Highly sensitive detection of DNA using enzyme-linked DNA-probe. 1. Colorimetric and fluorometric detection.
Highly sensitive detection of DNA using enzyme-linked DNA-probe. 1. Colorimetric and fluorometric detection.
复制标题
使用酶联 DNA 探针对 DNA 进行高灵敏度检测。
作者：
A. Murakami;J. Tada;K. Yamagata;J. Takano
为了开发非放射性寡核苷酸衍生物，并检验其作为诊断工具（即dna探针）的实用性，合成了一种酶联寡核苷酸。与M13mp8噬菌体DNA互补的寡核苷酸通过交联剂和间隔剂与碱性磷酸酶连接。将固定在硝化纤维素膜上的M13mp8噬菌体DNA（单链）与酶联寡核苷酸杂交。采用三种检测方法对杂种进行检测；(1)溶液比色法，(2)膜比色法，(3)溶液荧光法。方法(2)和方法(3)对低至几到几十个原子分子的DNA具有很高的灵敏度，从自动化的角度来看，这些酶联寡核苷酸方法对DNA探针方法是有效的。
In order to develop non-radioactive oligonucleotide derivatives and to examine their utility as a diagnostic tool, namely as DNA-probe, an enzyme-linked oligonucleotide was synthesized. Oligonucleotide complementary to M13mp8 phage DNA was linked to alkaline phosphatase via a crosslinker and a spacer. M13mp8 phage DNA (single strand) immobilized on the nitrocellulose membrane was hybridized with the enzyme-linked oligonucleotide. The hybrid was detected with three detection methods; (1)colorimetric detection in solution, (2)colorimetric one on membranes, and (3)fluorometric one in solution. Methods(2) and (3) gave high sensitivities to detect as low as several to several tens attomoles of DNA and it was found that those methods with enzyme-linked oligonucleotides are potent for DNA-probe methodology from the viewpoint of automation.