title: Mismatch cleavage detects base deletion in cystic fibrosis gene.
abstract: The delta F508 is the most common defect in the cystic fibrosis (CF) gene; it involves in a 3-base deletion in codon 508 and results in the loss of a phenylalanine residue at amino acid position 508. Our previous results have shown the mismatch enzyme cleavage at the mismatch of a DNA duplex in identifying a specific DNA sequence or a point mutation. The assay is simple and reliable. By manipulating the melting temperature (Tm) for the hybrids of the DNA targets and the deoxynucleotide probes, the mismatch cleavage assays are able to detect the most common defective CF gene, delta F508. The assays with a delta F508 and a normal wild-type probe can differentiate the three genotypes, i.e., delta F508/delta F508, delta F508/normal and normal/normal. Furthermore, the addition of ammonium acetate amplifier to the assay for recycling the target DNA can increase the sensitivity to a level that is sufficient to detect the mutated target in a few micrograms of genomic DNA without the aid of PCR amplification. The detection of the base deletion, the amplification of sensitivity and the differentiation among the genotypes of normal, carrier delta F508 and mutant delta F508 suggest the useful application of mismatch cleavage in genetic diagnosis at the DNA level.
authors.affiliation: Greenebaum Cancer Center, University of Maryland, Baltimore, USA.
authors.forename: I C
authors.initials: IC
authors.lastname: Hsu
authors.forename: W E
authors.initials: WE
authors.lastname: Highsmith
authors.forename: J
authors.initials: J
authors.lastname: Xu
authors.forename: D
authors.initials: D
authors.lastname: Kong
mesh_terms: D020137:Base Pair Mismatch / Q000235:genetics*; D003550:Cystic Fibrosis / Q000175:diagnosis* / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D045647:DNA Glycosylases; D004252:DNA Mutational Analysis / Q000379:methods*; D015342:DNA Probes; D004260:DNA Repair / Q000235:genetics; D005820:Genetic Testing; D005838:Genotype; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017384:Sequence Deletion / Q000235:genetics*; D013696:Temperature
chemical_list: C505032:CFTR protein, human; D015342:DNA Probes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase