EFFECTIVE AMPLIFICATION OF 20-KB DNA BY REVERSE TRANSCRIPTION PCR

Polymerase chain reaction has been applied to the amplification of lon g DNA fragments from a variety of sources, including genomic, mitochon drial, and viral DNAs. However, polymerase chain reaction amplificatio n from cDNA templates produced by reverse transcription has generally been restricted to products of less than 10 kilobases. In this paper, we report a system to effectively amplify fragments up to 20 kilobases from human coronavirus 229E genomic RNA. We demonstrate that the inte grity of the RNA template and the prevention of false priming events d uring reverse transcription are the critical parameters to achieve the synthesis of long cDNAs. The optimization of the polymerase chain rea ction conditions enabled us to improve the specificity and yield of pr oduct but they were not definitive. Finally, we have shown that the sa me reverse transcription polymerase chain reaction technology can be u sed for the amplification of extended regions of the dystrophin mRNA, a cellular RNA of relatively low abundance. (C) 1997 Academic Press.