POLYMERASE CHAIN-REACTION FINGERPRINTING IN FUNGI USING SINGLE PRIMERS SPECIFIC TO MINISATELLITES AND SIMPLE REPETITIVE DNA-SEQUENCES - STRAIN VARIATION IN CRYPTOCOCCUS-NEOFORMANS

Minisatellites and simple repetitive DNA sequence motifs are used as c onventional oligonucleotide probes in DNA-hybridization-based fingerpr inting. The same oligonucleotides can be used as single primers in the polymerase chain reaction (PCR) to generate individual PCR fingerprin ts. In this study, the simple repetitive sequences, (CA)(8), (CT)(8), (CAC)(5), (GTG)(5), (GACA)(4) and (GATA)(4), and a minisatellite core sequence derived from the wild-type phage M13 (5' GAGGGTGGCGGTTCT 3') were used as specific, single primers to amplify hypervariable repetit ive DNA sequences during PCR analysis. The potential applications of t his technique are demonstrated with clinical isolates of the human pat hogenic yeest, Cryptococcus neoformans. PCR fingerprint patterns have remained stable after long-term in vitro passage (>21/2 years to date) . Hybridization of the primers to blots of electrophoretically separat ed chromosomes demonstrated that the target sequences recognized by mo st of the primers are dispersed through the entire yeast genome. Seque nce analysis of the cloned bands obtained by PCR fingerprinting indica ted that if the same or extremely similar, inversely oriented tandem r epeats are located close to each other, when only one repeat-specific primer is used in the PCR, the region between these repeats is amplifi ed. PCR fingerprinting has a wide range of current and potential appli cations to fungi, such as clarifying taxonomic questions, facilitating epidemiological studies and improving the diagnosis of mycotic diseas es.