Custom polymerase-chain-reaction engineering of a plant expression vector

Abstract

Polymerase-chain-reaction (PCR) amplification combined with custom-synthesized oligodeoxyribonucleotide (oligo) primers can be used to make complex genetic engineering steps (e.g., translational fusions) easy. Much of the complexity of the engineering steps can be incorporated into the custom oligo primers. Using this technique, a plant constitutive expression vector, pUC18cpexp, was constructed. This vector is based on the cauliflower mosaic virus 35S gene-regulatory elements and the cucumber mosaic virus coat protein-encoding gene (cp) 5'-untranslated region. Use of this vector is demonstrated by modifying the cp genes of several plant viruses and cloning them into pUC18cpexp. Because the construction and use of this vector system require custom oligo primer synthesis and PCR amplification, the technique is referred to as custom PCR engineering.