Synthesis of long viral complementary DNA from 7.5 Kb poly A+ RNA templates.

The poly A+ RNA of the WW and GDVII virus isolates, belonging to the Theiler's murine encephalomyelitis virus group, were used as templates for cDNA synthesis. Since several secondary structures were present along these viral RNAs the reverse transcriptase was prematurely displaced from the RNA templates and only short cDNA molecules could be synthesized. Therefore a reliable and reproducible procedure for the synthesis of long cDNA transcripts, that can be directly used for cloning into respective plasmid or phage vectors, was developed. The precise conditions and kinetics of the several enzymatic reactions were studied. The use of methylmercury hydroxide for first strand synthesis, a correct choice of Klenow polymerase for second strand synthesis and the use of vertical gel electrophoresis in combination with zone centrifugation for removal of the excess linkers were found to be of paramount importance for the synthesis of long, up to intact, 7.5 Kb cDNA transcripts.