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Polymerase chain reaction fingerprinting in fungi using single primers specific to minisatellites and simple repetitive DNA sequences: Strain variation in Cryptococcus neoformans
Author(s) -
Meyer Wieland,
Mitchell Thomas G.
Publication year - 1995
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.11501601273
Subject(s) - minisatellite , biology , dna profiling , polymerase chain reaction , primer (cosmetics) , genetics , in silico pcr , repeated sequence , primer dimer , inverse polymerase chain reaction , dna , microbiology and biotechnology , microsatellite , genome , multiplex polymerase chain reaction , gene , allele , chemistry , organic chemistry
Minisatellites and simple repetitive DNA sequence motifs are used as conventional oligonucleotide probes in DNA‐hybridization‐based fingerprinting. The same oligonucleotides can be used as single primers in the polymerase chain reaction (PCR) to generate individual PCR fingerprints. 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 repetitive DNA sequences during PCR analysis. The potential applications of this technique are demonstrated with clinical isolates of the human pathogenic yeest, Cryptococcus neoformans . PCR fingerprint patterns have remained stable after long‐term in vitro passage (>2½ years to date). Hybridization of the primers to blots of electrophoretically separated chromosomes demonstrated that the target sequences recognized by most of the primers are dispersed through the entire yeast genome. Sequence analysis of the cloned bands obtained by PCR fingerprinting indicated that if the same or extremely similar, inversely oriented tandem repeats are located close to each other, when only one repeat‐specific primer is used in the PCR, the region between these repeats is amplified. PCR fingerprinting has a wide range of current and potential applications to fungi, such as clarifying taxonomic questions, facilitating epidemiological studies and improving the diagnosis of mycotic diseases.