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Ashulin, L., S. Tolkowsky Laboratory, A.R.O., The Volcani Center, Bet Dagan 50250, Israel
Mawassi, M., S. Tolkowsky Laboratory, A.R.O., The Volcani Center, Bet Dagan 50250, Israel
Bar-Joseph, M., S. Tolkowsky Laboratory, A.R.O., The Volcani Center, Bet Dagan 50250, Israel
We have devised a method to amplify complementary DNA (cDNA) from enzymatically polyadenylated citrus bent leaf viroid (CBLVd) RNA fragments. A 30 mer polylinked-DT15 oligodeoxynucleotide (P-dT) was used as a primer for cDNA synthesis and the resulting cDNAs were poly A tailed and TCR amplified using P-dT as a bidirectional primer. The PCR products were restricted enzymatically and cloned in pBluescript vector. Five out of 16 colonies analyzed were found to contain CBLVd specific cDNA inserts. Based on sequencing information derived from the central parts of three separate CBLVd inserts, two end-to-end 18 mer primers, oC and oS, with complementary and sense orientation, respectively, to the circular viroid RNA template were synthesized and used for reverse transcription and PCR amplification of full length cDNA molecules. The application of this method for rapid cloning and characterization of CBLVd RNA molecules is described.
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Procedure to amplify cDNA from viroid RNA templates using the polymerase chain reaction
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Ashulin, L., S. Tolkowsky Laboratory, A.R.O., The Volcani Center, Bet Dagan 50250, Israel
Mawassi, M., S. Tolkowsky Laboratory, A.R.O., The Volcani Center, Bet Dagan 50250, Israel
Bar-Joseph, M., S. Tolkowsky Laboratory, A.R.O., The Volcani Center, Bet Dagan 50250, Israel
Procedure to amplify cDNA from viroid RNA templates using the polymerase chain reaction
We have devised a method to amplify complementary DNA (cDNA) from enzymatically polyadenylated citrus bent leaf viroid (CBLVd) RNA fragments. A 30 mer polylinked-DT15 oligodeoxynucleotide (P-dT) was used as a primer for cDNA synthesis and the resulting cDNAs were poly A tailed and TCR amplified using P-dT as a bidirectional primer. The PCR products were restricted enzymatically and cloned in pBluescript vector. Five out of 16 colonies analyzed were found to contain CBLVd specific cDNA inserts. Based on sequencing information derived from the central parts of three separate CBLVd inserts, two end-to-end 18 mer primers, oC and oS, with complementary and sense orientation, respectively, to the circular viroid RNA template were synthesized and used for reverse transcription and PCR amplification of full length cDNA molecules. The application of this method for rapid cloning and characterization of CBLVd RNA molecules is described.
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