A strategy for rapid cDNA cloning from double-stranded RNA templates isolated from plants infected with RNA viruses by using Taq DNA polymerase

Abstract

A fast and efficient cDNA cloning procedure for plant RNA viruses was developed. In this procedure, double-stranded RNA (dsRNA) was used as a template source. Standard cDNA synthesis reagents and random hexamers were then used for making cDNAs. Taq DNA polymerase was used to add additional (A) at the ends of cDNAs, a TA cloning kit to ligate the cDNAs to vectors, and an electroporator to transform the DNAs to E. coli cells. dsRNAs were extracted from grapevine tissues infected with four different viruses and used for cloning. These viruses included grapevine rupestris stem pitting associated virus, grapevine leafroll associated virus 5, and two uncharacterized grapevine viruses, one each closely related to marafivirus and vitivirus groups. Selected cDNA clones were sequenced and PCR primers were developed for RT-PCR detection of these viruses in host plants.