Nonisotopic detection of mutations using a modified single-strand conformation polymorphism analysis.
This report describes a rapid convenient screening system with improved sensitivity to detect mutations in the cystic fibrosis transmembrane regulator (CFTR) gene based on nonisotopic SSCP analysis. Because conventional SSCP analysis is often hampered by poor yield of single-stranded DNA, we applied the well-established solid-phase technique in which streptavidin-coated magnetic beads are used to immobilize biotinylated polymerase chain reaction (PCR) products. High yield of single-stranded DNA can be eluted from the solid phase by denaturation and used for SSCP analysis. An additional advantage of this procedure is that the immobilized single strand is available without any further purification steps for solid-phase sequencing.
Weidner
J
J
Institut für Strahlenbiologie, Westfälische Wilhelms-Universität, Münster, Germany.
Eigel
A
A
Horst
J
J
Köhnlein
W
W
D001483:Base Sequence; D003550:Cystic Fibrosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
C505032:CFTR protein, human; D017931:DNA Primers; D004277:DNA, Single-Stranded; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator