Yoshimura Takeo, Suzuki Takamasa, Mineki Shigeru, Ohuchi Shokichi
Department of Applied Biological Science, Tokyo University of Science, 264 Yamazaki, Noda, Chiba 305-8506, Japan.
Graduate School of Life Science and System Engineering, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka, Fukuoka 820-8502, Japan.
PLoS One. 2015 Sep 8;10(9):e0136532. doi: 10.1371/journal.pone.0136532. eCollection 2015.
Rolling circle amplification (RCA) generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same.
滚环扩增（RCA）可生成单链DNA或RNA，这种等温技术的多种应用涵盖了从核酸的灵敏检测到单核苷酸多态性分析等领域。微波化学被广泛应用于提高反应速率以及产物的产率和纯度。本研究的目的是将微波加热应用于RCA，并指出促成微波选择性加热效应的因素。使用针对酶促规模反应优化的微波施加器严格控制微波反应温度。在此，我们表明，与传统RCA相比，由四种耐热DNA聚合酶中的任何一种催化的微波辅助RCA反应加速了4倍以上。此外，各个缓冲液成分的温度受到微波加热的特定影响。我们得出结论，尽管整体反应温度相同，但由于微波对反应成分温度的差异加热机制，微波加热加速了DNA的等温RCA。
