Citrus tristeza virus (CTV) is probably the most destructive viral pathogen of citrus. It causes chronic losses to commercial citrus production in all citrus-growing areas. The complete sequences of at least 42 genomes of different CTV strains have been obtained using different technologies including sequencing of multiple overlapped RT-PCR-amplified fragments with sizes of less than 4 kb, or from small viral RNA (svRNA), through next-generation high-throughput sequencing (NGS) technologies. The large size of CTV genome (>19.2 kb) makes it impractical to obtain and amplify full-length cDNA in a single step. The strategy of ligation of multiple cDNA fragments to assemble a full-length cDNA clone involves several serial cloning steps and sometimes subcloning phases using enzymatic digestion with restriction nucleases and ligation reactions. In this protocol, we describe a strategy to clone the entire genome of CTV obtained from two RT-PCR amplified products. These 5'- and 3'-genomic halves, which were designed to be overlapped in 15 nt in their 3'- and 5'-ends, respectively, were used as templates for further overlapped PCR to amplify the entire ~20 kb CTV genome. The resultant full cDNA PCR product was then inserted into pCAMBIA-binary vector.
Keywords: E. coli transformation; Large PCR fragments; Ligation; Overlapped PCR; RNA extraction; cDNA synthesis.