SYNTHESIS OF FULL-LENGTH POTYVIRUS CDNA COPIES SUITABLE FOR THE ANALYSIS OF GENOME POLYMORPHISM

New methods facilitating the synthesis and amplification of full-lengt h cDNA copies of single-stranded viral RNA genomes have been developed . A method is described for the efficient purification of potyviral RN A and total RNA from infected plants and it is shown that they can ser ve as templates for the efficient synthesis of a full-length, 10 kb lo ng, genomic cDNA. Two different reverse transcriptases were used (AMV- RT and MMLV-RT); only the first reverse transcriptase produced a good quality, full-length cDNA using viral RNA as a template. Surprisingly, MMLV-RT allowed for the full-length cDNA synthesis on virions rather than viral RNA. The PW cDNA, synthesized using either RNA or virions, can be amplified successfully by PCR with high yields of full-length p roducts. Such products are good substrates for the study by RFLP of th e total genome polymorphism of virus isolates. (C) 1997 Elsevier Scien ce B.V.