Simultaneous screening for 11 mutations in the cystic fibrosis transmembrane conductance regulator gene by multiplex amplification and reverse dot-blot.
An assay is described in which 11 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be screened simultaneously. Six different exons of the CFTR gene are amplified in a single multiplex amplification. Biotinylated dUTP is incorporated into the different fragments during the amplification process. A sample of this mixture is then hybridized to 21 different poly-dT tailed oligonucleotide probes which are bound to a nylon membrane. In order to screen the different mutations in a single step hybridization, the length of the different oligonucleotides and the amount used in the assay were optimized. The detection is performed by binding avidin-alkaline phosphatase to the biotin, followed by a chemiluminescent reaction. By means of this fast and sensitive assay, about 85% of all the cystic fibrosis mutations in the Belgian population can be detected.
Center for Human Genetics, University of Leuven, Belgium.
H
H
Cuppens
I
I
Buyse
M
M
Baens
P
P
Marynen
J J
JJ
Cassiman
D001483:Base Sequence; D003550:Cystic Fibrosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D015345:Oligonucleotide Probes / Q000235:genetics
C505032:CFTR protein, human; D008565:Membrane Proteins; D015345:Oligonucleotide Probes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator