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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.

Publication ,  Journal Article
Meyer, W; Mitchell, TG
Published in: Electrophoresis
September 1995

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 techniques are demonstrated with clinical isolates of the human pathogenic yeast, Cryptococcus neoformans. PCR fingerprint patterns have remained stable after long-term in vitro passage ( > 2 1/2 years to date). Hybridization of the primers to blots of electrophorectically 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.

Duke Scholars

Published In

Electrophoresis

DOI

ISSN

0173-0835

Publication Date

September 1995

Volume

16

Issue

9

Start / End Page

1648 / 1656

Location

Germany

Related Subject Headings

  • Time Factors
  • Species Specificity
  • Reproducibility of Results
  • Repetitive Sequences, Nucleic Acid
  • Polymerase Chain Reaction
  • Nucleic Acid Hybridization
  • Molecular Sequence Data
  • Microsatellite Repeats
  • Genome, Fungal
  • Genetic Variation
 
Journal cover image

Published In

Electrophoresis

DOI

ISSN

0173-0835

Publication Date

September 1995

Volume

16

Issue

9

Start / End Page

1648 / 1656

Location

Germany

Related Subject Headings

  • Time Factors
  • Species Specificity
  • Reproducibility of Results
  • Repetitive Sequences, Nucleic Acid
  • Polymerase Chain Reaction
  • Nucleic Acid Hybridization
  • Molecular Sequence Data
  • Microsatellite Repeats
  • Genome, Fungal
  • Genetic Variation