FAQ

In the McRAPD approach, melting data (melting curves or profiles) of RAPD can be compared in an automated way using the software tool QAS developed by our group. Then, McRAPD data acquired with an isolate of an unknown pathogenic yeast can be compared with the results of previously performed assays in known yeast species. In case of detecting a close similarity of the unknown sample to a known melting profile, this can be easily matched and the unknown yeast identified. In few cases where no such unequivocal similarity can be established, subsequent gel electrophoresis and comparison of fingerprinting patterns has been shown to unequivocally and correctly identify the species in our study including 300 unrelated strains of 9 yeast species most frequently isolated from patients.

However, the same standard protocol for PCR amplification and melting analysis has to be used to achieve inter-laboratory reproducibility of the data, enabling to use our database of McRAPD data as references samples. Also, the format of input melting data has to comply with that available in our database. For easier processing, we provide typical melting curves/profiles obtained by averaging data obtained with at least 40 unrelated strains of each species included in our database.

What steps have to be done to accomplish the analysis?

• perform McRAPD amplification using our optimised protocol
• download Java (in case not available at your PC)
• obtain QAS self-installing executable from us
• download reference average data from our database
• compile McRAPD data acquired with your unknown sample with our reference data in one file (MS-DOS text format; the number of lines has to be identical in all columns)
• import this file into QAS and process it
• based on the csv file generated by QAS, you can visually inspect the relatedeness of your unknown melting curve to the reference curves by construnting a plot using any common spreadsheet and plotting software (e.g. Excel)
• you can further evaluate the similarity/distance between all of the processed curves automatically using the distance matrix generated by QAS - this is available under the name "infile" (no extension), which can be further processed by Phylip software. Then, a phylogram/dendrogram can be ploted using resulting tree data (for details see the section on our software tool)

All of the tools need for data processing steps are freely available. When a simplified and rapid DNA extraction procedure is used (e.g. Steffan et al., 1997), the whole McRAPD identification can be accomplished within 3 hours after picking a sample from a single colony. Most of the steps are automated and require minimum labor time.

We welcome any cooperation to develop the technique further. We are particularly grateful for any gifts of strains of rare yeast species to further enrich the database of reference curves (list of species wanted). All of those, who request a copy of our software tool will be informed about major enrichments of the database and other improvements. We also aim to develop the QAS software tool further. Next steps should be:

• enable the import of HRMA data from RotorGene 6000
• implement the option of UPGMA analysis of differences into the tool
• include the option of automated comparison to reference data into the software