Long distance RT-PCRS of human coronavirus 229E RNA

Abstract

The generation and cloning of cDNA fragments longer than 10 kb is often a difficult and time consuming task. In this study, we have analysed the conditions necessary to produce reverse transcripts longer than 10 kb that can be amplified by polymerase chain reaction. Thus, we isolated poly(A)-RNA from human coronavirus 229E infected MRC-5 cells and did reverse transcription using a sequence-specific primer. Subsequently, we amplified PCR products of varying length upstream of the primer position. Optimisation of the poly(A)-RNA preparation, the reverse transcription protocol and the polymerase chain reaction cycle conditions enabled us to successfully amplify regions of the human coronavirus 229E genome between 11.5 and 20.3 kb in length.