One-step site-directed mutagenesis of viroid dimeric cDNA.

The study of viroids (plant pathogens constituted by small non-coding RNA) has greatly benefited from the use of site-directed mutagenesis tools. However, compared to viral systems, this technique is complicated by the fact that, usually, infectious cDNAs carry two copies of the viroid genome. A simple method for a one-step site-directed mutagenesis of viroids is described and tested by estimating the rate of mutation incorporation of three random nucleotide substitutions in each Chrysanthemum stunt viroid (CSVd) and Chrysanthemum chlorotic mottle viroid (CChMVd). The protocol is essentially based on the original QuickChange Stratagene methodology; dimeric cDNA templates are amplified directly using Pfu DNA polymerase and self-complementary mutagenic primers. The reaction typically yields dimeric, but also monomeric clones which can be easily distinguished by electrophoretic analysis. The data show that approximately 50% of the dimeric clones carry the desired mutation in both viroid copies. Since the proposed protocol is simple technically and rapid compared to previous methods, it could be applied routinely for site-directed mutagenesis studies in viroids.