1 2001 Vol: 81(10):1415-1427. DOI: 10.1038/labinvest.3780355

Peptide Nucleic Acids and Biosensor Technology for Real-Time Detection of the Cystic Fibrosis W1282X Mutation by Surface Plasmon Resonance

In this paper we demonstrate that peptide nucleic acids (PNAs) are excellent probes able to detect the W1282X point mutation of the cystic fibrosis (CF) gene when biospecific interaction analysis (BIA) by surface plasmon resonance (SPR) and biosensor technologies is performed. The results reported here suggest that BIA is an easy, fast, and automatable approach for detecting mutations of CF, allowing real-time monitoring of hybridization between 9-mer CF PNA probes and target biotinylated PCR products generated from healthy, heterozygous subjects and homozygous W1282X samples and immobilized on streptavidin-coated sensor chips. This method is, to our knowledge, the first application of PNAs, BIA, and SPR to a human hereditary mutation, and demonstrates the feasibility of these approaches for discriminating between normal and mutated target DNA. We like to point out that the procedure described in this paper is rapid and informative; results are obtained within a few minutes. This could be of great interest for molecular pre-implantation diagnosis to discriminate homozygous CF embryos from heterozygous and healthy embryos. Other advantages of the methodology described in the present paper are (a) that it is a nonradioactive methodology and (b) that gel electrophoresis and/or dot-spot analysis are not required. More importantly, the demonstration that SPR-based BIA could be associated with microarray technology allows us to hypothesize that the method described in the present paper could be used for the development of a protocol employing multispotting on SPR biosensors of many CF-PCR products and a real-time simultaneous analysis of hybridization to PNA probes. These results are in line with the concept that SPR could be an integral part of a fully automated diagnostic system based on the use of laboratory workstations, biosensors, and arrayed biosensors for DNA isolation, preparation of PCR reactions, and identification of point mutations.

Mentions
Figures
Figure 1: Map of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene region involved in the W1282X mutation causing CF and location of the CF1 (forward) and CF2 (reverse) PCR primers. The nucleotide sequences of the biotinylated target W1282X 9- and 21-mers injected overflow cells of SA5 sensor chips are indicated. The sequences of DNA and peptide nucleic acid (PNA) probes are also indicated. Figure 2: Melting curves and melting temperatures of (A) N-W1282X 9-mer PNA hybridized with the DNA 5'-AGTGGAGGA-3' (thick line, full match) and with the DNA 5'-AGTGAAGGA-3' (thin line, mismatch). B, M-W1282X 9-mer PNA hybridized with the DNA 5'-AGTGAAGGA-3' (thin line, full match) and with the DNA 5'-AGTGGAGGA-3' (thick line, mismatch). Figure 3: A, Scheme of DNA-DNA and DNA-PNA hybrids. B, Experimental strategy. C to E, Sensorgrams obtained after injection on a flow cell carrying M-W1282X 21-mer target DNA of 25 l containing 0.5 g of normal (dotted lines) and mutated (solid lines) W1282X 17-mer (C), 12-mer (D), and 9-mer (E) DNA probes. The oligonucleotide probes were dissolved in HEPES buffered saline-EP (HBS-EP). a, injection of the oligonucleotide probes; b, injection of HBS-EP. Figure 4: Sensorgrams obtained after injection of 25 l containing 0.5 g of normal (dotted lines) and mutated (solid line) W1282X 9-mer PNA (A–C), 9-mer DNA (D–F), and 12-mer DNA (G–I) probes to SA5 sensor chip flow cells carrying normal (A, D, G) or mutated (B, E, H) W1282X 12-mer target DNA. In Panels C, F, and I, the probes were injected to a flow cell carrying a 1:1 mixture of normal and mutated W1282X 12-mer target DNA. The oligonucleotide probes were dissolved in HBS-EP. Figure 5: A, Representative example of the increase of resonance units following injection on biotinylated CF1-CF2 PCR products from an homozygous W1282X sample. Three injections were consecutively performed (I–III). A total amount of 60 l of 2.5 m PCR products in HBS-EP was injected. After each injection (segments "a" of the panel), injections of HBS-EP (segments "b" of the panel) and 50 mm NaOH (segments "c" of the panel) were performed. No blank subtraction was performed. B, Comparison of the increase of resonance units following injection on biotinylated PCR products from normal subjects (open circles), homozygous W1282X samples (open squares), or heterozygous subjects (filled circles). Insert: Characterization by agarose gel electrophoresis of biotinylated PCR products from normal subjects (a), homozygous W1282X samples (b), or heterozygous subjects (c). Figure 6: A and B, Secondary structures of single-stranded CF1-CF2 CFTR PCR products carrying both normal (A) and mutated (B) W1282X sequences. The MFOLD software (version 3.0) developed by Zuker et al (1999) was used in this analysis. The experiments were performed at 25° C temperature and at 0.15 M NaCl. The nucleotide W1282X mutation is indicated by an arrow. C, Experimental strategy. D to N, Sensorgrams obtained after injection of 25 l containing 0.5 g of normal (dotted lines) and mutated (solid lines) W1282X 9-mer DNA (D–F), 9-mer PNA (G–I), and 12-mer DNA (L–N) probes onto flow cells carrying PCR products from normal subjects (D, G, L), heterozygous subjects (E, H, M), or homozygous W1282X samples (F, I, N). The probes were injected in HBS-EP. a, injection of the probes; b, injection of HBS-EP.
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References
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    • . . . Many diagnostic approaches have been recently introduced which are aimed at the development of high-throughput mutation detection and genotyping technologies, among which are gel-based methods such as PCR-restriction fragment length polymorphism (RFLP) (Shi et al, 1999), oligonucleotide ligation assay (OLA) (Baron et al, 1996; Eggerding, 2000; Rothschild et al, 1997), and minisequencing (Shi, 2001) . . .
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    • . . . Mutations leading to the CF phenotype are currently detected by a variety of PCR-based approaches (Saiki et al, 1985), including single-strand conformation polymorphism (SSCP/HA) (Wine et al, 2001), temporal temperature gradient gel electrophoresis (TTGE) (Wong et al, 2000), DNA sequencing (Bernardino et al, 2000), oligonucleotide ligation assay (OLA), sequence-coded separation (Brinson et al, 1997), and capillary zone electrophoresis combined with laser-induced fluorescence detection (Gelfi et al, 1998) . . .
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    • . . . The use of PNAs in SPR-based BIA has been recently explored by Sawata et al (1999), Kai et al (1997), and Burgener et al (2000) . . .
    • . . . The use of PNAs in SPR-based BIA has been recently explored by Sawata et al (1999), Kai et al (1997), and Burgener et al (2000). Sawata et al (1999) applied PNAs to the direct detection of deoxyribonucleic acid amplified by polymerase chain reaction, demonstrating that this method is a powerful tool for the diagnosis of pathologically significant DNA . . .
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    • . . . Although highly informative and successfully applied in the diagnosis of CF, some of these methodologies are tedious, technically complex, sometimes difficult to use routinely in a clinical context, when the most important requirements are quality of service, speed, accuracy, and transferability to high-throughput formats (Shi, 2001). . . .
    • . . . Many diagnostic approaches have been recently introduced which are aimed at the development of high-throughput mutation detection and genotyping technologies, among which are gel-based methods such as PCR-restriction fragment length polymorphism (RFLP) (Shi et al, 1999), oligonucleotide ligation assay (OLA) (Baron et al, 1996; Eggerding, 2000; Rothschild et al, 1997), and minisequencing (Shi, 2001) . . .
    • . . . This novel high-throughput methodology allows simultaneous analysis of a large number of polymorphisms and is expected to be the major field of applied research (Eggers, 2000; Shi, 2001) . . .
  48. Shi MM, Bleavins MR, and de la Iglesia FATechnologies for detecting genetic polymorphisms in pharmacogenomics. Mol Diagn 4: 343-351 , (1999) .
    • . . . Many diagnostic approaches have been recently introduced which are aimed at the development of high-throughput mutation detection and genotyping technologies, among which are gel-based methods such as PCR-restriction fragment length polymorphism (RFLP) (Shi et al, 1999), oligonucleotide ligation assay (OLA) (Baron et al, 1996; Eggerding, 2000; Rothschild et al, 1997), and minisequencing (Shi, 2001) . . .
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    • . . . The W1282X mutation, located in exon 20, was first observed in a French patient with cystic fibrosis by Vidaud et al (1990) and was demonstrated to be the most common CF mutation in the Ashkenazi Jewish population (Shoshani et al, 1992). . . .
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    • . . . Among non–gel-based diagnostic technologies, fluorescence resonance energy transfer (FRET) (Clegg, 1992) has been employed for FRET-based technologies such as TaqMan (Livak et al, 1995; Tapp et al, 2000) and Invader assays (Lyamichev et al, 1999), molecular beacons (Tyagi et al, 1998), FRET-based PCR-OLA (Chen et al, 1998), and FRET-based rolling circle amplification (RCA)(Lizardi et al, 1998) . . .
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    • . . . Among non–gel-based diagnostic technologies, fluorescence resonance energy transfer (FRET) (Clegg, 1992) has been employed for FRET-based technologies such as TaqMan (Livak et al, 1995; Tapp et al, 2000) and Invader assays (Lyamichev et al, 1999), molecular beacons (Tyagi et al, 1998), FRET-based PCR-OLA (Chen et al, 1998), and FRET-based rolling circle amplification (RCA)(Lizardi et al, 1998) . . .
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    • . . . The recent development of biosensor technologies for biospecific interaction analysis (BIA) (Jonsson et al, 1991; Malmqvist, 1993; Vadgama and Crump, 1992) enables us to monitor DNA-DNA and DNA-RNA hybridization in real time by surface plasmon resonance (SPR) . . .
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    • . . . The W1282X mutation, located in exon 20, was first observed in a French patient with cystic fibrosis by Vidaud et al (1990) and was demonstrated to be the most common CF mutation in the Ashkenazi Jewish population (Shoshani et al, 1992). . . .
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    • . . . A final comment should be made on DNA microarray genotyping (Hacia et al, 1999; Wang et al, 1998) . . .
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    • . . . In this respect, peptide nucleic acids (PNAs) (Nielsen et al, 1991) are molecules of great interest, because they offer great advantages, in comparison with oligonucleotide probes, in molecular diagnosis (Dueholm and Nielsen, 1997; Hyrup and Nielsen, 1996; Wang, 1998) . . .
    • . . . This property has been used for the detection of point mutations in advanced diagnostic methods, by means of PCR clamping (Ørum et al, 1993), affinity electrophoresis (Igloi, 1999), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Griffin et al, 1997), electrochemical biosensors (Wang, 1998, Wang et al, 1997b), quartz crystal microbalance (QCM) (Wang et al, 1997a), and microarrays (Weiler et al, 1997). . . .
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    • . . . This property has been used for the detection of point mutations in advanced diagnostic methods, by means of PCR clamping (Ørum et al, 1993), affinity electrophoresis (Igloi, 1999), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Griffin et al, 1997), electrochemical biosensors (Wang, 1998, Wang et al, 1997b), quartz crystal microbalance (QCM) (Wang et al, 1997a), and microarrays (Weiler et al, 1997). . . .
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    • . . . This property has been used for the detection of point mutations in advanced diagnostic methods, by means of PCR clamping (Ørum et al, 1993), affinity electrophoresis (Igloi, 1999), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Griffin et al, 1997), electrochemical biosensors (Wang, 1998, Wang et al, 1997b), quartz crystal microbalance (QCM) (Wang et al, 1997a), and microarrays (Weiler et al, 1997). . . .
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    • . . . This property has been used for the detection of point mutations in advanced diagnostic methods, by means of PCR clamping (Ørum et al, 1993), affinity electrophoresis (Igloi, 1999), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Griffin et al, 1997), electrochemical biosensors (Wang, 1998, Wang et al, 1997b), quartz crystal microbalance (QCM) (Wang et al, 1997a), and microarrays (Weiler et al, 1997). . . .
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    • . . . Mutations leading to the CF phenotype are currently detected by a variety of PCR-based approaches (Saiki et al, 1985), including single-strand conformation polymorphism (SSCP/HA) (Wine et al, 2001), temporal temperature gradient gel electrophoresis (TTGE) (Wong et al, 2000), DNA sequencing (Bernardino et al, 2000), oligonucleotide ligation assay (OLA), sequence-coded separation (Brinson et al, 1997), and capillary zone electrophoresis combined with laser-induced fluorescence detection (Gelfi et al, 1998) . . .
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    • . . . By contrast, even short PNAs are expected to be efficient in hybridizing to target DNA, because they are not negatively charged and, therefore, no electrostatic repulsion occurs during PNA-DNA hybrid formation (Wittung et al, 1994) . . .
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    • . . . Mutations leading to the CF phenotype are currently detected by a variety of PCR-based approaches (Saiki et al, 1985), including single-strand conformation polymorphism (SSCP/HA) (Wine et al, 2001), temporal temperature gradient gel electrophoresis (TTGE) (Wong et al, 2000), DNA sequencing (Bernardino et al, 2000), oligonucleotide ligation assay (OLA), sequence-coded separation (Brinson et al, 1997), and capillary zone electrophoresis combined with laser-induced fluorescence detection (Gelfi et al, 1998) . . .
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    • . . . Because the changes in refractive index are proportional to the changes in absorbed mass (Malmqvist, 1993; Vadgama and Crump, 1992), the SPR technology allows the monitoring of DNA-DNA hybridization while it is occurring (Nilsson et al, 1995; Wood, 1993) . . .
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    • . . . This was analyzed by using the MFOLD software (version 3.0) developed by Zuker et al (1999), and Mathews et al (1999) . . .
    • . . . The MFOLD software (version 3.0) developed by Zuker et al (1999) was used in this analysis . . .
    • . . . 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) . . .
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