Efficient reverse transcription of cowpea mosaic virus RNAs

Abstract

Conditions are described which give an efficient synthesis of DNA copies of cowpea mosaic virus (CPMV) RNAs, using avian myeloblastosis reverse transcriptase and oligo (dT) primers. Maximum incorporation of dAMP into cDNA is attained with 0.4 to 0.8 mM of each deoxynucleoside triphosphate, 12 mM Mg++ and 60 mM K+ ion concentrations. High enzyme concentrations (up to 100 units/ml) were used. Under these conditions over 1000 pmoles of dAMP were incorporated per reaction. The cDNA:RNA molar ratio approached 0.3 when 1 pmole CPMV RNA was used as template. The products were heterogeneous but large. Bottom component RNA (about 6000 nucleotides long) was copied into cDNA molecules ranging from about 1000 to 4000 nucleotides, and middle component RNA (about 4000 nucleotides long) was copied into cDNA mostly between 500 and 2000 nucleotides long, on average about 1500, which can be cleaved by restriction endonuclease Hae III into two fragments of 880 and 540 nucleotides.