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

脱钙是骨DNA提取中的一个重要过程，因为它使骨标本在蛋白酶K消化时可溶。这需要很长的时间，因此一些提高其速度的技术将带来高吞吐量的案例。在这项研究中，我们研究了微波（MW）对脱钙的加速作用。将牛密质骨成型为立方体，每边2.5 mm，在温度控制下（37℃、56℃和80℃），用0.5 M EDTA溶液脱钙，有或没有间歇MW照射。通过蛋白酶K消化，然后通过酚-氯仿提取从每个样品中提取DNA。采用显微X射线CT成像系统测量脱钙前和酶消化后骨块的体积，计算溶解度值，评价骨块的脱钙进程。还通过靶向牛线粒体DNA控制区的419 bp的竞争性PCR定量可扩增DNA的量，以评估通过每次处理获得的DNA的质量。在非MW辐照样品中，在37℃和56℃之间明显观察到温度依赖性加速。然而，在80℃下，这种效应显著降低。即使在处理24小时的样品中也没有观察到由于MW照射而导致的脱钙作用的明显加速。在56℃下，非MW样品获得最佳结果。可降解DNA的量与MW照射时间成比例地下降，表明MW照射会降解DNA对PCR的适用性。从迄今为止获得的结果，这表明，微波照射对骨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其他实验。

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.