66] Mapping of gene transcripts by nuclease protection assays and cDNA primer extension

Abstract

An important problem often faced in the molecular characterization of genes is the precise mapping of those genomic sequences transcribed into RNA. This requires identification of the genomic site initiating gene transcription, the location of genomic sequences removed from the primary gene transcript during RNA processing, and knowledge of the sequences terminating the processed gene transcript. The objective of the protocols described here is the generation of transcription maps utilizing relatively uncharacterized gene fragments. The basic approach is hybridization of a single-stranded DNA probe with cellular RNA, followed by treatment with a single-strand-specific nuclease that does not attack DNA-RNA hybrids, in order to destroy any unreacted probe sequences. Thus the probe sequences included in the hybrid duplexes are protected from nuclease digestion. The sizes of the protected probe fragments determined by gel electrophoresis correspond to the lengths of the hybridized sequence elements. Methodology for nuclease mapping of RNA transcripts with genomic DNA probes was first introduced by Berk and Sharp. We have adapted this technology for the purpose of obtaining transcription maps of new gene isolates that include 20-30 kb of genomic DNA, using probes as large as 2.5 kb. The information provided here will also be useful for less demanding mapping experiments. The use of RNA rather than DNA probes to map gene transcripts is presented in this volume.