Takenaka S, Uto Y, Kondo H, Ihara T, Takagi M
Department of Biochemical Engineering and Science, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology, Iizuka, Japan.
Anal Biochem. 1994 May 1;218(2):436-43. doi: 10.1006/abio.1994.1203.
Electrochemically active DNA probes were prepared by linking a ferrocene unit with 5'-aminohexyl-terminated oligonucleotides. The DNA sequences of probes 5a, 5b, and 5c were 5'-T12-3', 5'-T20-3', and 5'-TGCAG TTCCG GTGGC TGATC-3', respectively. Probe 5a could form a complex selectively with a single-strand poly(A) and a double-strand DNA fragment containing an A13 sequence and these complexes could be detected at femtomole levels by an electrochemical detector (ECD) on HPLC. The observed ECD response was proportional to the amount of the complex over the range 20-100 fmol. Probe 5c was capable of detecting femtomole levels of a restriction DNA fragment having oncogene v-myc. Moreover, probe 5b was able to detect picogram levels of mRNA taken from rat brain or yeast total cellular RNA. This proves that the electrochemically active DNA probes are useful in analyzing traces of DNA and RNA carrying the complementary sequence.
通过将二茂铁单元与5'-氨基己基末端的寡核苷酸相连，制备了具有电化学活性的DNA探针。探针5a、5b和5c的DNA序列分别为5'-T12-3'、5'-T20-3'和5'-TGCAG TTCCG GTGGC TGATC-3'。探针5a可以与单链聚（A）以及含有A13序列的双链DNA片段选择性地形成复合物，并且这些复合物可以通过HPLC上的电化学检测器（ECD）在飞摩尔水平进行检测。观察到的ECD响应在20-100飞摩尔范围内与复合物的量成正比。探针5c能够检测到具有癌基因v-myc的限制性DNA片段的飞摩尔水平。此外，探针5b能够检测从大鼠脑或酵母总细胞RNA中提取的皮克水平的mRNA。这证明了具有电化学活性的DNA探针可用于分析携带互补序列的痕量DNA和RNA。
