56] RNA polymerase nascent product analysis

Abstract

Publisher Summary This chapter discusses the RNA polymerase nascent product analysis. Synthesis of RNA with RNA polymerase is initiated with purine nucleoside triphosphates. Under a reduced NTP concentration, RNA synthesis can be initiated with oligonucleotides as primer at defined sites of single-stranded DNA. By applying this principle, the entire region of a DNA segment is able to be transcribed into RNA sequences from which the sequence of the template is deduced. This procedure is applied for the sequence determination of the lac operator and of fd promoters. As new methods for direct DNA sequencing were developed, the primed RNA synthesis method is used because of complexity of the sequencing procedure. This method may be effective for sequence analysis of DNA segments containing heterogeneous ends and no substrate sites of known restriction endonucleases. The new sequencing procedure that resolves partial nuclease digests of terminally labeled RNA by gel electrophoresis would be more useful because oligonucleotides with 5'-OH ends can be used as primer, and RNA chains primed with such oligonucleotides are selectively labeled with 32 P in the polynucleotide kinase reaction.