Podell S, Maske W, Ibañez E, Jablonski E
Molecular Biosystems, Inc., San Diego, CA 92121.
Mol Cell Probes. 1991 Apr;5(2):117-24. doi: 10.1016/0890-8508(91)90005-5.
The hybridization efficiencies of oligonucleotide probes directly labelled with alkaline phosphatase and probes labelled with 32P were compared by quantitating the enzyme activity or radioactivity associated with hybridization targets over time. The targets tested included both synthetic oligonucleotides (53 bases in length) and single-stranded and double-stranded cloned M13 DNA (7350 bases long). Hybrid molecules were separated from unhybridized probes using size exclusion FPLC. This system allowed quantitative analysis of the time course and efficiency of hybridization for both probes and targets in complex hybridization media containing protein blocking agents, formamide, and carrier DNA. Similar maximum hybridization efficiencies were attained for probes labelled with either radioactivity or alkaline phosphatase as a marker. The reaction rate constant for oligonucleotide hybridization to long M13 targets was 3.6 x 10(5) mol-1 s-1 for a probe labelled with alkaline phosphatase, and 5.8 x 10(5) mol-1 s-1 for the same probe labelled with 32P.
通过对与杂交靶标相关的酶活性或放射性随时间进行定量，比较了直接用碱性磷酸酶标记的寡核苷酸探针和用32P标记的探针的杂交效率。测试的靶标包括合成寡核苷酸（长度为53个碱基）以及单链和双链克隆的M13 DNA（长度为7350个碱基）。使用尺寸排阻快速蛋白质液相色谱法从未杂交的探针中分离杂交分子。该系统允许对含有蛋白质封闭剂、甲酰胺和载体DNA的复杂杂交介质中探针和靶标的杂交时间进程和效率进行定量分析。以放射性或碱性磷酸酶作为标记的探针获得了相似的最大杂交效率。对于用碱性磷酸酶标记的探针，寡核苷酸与长M13靶标的杂交反应速率常数为3.6×10(5) mol-1 s-1，对于用32P标记的相同探针，该反应速率常数为5.8×10(5) mol-1 s-1。
