Articles
COMPLEMENTARY DNA PROBES GENERATED FROM DOUBLE-STRANDED RNAS OF A RUBUS VIRUS PROVIDE THE POTENTIAL FOR RAPID IN VITRO DETECTION
Article number
236_13
Pages
103 – 110
Language
Abstract
DsRNA extractions of an aphid-borne virus from virus-infected red raspberry cultivars revealed three major dsRNA bands (1, 2 and 3) with sizes of about 3.7, 2.6 and 1.8 kilobase pairs, respectively.
To provide highly purified nucleic acid templates for cDNA synthesis, the dsRNA was reextracted from low-melting-temperature agarose gels.
DsRNA templates were denatured with methylmercuric hydroxide, first-strand cDNA synthesis initiated with random primers and second-strand synthesis carried out by standard methods.
The double-stranded DNA was size fractionated through a CL-4B Sepharose column and larger cDNA recovered by ethanol precipitation.
After C-tailing, the cDNA was annealed into G-tailed pUC9 and used to transform Escherichia coli DH5a.
Colony filter hybridization was used to select 180 clones that were then grown in overnight cultures and analysed by rapid plasmid isolation.
Inserts ranging from 300 to 1,600 base pairs were found.
Hybridizations of 32P-dATP oligo-labeled cDNA clones to Northern blots with dsRNA demonstrate that clones to all three dsRNAs were generated.
Sequence homologies between RNA-1 and RNA-2 were observed by Northern hybridization with clone p195. Dot blot hybridizations with 32P-labeled clones to total RNA from virus-infected plants indicate their usefulness for virus detection.
To provide highly purified nucleic acid templates for cDNA synthesis, the dsRNA was reextracted from low-melting-temperature agarose gels.
DsRNA templates were denatured with methylmercuric hydroxide, first-strand cDNA synthesis initiated with random primers and second-strand synthesis carried out by standard methods.
The double-stranded DNA was size fractionated through a CL-4B Sepharose column and larger cDNA recovered by ethanol precipitation.
After C-tailing, the cDNA was annealed into G-tailed pUC9 and used to transform Escherichia coli DH5a.
Colony filter hybridization was used to select 180 clones that were then grown in overnight cultures and analysed by rapid plasmid isolation.
Inserts ranging from 300 to 1,600 base pairs were found.
Hybridizations of 32P-dATP oligo-labeled cDNA clones to Northern blots with dsRNA demonstrate that clones to all three dsRNAs were generated.
Sequence homologies between RNA-1 and RNA-2 were observed by Northern hybridization with clone p195. Dot blot hybridizations with 32P-labeled clones to total RNA from virus-infected plants indicate their usefulness for virus detection.
Authors
W. Jelkmann, R.R. Martin
Keywords
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