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Related Experiment Videos

Allelic discrimination by nick-translation PCR with fluorogenic probes

L G Lee1, C R Connell, W Bloch

  • 1Applied Biosystems, Division of Perkin-Elmer, Foster City, CA 94404.

Nucleic Acids Research
|August 11, 1993
PubMed
Summary
This summary is machine-generated.

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This study introduces a novel nick-translation PCR method using dual fluorogenic probes for precise cystic fibrosis (CF) gene mutation detection. The technique accurately identifies wild-type and delta F508 deletion mutations in DNA samples.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Cystic Fibrosis (CF) is a genetic disorder caused by mutations in the CFTR gene.
  • Accurate detection of CF-causing mutations, such as the common delta F508 deletion, is crucial for diagnosis and genetic counseling.
  • Existing methods may require complex procedures or lack high specificity for certain mutations.

Purpose of the Study:

  • To develop a sensitive and specific method for detecting the delta F508 mutation in the cystic fibrosis gene.
  • To utilize fluorogenic probes in a Polymerase Chain Reaction (PCR) assay for real-time mutation identification.
  • To differentiate between wild-type (wt) and mutated (mut) DNA sequences containing the F508 codon or the delta F508 deletion.

Main Methods:

  • Nick-translation PCR was employed using two distinct fluorogenic probes.

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  • One probe was designed for wild-type (wt) DNA (F508 codon), and the other for mutated (mut) DNA (delta F508 deletion).
  • Each probe featured a unique 5' fluorescent dye and a quencher, with the target site located between them. Probe degradation during PCR released fluorescence upon target sequence complementarity.

    • Main Results:

    • Both probes generated fluorescence only when perfectly complementary to the target DNA sequence between the indicator and quencher dyes.
    • The method successfully distinguished between wt DNA, mut DNA, and heterozygous DNA samples.
    • Post-PCR fluorescence emission spectra allowed for the definitive identification of the target DNA sequence.

    Conclusions:

    • This nick-translation PCR assay with dual fluorogenic probes provides a reliable method for detecting the delta F508 cystic fibrosis mutation.
    • The technique offers high specificity and allows for direct identification of target DNA sequences based on fluorescence.
    • This approach has potential applications in genetic diagnostics and research for cystic fibrosis.