Detection of mutation delta F508 in the cystic fibrosis gene using allele-specific PCR primers and time-resolved fluorometry

PCR Methods Appl. 1992 Nov;2(2):157-62. doi: 10.1101/gr.2.2.157.

Abstract

A method to detect the main cystic fibrosis (CF) mutation delta F508 from dried blood spots, whole blood, or saliva using the polymerase chain reaction (PCR) and time-resolved fluorometry (TRF) is described. Samples are treated by boiling in mild alkaline solution, after which two allele-specific PCR reactions are performed. Allele-specific primers and a common biotinylated primer are used in the amplification reactions. To detect the PCR product, an europium-labeled oligonucleotide, complementary to the biotinylated strand of the PCR product, is used in a solution hybridization. Hybridization is done in streptavidin-coated microtitration wells, making the detection easy to perform. After a washing step, the bound label is detected using a time-resolved fluorometer. To analyze function of the assay, 20 dried blood spot samples were tested. PCR amplification of the deletion region combined with gel retardation assay was used as a control method. In the initial testing, 2 samples giving discrepant results in the two assays were found. In addition, 17 samples from known CF patients together with 6 normal control samples were analyzed. Among these patient samples, 10 homozygotes and 6 carriers for mutation delta F508 were found.

MeSH terms

  • Alleles
  • Base Sequence
  • Blood Proteins / genetics*
  • Blood Specimen Collection
  • Cystic Fibrosis / blood
  • Cystic Fibrosis / genetics*
  • Cystic Fibrosis Transmembrane Conductance Regulator
  • DNA / blood
  • DNA Mutational Analysis*
  • Fluorometry / methods*
  • Heterozygote
  • Hot Temperature
  • Humans
  • Hydrogen-Ion Concentration
  • Molecular Sequence Data
  • Nucleic Acid Hybridization
  • Oligodeoxyribonucleotides
  • Oligonucleotide Probes
  • Polymerase Chain Reaction* / methods*
  • Sensitivity and Specificity
  • Sequence Deletion

Substances

  • Blood Proteins
  • CFTR protein, human
  • Oligodeoxyribonucleotides
  • Oligonucleotide Probes
  • Cystic Fibrosis Transmembrane Conductance Regulator
  • DNA