Abstract
Molecular cloning, a crucial prerequisite for engineering plasmid constructs intended for functional genomic studies, relies on successful restriction and ligation processes. However, the lack of unique restriction sites often hinders construct preparation, necessitating multiple modifications. Moreover, achieving the successful ligation of large plasmid constructs is frequently challenging. To address these limitations, we present a novel PCR strategy in this study, termed 'long-fragment circular-efficient PCR' (LC-PCR). This technique involves one or two rounds of PCR with an additional third-long primer that complements both ends of the newly synthesized strand of a plasmid construct. This results in self-circularization with a nick-gap in each newly formed strand. The LC-PCR technique was successfully employed to insert a partial sequence (210 nucleotides) of the phytoene desaturase gene from Nicotiana benthamiana and a full capsid protein gene (770 nucleotides) of a begomovirus (tomato leaf curl New Delhi virus) into a 16.4 kb infectious construct of a tobamovirus, cucumber green mottle mosaic virus (CGMMV), cloned in pCambia. This was done to develop the virus-induced gene silencing vector (VIGS) and an expression vector for a foreign protein in plants, respectively. Furthermore, the LC-PCR could be applied for the deletion of a large region (replicase enzyme) and the substitution of a single amino acid in the CGMMV genome. Various in planta assays of these constructs validate their biological functionality, highlighting the utility of the LC-PCR technique in deciphering plant-virus functional genomics. The LC-PCR is not only suitable for modifying plant viral genomes but also applicable to a wide range of plant, animal, and human gene engineering under in-vitro conditions. Additionally, the LC-PCR technique provides an alternative to expensive kits, enabling quick introduction of modifications in any part of the nucleotide within a couple of days. Thus, the LC-PCR proves to be a suitable 'all in one' technique for modifying large plasmid constructs through site-directed gene insertion, deletion, and mutation, eliminating the need for restriction and ligation.