[68] Transcription maps of polyoma virus-specific RNA: Analysis by two-dimensional nuclease S1 gel mapping

This chapter describes the analysis of transcription maps of polyoma virus-specific RNA by 2-D nuclease S1 gel mapping and the standard procedures used for the preparation of viral nucleic acids and hybridization probes.

Abstract

Publisher Summary This chapter describes the analysis of transcription maps of polyoma virus-specific RNA by 2-D nuclease S1 gel mapping. The chapter describes the methodology used to map polyoma virus transcripts on the physical map of the viral genome. The ultimate characterization of viral RNA is the determination of the complete nucleotide sequence of individual RNA species. A rapid procedure for the analysis of labeled viral RNA using an adaptation of the Southern transfer technique (mini-blot hybridization) has also been described. The hybrids formed between polyoma virus RNA and DNA are digested with single-strand-specific endonuclease S1 and the resulting duplexes or their component DNA strands are sized by gel electrophoresis. The 2-D variation of this basic procedure is a general diagonal assay for splicing within RNA molecules. The chapter describes the standard procedures used for the preparation of viral nucleic acids and hybridization probes.