Search

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.

Relative fitness determination has become a standard tool in experimental virus evolution studies. In this type of studies, the tested strain is mixed with a reference strain, which differs in an easy-to-score and genetically stable marker, and allowed to compete for a limited common pool of resources during a given number of generations. In this report, a TaqMan real-time PCR methodology is proposed for quantifying the relative fitness of tobacco etch potyvirus strains (TEV) in in planta mixed infections with a reference TEV strain. Two different forward primers along with a common reverse one are used into separated reactions mixes from the same RNA preparation. The reference strain, named TEV-PC1, was genetically engineered to carry a neutral marker in a highly conserved region of the RNA polymerase NIb gene. This marker allows tracking the frequency of both competitors during competition experiments by real-time quantitative PCR using specific primers. Both the reproducibility and sensitivity of the method have been explored. Reproducibility was assessed by running multiple competition experiments for the same genotype. Sensitivity was assessed by comparing the results of competition experiments against TEV-PC1 of 24 single-nucleotide substitutions mutants.

A protocol is described for general application for cloning small circular RNAs which requires only minimal amounts of template (approximately 50 ng) of unknown sequence. Both cDNA strands are synthesized with a 26-mer primer whose six 3'-terminal positions are totally degenerate in two consecutive reactions catalyzed by reverse transcriptase and DNA polymerase, respectively. The cDNAs are then PCR-amplified, using a 20-mer primer with the non-degenerate sequence of the previous primer, cloned and sequenced. This information permits the synthesis of one or more pairs of specific and adjacent primers for obtaining full-length cDNA clones by a protocol which is also described.

Two PCR-based methods are described for obtaining clones of small circular RNAs of unknown sequence and for which only minute amounts are available. To avoid introducing any assumption about the RNA sequence, synthesis of the cDNAs is initiated with random primers. The cDNA population is then PCR-amplified using a primer whose sequence is present at both sides of the cDNAs, since they have been obtained with random hexamers and then a linker with the sequence of the PCR primer has been ligated to their termini, or because the cDNAs have been synthesized with an oligonucleotide that contains the sequence of the PCR primer at its 5' end and six randomized positions at its 3' end. The procedures need only approximately 50 ng of purified RNA template. The reasons for the emergence of cloning artifacts and precautions to avoid them are discussed.