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

脱钙是骨DNA提取中必不可少的过程，因为它使骨标本在蛋白酶K消化中溶解。这需要很长时间，因此一些技术可以提高其速度，从而提高案件的吞吐量。在本研究中，我们考察了微波（MW）对脱钙的加速作用。分别在37℃、56℃和80℃的温度控制下，用0.5 M EDTA溶液进行脱钙处理，使牛致密骨呈立方状，每边2.5 mm。每个样品的DNA通过蛋白酶K酶切提取，然后用苯酚-氯仿萃取。采用微x线CT成像系统测定骨块脱钙前和酶解后的体积，计算溶解度评价骨块脱钙的进展。以牛线粒体DNA控制区419 bp为靶点，采用竞争性PCR法测定可扩增DNA的数量，以评价各处理获得的DNA质量。在非毫瓦辐照样品中，在37℃~ 56℃之间明显观察到温度相关的加速。但在80℃时，这一效果显著降低。即使在处理了24小时的标本中，也没有观察到由于毫瓦辐射而导致脱钙的明显加速。在温度为56℃时，无mw的样品效果最好。可扩增的DNA数量随辐照时间的增加呈比例下降，表明辐照会降低DNA的PCR适宜性。从目前得到的结果来看，MW辐照对骨DNA提取的积极影响不大。目前正在进行对微波功率和反应溶液温度进行严格控制的附加实验。

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.