Detection of the deltaF508 (F508del) mutation of the cystic fibrosis gene by surface plasmon resonance and biosensor technology.
In the present paper, we applied surface plasmon resonance (SPR) and biosensor technologies for biospecific interaction analysis (BIA) to detect deltaF508 mutation (F508del) of the cystic fibrosis transmembrane regulator (CFTR) gene in both homozygous as well as heterozygous human subjects. The proposed method is divided into three major steps. The first step is the immobilization on a SA5 sensor chip of two biotinylated oligonucleotide probes (one normal, N-508, and the other mutant, deltaF508) that are able to hybridize to the CFTR gene region involved in F508del mutation. The second step consists of the molecular hybridization between the oligonucleotide probes immobilized on the sensor chips and (1) wild-type or mutant oligonucleotides, as well as (2) single-stranded DNA obtained by asymmetric polymerase chain reaction (PCR), performed using genomic DNA from normal individuals and from F508del heterozygous and F508del homozygous patients. The third, and most important, step consists of the evaluation of differential stabilities of DNA/DNA molecular complexes generated after hybridization of normal and deltaF508 probes immobilized on the sensor chips. The results obtained strongly suggest that the proposed procedure employing SPR technology enables a one-step, nonradioactive protocol for the molecular diagnosis of F508del mutation of the CFTR gene. This approach could be of interest in clinical genetics, as the hybridization step is oftenly required to detect microdeletions present within PCR products.
Feriotto
G
G
Department of Biochemistry and Molecular Biology, Ferrara University, Italy.
Lucci
M
M
Bianchi
N
N
Mischiati
C
C
Gambari
R
R
D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D003550:Cystic Fibrosis / Q000175:diagnosis* / Q000235:genetics*; D004252:DNA Mutational Analysis; D019075:Electrophoresis, Capillary; D006801:Humans; D008957:Models, Genetic; D009154:Mutation; D009841:Oligonucleotides; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*; D013997:Time Factors
D009841:Oligonucleotides