Sasaki Naoki, Kase Chikako, Sato Kae
Department of Chemistry College of Science, Rikkyo University, 3-34-1 Nishi-Ikebukuro, Toshima, Tokyo, 171-8501, Japan.
Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, 2-8-1 Mejirodai, Bunkyo, Tokyo, 112-8681, Japan.
Anal Sci. 2021 May 10;37(5):727-732. doi: 10.2116/analsci.20SCP14. Epub 2021 Jan 22.
Bead-based padlock rolling circle amplification under molecular crowding conditions, which we have developed for ultrasensitive detection of DNA, is examined to improve the detection efficiency and sensitivity of the method as well as to gain insight into the mechanism of the method. Both non-magnetic and magnetic sepharose microbeads were employed. Biotinylated DNA had to be pre-immobilized onto the microbeads in order to obtain products on the magnetic beads. The optimal concentration of biotinylated DNA was found to be about 5 μM, above which the number of products decreased. The effect of the crowder charge was examined, and neutral polymers were found to be effective on ligation and the hybridization step, while charged polymers were only effective on the hybridization step and inhibited the ligation and primer extension. The effect of the molecular weight of neutral dextran on the number of products was investigated, and the number of products was found to be increased with an increase in the molecular weight of dextran.
我们开发的用于DNA超灵敏检测的基于珠子的锁式滚环扩增技术，在分子拥挤条件下进行了研究，以提高该方法的检测效率和灵敏度，并深入了解其机制。使用了非磁性和磁性琼脂糖微珠。为了在磁珠上获得产物，生物素化的DNA必须预先固定在微珠上。发现生物素化DNA的最佳浓度约为5μM，高于此浓度产物数量会减少。研究了拥挤剂电荷的影响，发现中性聚合物对连接和杂交步骤有效，而带电聚合物仅对杂交步骤有效，并抑制连接和引物延伸。研究了中性葡聚糖分子量对产物数量的影响，发现产物数量随葡聚糖分子量的增加而增加。
