Chapter 22. In Vitro Synthesis of Single-Stranded DNA Complementary to Histone Messenger RNAs

Abstract

Publisher Summary This chapter describes a procedure for the synthesis of a single-stranded DNA complementary to histone messenger RNAs. The histone mRNAs when isolated from the polyribosomes of S -phase HeLa cells lack poly(A) at their 3’-OH termini. However, adenosine monophosphate (AMP) residues can be added to these messages, and as such they become effective templates for transcription by RNA-dependent DNA polymerase. Characterization and specificity of the histone complementary DNA (cDNA) probe are considered in this chapter. The discovery and demonstration of RNA-dependent DNA polymerase (reverse transcriptase) have provided a very useful approach for the study of specific messenger RNAs. The enzyme makes possible the synthesis of high-resolution probes, cDNA molecules for the detection and quantitation of specific RNA sequences by nucleic acid hybridization techniques. As RNA-dependent DNA polymerase requires a primer, the presence of 150–200 AMP residues at the 3'-OH termini of most eukaryotic mRNAs has facilitated transcription by the enzyme. Exogenous oligo(dT) readily anneals to these terminal poly(A) sequences, thus providing an effective primer for transcription of cDNA.