Previous View
APSnet Home
Phytopathology Home



Cloning of Four Plant Viruses from Small Quantities of Double-Stranded RNA. Wilhelm Jelkmann, Agriculture Canada Research Station, 6660 N.W. Marine Drive, Vancouver B.C. V6T 1X2, Canada.; Robert R. Martin(2), and Edgar Maiss(3). (2)Agriculture Canada Research Station, 6660 N.W. Marine Drive, Vancouver B.C. V6T 1X2, Canada; (3)Biologische Bundesanstalt, Messeweg 11/12, 3300 Braunschweig, West-Germany. Phytopathology 79:1250-1253. Accepted for publication 21 June 1989. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 1989. DOI: 10.1094/Phyto-79-1250.

The cloning of less than microgram amounts of dsRNA of four plant viruses, cucumber mosaic virus (CMV), plum pox virus (PPV), raspberry leaf spot virus (RLSV), and strawberry mild yellow-edge virus (SMYEV), by using three methods of denaturation was accomplished. PPV and RLSV clones were up to 1,600 bp in length while the largest insert of SMYEV was 3,000 bp. Denaturation methods used were: boiling for 5 min; incubation in 90% dimethyl sulfoxide (DMSO) at 65 C for 30 min; and incubation at room temperature in 20 mM methylmercuric hydroxide (MeHg) for 10 min. The first strand and second strand synthesis were basically that of Gubler and Hoffman with the omission of mung bean nuclease digestion. After second strand synthesis, the DNA was size-fractionated on a Sepharose CL-4B column, C-tailed, annealed to G-tailed pBR322/pUC9, and Escherichia coli DH1/DH5a cells transformed. No differences in length of cDNA and size of inserts was observed when heat- and DMSO-denaturation were used. However, use of MeHg yielded greater incorporation of 32P-dATP into first strand when compared to the boiling method of denaturation.