Detection of mutation delta F508 in the cystic fibrosis gene using allele-specific PCR primers and time-resolved fluorometry.
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.
Iitiä
A
A
Wallac Oy, Turku, Finland.
Høgdall
E
E
Dahlen
P
P
Hurskainen
P
P
Vuust
J
J
Siitari
H
H
D000483:Alleles; D001483:Base Sequence; D001798:Blood Proteins / Q000235:genetics*; D001800:Blood Specimen Collection; D003550:Cystic Fibrosis / Q000097:blood / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA / Q000097:blood; D004252:DNA Mutational Analysis; D005470:Fluorometry / Q000379:methods*; D006579:Heterozygote; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017384:Sequence Deletion
D001798:Blood Proteins; C505032:CFTR protein, human; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA