In vitro Synthesis of High Specific Activity 32P-labeled RNA using Bacteriophage T7 RNA Polymerase, and use as Nucleic Acid Hybridization Probes.

Abstract

This study was undertaken to investigate the optimal conditions for in vitro transcription of RNA probes using bacteriophage T7 RNA polymerase. The reaction conditions were studies so that radiolabelled RNA probes of high specific activity could be synthesized in excess of 8 Kb in length. A series of recombinant plasmids were constructed by subcloning of different HPV types and a part of cytomegalovirus DNA into an appropriate site in a vector containing RNA polymerase promoter sequence. These plasmids were used to study the optimal conditions for in vitro transcription with regard to concentration of template nucleotides, 17 RNA polymerase, NaCl and source of labelled nucleotide. Effect of each of these parameters on the kinetics of RNA synthesis and the physical characteristics of the synthesized RNA was studies. It was shown that under optimal conditions RNA probes of high specific activity (1 - 9 x 10⁸dpm/μg) could be synthesized and successfully used in hybridization experiments. It was also shown that using RNA probe HPV DNA could be easily detected in clinical specimens immobilized on membrane filters. Excellent correlation was observed between the dot blots probed with RNA and Southern blots probed with standard DNA probes. RNA probes of high specific activity are flexible tools and good alternative to DNA probes for detection of various nucleic acids by hybridization.