Hanami Takeshi, Oyama Rieko, Itoh Masayoshi, Yasunishi-Koyama Ayako, Hayashizaki Yoshihide
Omics Science Center, RIKEN Yokohama Institute, Tsurumi-ku, Yokohama, Japan.
Nucleosides Nucleotides Nucleic Acids. 2012;31(8):608-15. doi: 10.1080/15257770.2012.714516.
The incorporation of deoxynucleoside triphosphates (dNTPs) catalyzed by polymerases is conventionally examined using gel electrophoresis autoradiography. Here, we studied an alternative method, pyrosequencing, to verify the incorporation of dNTPs containing unnatural nucleotides by polymerases. We found that the pyrosequencing method more rapidly and easily confirmed the incorporation of dNTPs than the conventional method, especially in the presence of low-efficiency dNTP polymerases. Furthermore, the method can detect the pyrophosphorolysis reaction just before the position of the unnatural nucleic acid, and the efficiency of incorporation just after it.
传统上，通过凝胶电泳放射自显影法来检测聚合酶催化的脱氧核苷三磷酸（dNTP）的掺入情况。在此，我们研究了一种替代方法——焦磷酸测序法，以验证聚合酶对含非天然核苷酸的dNTP的掺入情况。我们发现，焦磷酸测序法比传统方法能更快速、轻松地确认dNTP的掺入，尤其是在存在低效dNTP聚合酶的情况下。此外，该方法能够在非天然核酸位置之前检测焦磷酸解反应，并在其之后检测掺入效率。
