Hirata Kazuya, Ishii Daisuke, Kano Arihiro, Yamayoshi Asako, Akaike Toshihiro, Maruyama Atsushi
Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
Nucleic Acids Symp Ser (Oxf). 2005(49):223-4. doi: 10.1093/nass/49.1.223.
Previously we designed the partially double-stranded (PDS) probes that have protruding single-stranded portion for a single-base mismatch analysis. The single-stranded portion is engineered to sense existences of mismatches in the counterparts and to transduce it in strand exchange rates. Here we report the influence of probe length and operating conditions, such as temperature and buffer conditions, on the mismatch resolution using the PDS probes. Reliable detection of single-base mismatches was achieved even with a 45mer-long probe. By lowering operating temperature, the higher and faster discrimination of the mismatches was demonstrated. Addition of cationic comb-type copolymers (CCCs) in the buffer increased the reaction rate 3-4 orders without disordering the resolving power.
此前，我们设计了部分双链（PDS）探针，其具有突出的单链部分用于单碱基错配分析。单链部分经设计可感知对应链中错配的存在，并将其转化为链交换速率。在此，我们报告了探针长度以及温度和缓冲条件等操作条件对使用PDS探针进行错配分辨率的影响。即使使用45个碱基对长的探针也能实现对单碱基错配的可靠检测。通过降低操作温度，可实现对错配更高且更快的区分。在缓冲液中添加阳离子梳型共聚物（CCC）可使反应速率提高3 - 4个数量级，同时不影响分辨能力。
