A coupled one-step reverse transcription PCR procedure for generation of full-length open reading frames

Abstract

A one-step (all reactants added simultaneously) reverse transcription and polymerase chain reaction (RT-PCR) procedure for amplification of full-length open reading frames (ORFs) of relatively rare transcripts was developed. It was applied for cloning rat luteinizing hormone/chorionic gonadotropin receptor cDNA isoforms larger than two kb. In the procedure developed, manual work is minimized, thus large numbers of samples can be handled, since after denaturation of template RNA and the primers and addition of other reagents, no further manual steps are needed. No inhibitory effect of avian myeloblastosis virus (AMV) reverse transcriptase (RT) on Thermus aquaticus (Taq) DNA Polymerase was found. This was because, under the conditions described, Taq DNA Polymerase effectively amplified picogram amounts of plasmid DNA or template reverse transcribed from nanograms of total ovarian RNA in the presence of AMV-RT. Even a large excess of AMV-RT did not inhibit Taq DNA Polymerase. Thus, our coupled one-step RT-PCR procedure amplifies fast and reproducibly full-length ORFs from nanogram amounts of total RNA.