard to the suitability for PCR. From the results obtained so far, it was suggested that MW irradiation has little positive effect on bone DNA extraction. The additional experiments with strict controls on the power of MW and on the temperature of reacting solution are now ongoing.