Comparison of solution hybridization efficiencies using alkaline phosphatase-labelled and 32P-labelled...

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.