ABCMdb

PMID: 11438995

Feriotto G, Ferlini A, Ravani A, Calzolari E, Mischiati C, Bianchi N, Gambari R
Biosensor technology for real-time detection of the cystic fibrosis W1282X mutation in CFTR.
Hum Mutat. 2001;18(1):70-81., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Trp1282* ABCC7 p.Trp1282* HUMAN MUTATION 18:7081 (2001) (c) 2001 WILEY-LISS, INC. METHODS Biosensor Technology for Real-Time Detection of the Cystic Fibrosis W1282X Mutation in CFTR Giordana Feriotto,1 Alessandra Ferlini,2 Anna Ravani,2 Elisa Calzolari,2 Carlo Mischiati,3 Nicoletta Bianchi,3 and Roberto Gambari1,3* 1 Biotechnology Center, Ferrara University, Ferrara, Italy 2 Department of Experimental and Diagnostic Medicine, Ferrara University, Ferrara, Italy 3 Department of Biochemistry and Molecular Biology, Ferrara University, Ferrara, Italy Communicated by Richard G.H. Cotton 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. Login to comment 2 ABCC7 p.Trp1282* 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. Login to comment 3 ABCC7 p.Trp1282* 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. Login to comment 4 ABCC7 p.Trp1282* 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. Login to comment 7 ABCC7 p.Trp1282* 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. Login to comment 11 ABCC7 p.Trp1282* The W1282X mutation (MIM# 602421.0022), located in exon 20, was first observed in a French patient with cystic fibrosis by Vidaud et al. [1990]. Login to comment 16 ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Trp1282* Shoshani et al. [1992] cited evidence that the W1282X mutation is the most common CF mutation in the Ashkenazi Jewish population, where it ispresenton50-60%ofCFchromosomes.Patients homozygous for the W1282X mutation and patients heterozygous for the F508del and W1282X mutations had similarly severe disease reflected by pancreatic insufficiency, high incidence of meconium ileum, poor nutritional status, and variable pulmonary function and complications [Shoshani et al., 1992]. Login to comment 23 ABCC7 p.Trp1282* In the present paper, we first immobilized on a SA5 sensor chip a single stranded biotinylated oligonucleotide containing the sequence involved in the tryptophan 1282 -> TER mutation (W1282X) of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (Online Mendelian Inheritance in Man, OMIM# 602421, according to Antonarakis and Nomenclature Working Group [1998]). Login to comment 25 ABCC7 p.Trp1282* Second, we employed the most efficient oligonucletide probes to determine the presence of the W1282X mutations within PCR products from normal subjects, as well as from heterozygous and homozygous samples. Login to comment 26 ABCC7 p.Trp1282* MATERIALS AND METHODS Synthetic Oligonucleotides The CFTR W1282X region was amplified with the primers CF1, 5'-TGA GAC TAC TGA ACA CTGAA-3', CF2, 5'-GCT CAC CTG TGG TAT CAC T-3', and 5'-end biotinylated CF3 oligonucleotide, 5'- AAG GAG AAA TCC AGA TCG A-3'. Login to comment 27 ABCC7 p.Trp1282* The target oligonucleotide (M- CFt-21) and the normal (N-CFp) and the mutated (M-CFp) W1282X oligonucleotide probes (see Fig. 1 for nucleotide sequences) were purchased from Pharmacia (Uppsala, Sweden) and purified by HPLC. Login to comment 28 ABCC7 p.Trp1282* Polymerase-Chain Reaction In each PCR reaction, 100 ng of human genomic DNA was amplified by Taq DNA polymerase using the CF1 and CF2 primers, amplifying the W1282X region of the CFTR gene. Login to comment 32 ABCC7 p.Trp1282* Map of the CFTR gene region involved in the W1282X mutation causing CF and location of the CF1, CF3 (forward), and CF2 (reverse) PCR primers. Login to comment 34 ABCC7 p.Trp1282* The DNA probes (N-CFp and M-CFp) recognizing normal and mutated W1282X sequences are also shown. Login to comment 52 ABCC7 p.Trp1282* Computer-Assisted Prediction of Secondary Structure of Single Stranded PCR Products Secondary structures of single stranded CFTR PCR products carrying both normal and mutated W1282X sequences were determined using the MFOLD software (version 3.0) developed by Zuker et al. [1999] and Mathews et al. [1999]. Login to comment 69 ABCC7 p.Trp1282* Taken together, these results conclusively demonstrate that, with our BIA experimental conditions, the 17-mer DNA probes are not suitable for identification of the W1282X mutation. Login to comment 71 ABCC7 p.Trp1282* However, the use of 9-10 mers DNA probes allows identification of the W1282X mutation during the association phase; whereas contrary, longer DNA probes (12-13 mers) allow identification of the mutation during the dissociation phase. Login to comment 72 ABCC7 p.Trp1282* ABCC7 p.Trp1282* Immobilization on a SA5 Sensor Chip of Target CF PCR Products From a Normal Subject or Heterozygous and Homozygous W1282X CF Samples Figure 1 shows the location of the CF1, CF2, and CF3 PCR primers within the exon 20 portion of the CFTR gene carrying the W1282X mutation [Riordan et al., 1989]. Login to comment 89 ABCC7 p.Trp1282* ABCC7 p.Trp1282* Increase of Resonance Units (RU) Following Injections of CFp Probes to SA5-Sensor Chips Carrying Target M-CFt-21 Mer Target: M-CFt-21 mer N-W1282X M-W1282X CFp probe RUfin-RUi RUres-RUi RUfin-RUi RUres-RUi 9-mer 2 5 145 13 10B-mer 7 12 179 10 10A-mer 9 13 218 32 11-mer 92 21 261 144 12-mer 197 14 299 242 13-mer 246 42 342 322 17-mer 556 509 566 550 (Dynabeads, DYNAL, Oslo, Norway). Login to comment 91 ABCC7 p.Trp1282* In both cases, injections of appropriate single-stranded PCR products to sensor chips carrying immobilized N-CFp and M-CFp probes caused very low increase of RU; in addition non-satisfactory discrimination between normal and homozygous W1282X samples was observed (data not shown). Login to comment 93 ABCC7 p.Trp1282* ABCC7 p.Trp1282* This is expected to be the case in the CF W1282X PCR system, as suggested by the analysis shown in Figure 4, showing the theorical secondary structures of the single-stranded CFTR PCR products carrying normal (Fig. 4A) or mutated (Fig. 4B) W1282X sequences. Login to comment 96 ABCC7 p.Trp1282* The ∆G° was found to be -7.9 kcal/mole and -8.6 kcal/mole for normal and W1282X mutant CF PCR products, respectively. Login to comment 97 ABCC7 p.Trp1282* Tm was 43.6 and 44.3 for normal and W1282X mutant CF PCR products, respectively. Login to comment 98 ABCC7 p.Trp1282* Accordingly to these preliminary experiments, we decided 1) to immobilize on the sensor chips the W1282X PCR products from normal subjects, as well as from heterozygous and homozygous samples and 2) to inject the N-CFp and M-CFp DNA probes. Login to comment 101 ABCC7 p.Trp1282* The final W1282X PCR products were in any case further purified with Microcon-30. Login to comment 102 ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Trp1282* Direct sequencing of the PCR products confirmed the presence of 1) normal CF sequence (Fig. 5A), 2) mixed normal and mutated W1282X sequences (Fig. 5C), and 3) mutated W1282X sequences (Fig. 5B) in PCR products obtained by using as target DNA isolated from normal subject or heterozygous and homozygous W1282X CF samples. Login to comment 106 ABCC7 p.Trp1282* Hybridization of DNA Probes to Target CF PCR Products A representative example of the binding of N-CFp and M-CFp 12-mer DNA probes to CF PCR products from normal, heterozygous, and homozygous CF W1282X samples is shown in Figure 5D-G. Login to comment 110 ABCC7 p.Trp1282* By contrast, the complexes generated after the binding of the same probe to PCR products from a homozygous W1282X sample were found to be unstable (Fig. 5G, dotted line). Login to comment 111 ABCC7 p.Trp1282* Intermediate stability (50% decrease of RUres-RUi) was found for complexes generated after binding of the N- CFp12-mer DNA probes to PCR products from heterozygous W1282X samples (Fig. 5F, dotted line). Login to comment 114 ABCC7 p.Trp1282* By contrast, the complexes generated after the binding of the same probe to PCR products from homozygous W1282X sample were found to be stable (Fig. 5G). Login to comment 115 ABCC7 p.Trp1282* Intermediate stability (50% decrease of RUres-RUi) was found for complexes generated after binding of the M-CFp12-mer DNA probes to PCR products from heterozygous W1282X samples (Fig. 5F). Login to comment 117 ABCC7 p.Trp1282* Secondary structures of single stranded CFTR PCR products carrying both normal (A) and mutated (B) W1282X sequences. Login to comment 121 ABCC7 p.Trp1282* The nucleotide W1282X mutation is arrowed. Login to comment 123 ABCC7 p.Trp1282* A-C: Characterization of biotinylated PCR products from normal subjects(A), homozygous W1282X samples (B) or heterozygous subjects (C). Login to comment 125 ABCC7 p.Trp1282* D: Experimental strategy for detecting the W1282X mutation. Login to comment 127 ABCC7 p.Trp1282* In this experiment 25 µl of normal (dotted lines) and mutated (solid line) 6 µM CFp-12 mers solutions were injected to flow cells carrying PCR products from normal subjects (E), heterozygous subjects (F) or homozygous W1282X samples (G). Login to comment 129 ABCC7 p.Trp1282* The sensorgrams shown in this figure have been obtained by subtracting to the experimental sensorgrams, sensorgrams obtained by injecting HBS-EP. A comparison of the binding efficiencies of the CFp-9 mer, 10A mer, and 12 mer DNA probes to CF PCR products from a normal subject or heterozygous and homozygous CF W1282X samples are shown in Table 2. Login to comment 132 ABCC7 p.Trp1282* ABCC7 p.Trp1282* On the contrary, binding of the N-CFp-10A probe was detectable to flow cells carrying PCR products from normal and heterozygous W1282X samples. No hybridization of N-CFp-10A probe was found to the flow cells carrying CF PCR products from homozygous W1282X sample. Login to comment 133 ABCC7 p.Trp1282* Conversely, binding of the M-CFp-10A mer probe was detectable to flow cells carrying PCR products from heterozygous and homozygous W1282X samples. No hybridization of M-CFp-10A probe was found to the flow cells carrying CF PCR products from a normal subject. Login to comment 134 ABCC7 p.Trp1282* It should be noted that the values of RUfin obtained by injecting the same CF probe on flow cells carrying different W1282X PCR products could be different, depending on the amounts of immobilized PCR products and to the secondary structures of the single-stranded PCR product itself. Login to comment 142 ABCC7 p.Trp1282* Taken together, the obtained results suggest that the CF index could be introduced to discriminate between CF PCR products from normal subject and heterozygous or homozygous W1282X samples. Login to comment 143 ABCC7 p.Trp1282* DISCUSSION In the present paper, we demonstrate that allele-specific 10 and 12 mer oligonucleotides are suitable probes to detect the W1282X point mutation of the cystic fibrosis gene by performing biospecific interaction analysis (BIA) and employing surface plasmon resonance (SPR) [Malmqvist, 1993] and biosensor technologies. Login to comment 145 ABCC7 p.Trp1282* This procedure makes it possible to monitor the real-time hybridization between target single stranded PCR products obtained by using as substrates DNA isolated from normal subject or heterozygous and homozygous W1282X CF samples and oligonucleotide probes, therefore enabling a one-step, non-radioactive protocol to perform diagnosis. Login to comment 147 ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Trp1282* Increase of Resonance Units (RU) Following Injections of W1282X Probes to SA5-Sensor Chips Carrying Single Stranded Target PCR Products Target: PCR products Normal Heterozygous Homozygous (-/-) (-/M-W1282X) (M-W1282X/M-W1282X) W1282X probe RUfin-RUi RUres-RUi RUfin-RUi RUres-RUi RUfin-RUi RUres-RUi N-CFp-9 mer 17 9 12 10 11 10 M-CFp-9 mer 10 11 10 9 9 11 N-CFp-10A mer 38 26 29 20 7 10 M-CFp-10A mer 10 12 36 15 29 13 N-CFp-12 mer 52 51 78 36 37 10 M-CFp-12 mer 59 16 70 32 39 41 is readily and reproducibly observed by using the CFp-10 mer probes. Login to comment 152 ABCC7 p.Trp1282* Increased hybridization efficiency could be obtained either by using PCR primers amplifying CF sequences lacking secondary structures at the level of W1282X mutation, or by using as molecular probes molecules able to hybridize with target DNA independently of secondary structures, such as the recently described peptide nucleic acids (PNAs) [Egholm et al., 1993; Sawata et al., 1999]. Login to comment 162 ABCC7 p.Trp1282* CF W1282X index in six different experiments. Login to comment