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Biosensor technology for real‐time detection of the cystic fibrosis W1282X mutation in CFTR
Author(s) -
Feriotto Giordana,
Ferlini Alessandra,
Ravani Anna,
Calzolari Elisa,
Mischiati Carlo,
Bianchi Nicoletta,
Gambari Roberto
Publication year - 2001
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.1151
Subject(s) - biotinylation , oligonucleotide , microbiology and biotechnology , cystic fibrosis transmembrane conductance regulator , cystic fibrosis , biology , mutation , dna , gene , biochemistry , genetics
In the present paper, biospecific interaction analysis (BIA) was performed using surface plasmon resonance (SPR) and biosensor technologies to detect the Trp1282Ter mutation (W1282X) of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene. We first immobilized on a SA5 sensor chip a single‐stranded biotinylated oligonucleotide containing the sequence involved in this mutation, and the efficiency of hybridization of oligonucleotide probes differing in length was determined. Second, we immobilized on different SA5 sensor chips biotinylated polymerase‐chain reaction (PCR) products from a normal subject as well as from heterozygous and homozygous W1282X samples. The results obtained show that both allele‐specific 10‐ and 12‐mer oligonucleotides are suitable probes to detect W1282X mutations of the cystic fibrosis gene under standard BIA experimental conditions. During the association phase performed at 25°C, discrimination between mismatched and full matched hybrids was readily and reproducibly observed by using the 10‐mer W1282X probes. By contrast, when the 12‐mer DNA probes were employed, discrimination between mismatched and full matched hybrids was observed during the dissociation phase. Taken together, the results presented suggest that BIA is an easy, speedy, and automatable approach to detect point mutations leading to cystic fibrosis. By this procedure, it is possible to perform real‐time monitoring of hybridization between target single stranded PCR products obtained by using as substrates DNA isolated from normal or heterozygous subjects, and homozygous W1282X CF samples and oligonucleotide probes, therefore enabling a one‐step, non‐radioactive protocol to perform diagnosis. Hum Mutat 18:70–81, 2001. © 2001 Wiley‐Liss, Inc.