Synthesis of full-length potyvirus cDNA copies suitable for the analysis of genome polymorphism.
New methods facilitating the synthesis and amplification of full-length cDNA copies of single-stranded viral RNA genomes have been developed. A method is described for the efficient purification of potyviral RNA and total RNA from infected plants and it is shown that they can serve as templates for the efficient synthesis of a full-length, 10 kb long, 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 PVY cDNA, synthesized using either RNA or virions, can be amplified successfully by PCR with high yields of full-length products. Such products are good substrates for the study by RFLP of the total genome polymorphism of virus isolates.
Chachulska
A M
AM
Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland.
Fakhfakh
H
H
Robaglia
C
C
Granier
F
F
Zagórski
W
W
Vilaine
F
F
D018076:DNA, Complementary / Q000138:chemical synthesis* / Q000378:metabolism; D016679:Genome, Viral; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D017800:Potyvirus / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D012367:RNA, Viral / Q000138:chemical synthesis; D011198:Solanum tuberosum / Q000821:virology
D018076:DNA, Complementary; D012367:RNA, Viral