Synthesis of Double-Stranded Complementary DNA from Poly(A)⁺mRNA

Abstract

The use of avian myeloblastosis virus reverse transcriptase (AMV RTase) to produce DNA copies of mRNA templates is a common and well-documented method (1–3). Briefly, the method involves synthesis of a complementary DNA strand to the mRNA from a short double-stranded region, usually provided by using an oligo(dT) primer on poly(A)⁺RNA. The enzyme does not always produce full length transcripts, but all the complementary strands are finished off with a short hairpin loop. This provides a ready-made primer for second strand synthesis, useful whether this is to be performed by more reverse transcriptase or by E. coli DNA polymerase 1 (pol 1). An idealized picture is shown in Fig. 1. Before the double-stranded cDNA (ds cDNA) copy can be cloned it is necessary to remove this hairpin loop using the single-strand specific nuclease S1.