Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species

TLDR

Detailed restriction analyses of many samples often require substantial time and effort for DNA extraction, restriction digests, Southern blotting, and hybridization. The study presents a novel PCR‑based approach for rapid simplified restriction typing and mapping of DNA from diverse isolates. The method amplifies rDNA fragments by PCR, digests them with frequent‑cutting restriction enzymes, and analyzes the resulting fingerprints by electrophoresis to map restriction sites across progressively larger fragments sharing a common primer. The PCR approach efficiently amplified up to 2‑kb rDNA fragments from several pathogenic Cryptococcus species, revealed no restriction variation among 19 C. neoformans isolates for four enzymes, and identified distinct restriction patterns in other Cryptococcus spp., demonstrating genetic differentiation and broad applicability to other fungi.

Abstract

Detailed restriction analyses of many samples often require substantial amounts of time and effort for DNA extraction, restriction digests, Southern blotting, and hybridization. We describe a novel approach that uses the polymerase chain reaction (PCR) for rapid simplified restriction typing and mapping of DNA from many different isolates. DNA fragments up to 2 kilobase pairs in length were efficiently amplified from crude DNA samples of several pathogenic Cryptococcus species, including C. neoformans, C. albidus, C. laurentii, and C. uniguttulatus. Digestion and electrophoresis of the PCR products by using frequent-cutting restriction enzymes produced complex restriction phenotypes (fingerprints) that were often unique for each strain or species. We used the PCR to amplify and analyze restriction pattern variation within three major portions of the ribosomal DNA (rDNA) repeats from these fungi. Detailed mapping of many restriction sites within the rDNA locus was determined by fingerprint analysis of progressively larger PCR fragments sharing a common primer site at one end. As judged by PCR fingerprints, the rDNA of 19 C. neoformans isolates showed no variation for four restriction enzymes that we surveyed. Other Cryptococcus spp. showed varying levels of restriction pattern variation within their rDNAs and were shown to be genetically distinct from C. neoformans. The PCR primers used in this study have also been successfully applied for amplification of rDNAs from other pathogenic and nonpathogenic fungi, including Candida spp., and ought to have wide applicability for clinical detection and other studies.