Amplification and Cloning of Large cDNA Fragments of the Citrus tristeza virus Genome

Abstract

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.