Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification

The study of functional RNAs of various sizes and structures requires efficient methods for their synthesis and purification. Here, 23 group I intron variants ranging in length from 246 to 341 nucleotides—some containing exons—were subjected to a native purification technique previously applied only to shorter RNAs (<160 nucleotides). For the RNAs containing both exons, we adjusted the original purification protocol to allow for purification of radiolabeled molecules. The resulting RNAs were used in folding assays on native gel electrophoresis and in self-splicing assays. The intron-only RNAs were subjected to the regular native purification scheme, assayed for folding and employed in crystallization screens. All RNAs that contained a 3′ overhang of one nucleotide were efficiently cleaved off from the support and were at least 90% pure after the non-denaturing purification. A representative subset of these RNAs was shown to be folded and self-splicing after purification. Additionally, crystals were grown for a 286 nucleotide long variant of the Clostridium botulinum intron. These results demonstrate the suitability of the native affinity purification method for the preparation of group I introns. We hope these findings will stimulate a broader application of this strategy to the preparation of other large RNA molecules

Trichophyton species: use of volatile fingerprints for rapid identification and discrimination.

Background: Fungal infection of the skin is a common clinical problem, and laboratory confirmation of the diagnosis is important to ensure appropriate treatment. The identification of the species of fungus is also important, because different fungal species have different modes of transmission, and this may be of importance both in preventing re-infection or in avoidance of infection of others. Objective: This study examined the potential of using volatile production patterns for the detection and discrimination between four Trichophyton species (T. mentagrophytes, T. rubrum, T. verrucosum and T. violaceum) in vitro on solid media and in broth culture. Methods: Two different sensor array systems (conducting polymer and metal oxide sensors) were examined for comparing the qualitative volatile fingerprints produced by these species over periods of 24-120 hrs in the headspace. The relative sensitivity of detection of two of the species (T. mentagrophytes, T. rubrum) was determined for log1 to log7 inoculum levels over the same time period. Results: The conducting polymer based system was unable to differentiate between species based on volatile fingerprints over the experimental period. However, metal oxide-based sensor arrays were found to be able to differentiate between the four species within 96 hrs of growth using PCA analysis which accounted for approximately 93% of the data in PC1 and 2 based on the qualitative volatile production patterns. This differentiation was confirmed by the Cluster analysis of the data using Euclidean distance and Ward’s linkage. Studies of the sensitivity of detection showed that for T. mentagrophytes and T. rubrum it was possible to differentiate between log3, log5 and log7 inoculum levels within 96 hrs. Conclusions: This is the first detailed study of the use of qualitative volatile fingerprints for identification and discrimination of dermatophytes. This approach could have potential for rapid identification of patient samples reducing significantly the time to treatment

Identification of infectious agents in onychomycoses by PCR-terminal restriction fragment length polymorphism.

A fast and reliable assay for the identification of dermatophyte fungi and nondermatophyte fungi (NDF) in onychomycosis is essential, since NDF are especially difficult to cure using standard treatment. Diagnosis is usually based on both direct microscopic examination of nail scrapings and macroscopic and microscopic identification of the infectious fungus in culture assays. In the last decade, PCR assays have been developed for the direct detection of fungi in nail samples. In this study, we describe a PCR-terminal restriction fragment length polymorphism (TRFLP) assay to directly and routinely identify the infecting fungi in nails. Fungal DNA was easily extracted using a commercial kit after dissolving nail fragments in an Na(2)S solution. Trichophyton spp., as well as 12 NDF, could be unambiguously identified by the specific restriction fragment size of 5'-end-labeled amplified 28S DNA. This assay enables the distinction of different fungal infectious agents and their identification in mixed infections. Infectious agents could be identified in 74% (162/219) of cases in which the culture results were negative. The PCR-TRFLP assay described here is simple and reliable. Furthermore, it has the possibility to be automated and thus routinely applied to the rapid diagnosis of a large number of clinical specimens in dermatology laboratories