Labelless electrochemical melting curve analysis for rapid mutation detection

Abstract

In the post-genome era there is an increasing demand for cost effective and rapid methods for the detection of specific mutations and single nucleotide polymorphisms. Here we describe a method for the rapid detection of mutations exploiting labelless electrochemical melting curve analysis, using the detection of the cystic fibrosis associated DF508 mutant as a model. A 21-base long thiolated probe, complementary to the region of Exon 10 of the cystic fibrosis transmembrane regulatory gene where the DF508 mutation lies, was immobilised on a gold electrode and hybridised to a synthetic analogue of single stranded PCR products for each of the mutant (85 bases) and wild type (82 bases) targets. Experimental conditions were optimised to exploit the guanine-specific interaction of the electroactive indicator, methylene blue. Upon hybridisation of the immobilised probe to the target, the number of guanine bases present in close proximity with the sensor surface increased from 3 to 14, resulting in a significant increase in signal. Ramping of the temperature caused denaturation of the on-surface immobilised duplex and a concomitant reduction in signal. From the first derivative of the melting curves a clear differentiation between the mutant and wild-type target could be observed. The proposed approach can be extended to array based melting curve analysis, allowing the simultaneous detection of multiple mutations and SNPs, and moreover the melting properties observed can also be used to design genosensors for single target detection.

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1. Introduction

The completion of the Human Genome Project has resulted in the continuous identification of mutations associated with genetic diseases or disease pre-dispositions. The ability to simultaneously analyse many mutations in a simple, rapid, and inexpensive way is essential and this requirement has ushered the development of many techniques and technologies for genotyping.¹ DNA duplex stability can be used to identify mutations, for example, single-stranded conformation polymorphism,² denaturing gradient gel electrophoresis³ and temperature gradient gel electrophoresis⁴ as well as stability of different conformations.⁵ Hybridisation stability can also be monitored using fluorescence resonance energy transfer,⁶ and monitoring of melting curves has been reported as an additional discriminating dimension in sequencing by hybridisation techniques.⁷ PCR product melting analysis, first introduced in 1997,⁸ is a simple, rapid and inexpensive analytical tool but depends strongly on the dyes and instruments used.⁹ There have been advances in DNA melting techniques, e.g. improvements in instrumentation and data collection,¹⁰ more precise data analysis, development of fluorescent DNA binding dyes with improved properties¹¹ or FRET,¹² which have led to the development of high-resolution melting curve analysis,¹³ demonstrating several advantages and capabilities for genotyping and/or mutation scanning.¹⁴ This discrimination is based on the fact that each double-stranded DNA fragment has its own characteristic melting behaviour, dependent on GC content, length, and sequence of the product, among other factors.⁵ Sequence alterations lead to changes in duplex stability, and thus changing melting behaviour,¹⁵ which can be easily detected using the first derivative method, or by means of the shape of the generated melting curves.¹¹ Straightforward melting curve analysis has been used for genotyping of sequence changes such as single-nucleotide polymorphisms,¹⁶ insertion/deletion polymorphisms,¹⁷ deletions of varying lengths¹⁸ and internal tandem duplications.^13,19 However, fluorescence based melting curve analysis has some limitations: such as the effect of the fluorescent dye concentration and the temperature transition rates on the absolute position and width of melting curves,⁵ as well as the effect of the addition of intercalators, such as ethidium bromide, increasing the melting temperature and broadening the melting transition.²⁰ In this article, we report on a labelless approach for electrochemical melting curve analysis. Electrochemistry-based methods are an attractive alternative to conventional fluorescence methods due to their low-cost, high-sensitivity, simplicity, compatibility with microfabrication techniques and their miniaturisability. There have been reports of the electrochemical detection of melting temperature, such as those by Brewood et al.²¹ and Marquette et al.²² who developed impedance based approaches for the detection of the melting temperature; Xiaoteng et al.²³ reported on an immobilisation free method for the melting curve analysis of a DNA–PNA duplex and Surkus and Flechsig²⁴ used melting curve analysis to explore the effect of ionic strength and number of mismatches on melting temperature, observing that the melting temperature of the surface immobilised DNA duplex was significantly lower than in homogenous solution. Recently the authors reported on an electrochemical approach for melting curve analysis based on the labelling of the DNA target with an electroactive moiety (ferrocene).²⁵This approach, despite having shown the ability to discriminate between fully complementary and mismatched sequences, it has the drawback of requiring target DNA labelling.

Methylene blue (MB), an organic dye belonging to the phenothiazine family, is an example of a redox active non-metal molecule having high affinity for nucleic acids. Three different mechanisms of MB–DNA interaction have been recognised: (i) electrostatic interaction of the dye with the negatively charged DNA backbone,²⁶ (ii) intercalation within the DNA double helix²⁷ and (iii) preferential binding to free guanine bases present on single stranded DNA (ssDNA).^28,29 Recently Nasef et al. showed that the mechanism of the DNA–MB interaction is strongly dependent from the presence of cationic species in the solution.³⁰ Several reports on the use of MB as a reporting element in electrochemical genosensors can be found in the literature;^28–33 these were clearly showing the ability of this approach to detect hybridisation even at target concentration of the order of few nM.³⁰ Meunier-Prest et al.³⁴ studied the melting behaviour of a synthetic model DNA duplex immobilized onto gold electrodes using methylene blue as an electroactive intercalator; where duplex melting resulted in the liberation of methylene blue and a concomitant reduction in signal.

Herein, we report on a facile method for labelless electrochemical melting curve analysis using methylene blue as an electroactive indicator of hybridisation and denaturation. As a model target to demonstrate the approach, the cystic fibrosis associated DF508 mutation was used, which represents a 3 base mutation at the 508 position of the cystic fibrosis transmembrane regulator gene, resulting in deletion of a phenylalanine. A gold electrode was functionalised with thiolated probe complementary to either the wild type, or the mutant form, and was hybridised with single stranded synthetic analogues of PCR products being 82 and 85 bases in length for mutant and wild type target, respectively. The stability of the immobilised probes at elevated temperatures was evaluated and the melting curves obtained showed a clear differentiation between wild type and mutant targets.

2. Experimental

2.1. Materials and methods

All chemicals and reagents were of analytical grade and used without further purification: 2-amino-2-(hydroxymethyl)-1,3-propanediol, Trizma base (Sigma), hydrochloric acid (6 M, Scharlau), potassium dihydrogen phosphate KH₂PO₄ (Scharlau), sodium chloride NaCl (Scharlau), sodium hydroxide NaOH pellets (Panreac), sulfuric acid H₂SO₄ (Sigma), 6-mercaptohexanol, MCH (Fluka), and methylene blue (Fluka). All solutions were prepared in ultra pure water (18 MΩ cm) obtained using a “Simplicity Water Purification System” (Millipore, France). The oligonucleotides used in the study, optimised in a previous report,³⁵ were obtained from Biomers.net (Germany) and are detailed in Table 1.

Table 1 Details of oligonucleotides used in this study. The long sequences represent synthetic analogues of single stranded PCR amplicons. Underlined in the Mut sequence is the region reverse complementary to the probe. The 3 bases underlined and in italic in the WT sequence are those deleted in the Mut sequences

Mutant target amplicon, Mut (82 mer)	5′ GCC GCG AAT TCA CTA GTG TGG CAC CAT TAA AG̲A̲ A̲A̲A̲ T̲A̲T̲ C̲A̲T̲ T̲G̲G̲ T̲G̲T̲ T̲T̲C̲ C̲TA TGA TGA ATA TAA TCG AAT TCC CGC GGC C 3′
Wild type target amplicon, WT (85 mer)	5′ GCC GCG AAT TCA CTA GTG TGG CAC CAT TAA AGA AAA TAT CAT C̲T̲T̲ TGG TGT TTC CTA TGA TGA ATA TAA TCG AAT TCC CGC GGC C 3′
Mutant probe (21 mer)	5′ GGA AAC ACC AAT GAT ATT TTC-C6-SH 3′

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Electrochemical measurements were carried out using an Autolab model PGSTAT 12 potentiostat/galvanostat controlled with the General Purpose Electrochemical System (GPES) software (Eco Chemie B.V., The Netherlands). A classical three electrode configuration was used: a Ag/AgCl wire reference electrode, a Pt wire counter electrode and a thin film Au working electrode. Working electrodes were cleaned electrochemically by cycling (scan rate 100 mV s⁻¹) between −0.2 and 1.6 V in 0.5 M sulfuric acid until a peak-to-peak separation below 80 mV was recorded.

Thiolated probe (Mut or Wt) was self-assembled, via spotting of 10 µl of a 4 µM solution, freshly prepared in 1 M KH₂PO₄, onto the electrode surface and left to self-assemble for 3 hours, followed by thorough washing with ultra pure water and subsequent back-filled via spotting of the electrode with 10 µl of a 0.01 M aqueous solution of 1-mercaptohexanol. The backfiller was left to incubate for 30 minutes and the sensor was then again extensively washed via sequential immersion in 0.1 M NaOH, MilliQ water, 0.1 M HCl and MilliQ water, leaving an organised mixed SAM of chemisorbed DNA probe and MCH.

Effect of temperature on buffer pH and probe stability. An aliquot of the 10 mM Trizma buffer (pH 7.4) containing 0.5 M of NaCl was thermostatted, using a water bath, at fixed temperatures (between 30 and 65 °C with steps of 5 °C) and following temperature stabilisation the pH was measured. Additionally, the probe stability was measured over the same temperature interval, using differential pulse voltammetry and methylene blue as an electrochemical reporter.

Hybridisation with target sequences and electrochemical melting curve analysis. Target hybridisation was carried out via 1 hour incubation of a 1 µM solution of the desired target (Mut or WT synthetic PCR analogues) in 20 mM Trizma buffer (pH 7.4) in the presence of 0.5 M NaCl; following hybridisation the sensor was extensively washed with the hybridisation buffer. Estimation of the hybridisation level was performed by monitoring the electrochemical reduction of the MB. In more detail, an aliquot of a 20mM Trizma (pH 7.4) and 0.5 M NaCl buffer containing 20 µM methylene blue was added to the electrode surface and left to interact for 5 minutes, and subsequently a differential pulse voltammogram (DPV) was recorded. The resulting reduction wave at ca. −100 mV (vs. the Ag/AgCl reference) was measured.

It is known that the electrochemical behaviour of methylene blue is influenced by temperature.³² To address this, all electrochemical measurements were performed at a fixed temperature of 21 °C. Sensor heating was performed by immersion in an Eppendorf containing 500 µl of 20 mM Trizma buffer (pH 7.4) and 0.5 M NaCl buffer for 10 minutes previously stabilised to the desired temperature using a Thermomixer compact (Eppendorf Iberica, Spain). Following thermal treatment the sensor was washed within the same temperature via immersion in a second thermostatted Eppendorf containing the same buffer solution. Finally, the electrode was transferred to the measurement buffer and let to cool to room temperature before measurement.

Optimisation of salt concentration for maximal discrimination. In order to maximise the discrimination between mutant and wild type targets, an optimisation of the salt (NaCl) content in the hybridisation buffer was evaluated, by monitoring the voltammetric response as a function of three different NaCl concentrations (0.05, 0.2 and 0.5 M) in the hybridisation buffer.

3. Results and discussion

Previously, we have developed fluorescence based³⁶ and electrochemical²⁵ techniques for the measurement of the melting temperature of surface immobilised duplexes. These were based on the use of gold film immobilised probe and of fluorophore.³⁶ In the fluorescence approach³⁶ the gold film quenches the fluorescence upon hybridisation and when heated the duplex dissociates, with the fluorophore moving away from the gold film, thus giving an increase in fluorescent signal.³⁶ The melting temperatures obtained showed a significant difference as compared to those recorded for solution based studies, with the surface immobilised duplex being markedly less stable. This was further demonstrated in an extension of this work where the fluorescent label was replaced by a ferrocene electrochemical label, and electrochemical melting curve analysis was carried out to differentiate between the cystic fibrosis DF508 mutant and corresponding wild type.²⁵ Whilst this approach has extensive application for the melting curve analysis of surface tethered DNA duplexes, labelling of forward/reverse primer is required and the approach has not yet been demonstrated with full-length PCR amplicons. In the work we report here, we carry out labelless electrochemical melting curve analysis using an immobilised probe that is complementary to the DF508 region of Exon 10 of the cystic fibrosis transmembrane regulator. This method has the advantage of not requiring the labelling of the target during the amplification; moreover the electrochemical detection approach on which the method is based has already been shown to be suitable for the detection of synthetic PCR analogues.³⁰ Nasef et al.³⁰ showed that the electrochemical monitoring of the DNA–MB interaction was suitable to perform hybridisation studies on a large temperature window. The probe is hybridised to a synthetic analogue of single stranded PCR product of the complementary mutant target (85 bases) or non-complementary wild type target (82 bases), and the electrochemical indicator methylene blue is used to measure the levels of hybridisation. A schematic representation of the proposed approach is presented in Fig. 1.

Fig. 1 Schematic representation of sensor preparation (A) and duplex determination via methylene blue (represented as small blue spheres) approach (B). Inset: differential pulse voltammograms of mutant amplicon captured on the electrode surface. Hybridisation performed from a solution of 1 µM target amplicons. Measurement solution: 20 µM MB in 0.5 M NaCl and 20 mM Trizma, pH 7.4.

Hybridisation with the targets adds free guanine bases to the electrode surface duplex through the lateral sides of the amplicon which do not participate in hybridisation, increasing the number of guanines from 3, present in the immobilised probe, to 14 present in the duplex formed upon hybridisation. This increase in guanine content results in a significant increase in voltammetric signal; typical DPV results, obtained upon hybridisation, are presented in the inset of Fig. 1B, where a clear difference between probe and duplex signals can be seen.

Prior to carrying out the temperature ramping experiments, the probe stability over the temperature range of interest was determined. There are several articles outlining the thermal stability of alkanethiol and DNA monolayers, reporting desorption from gold surface, ranging from 55 °C^37,38 and 90 °C,³⁹ dependent on the surface chemistry and on the nature of the surface. An evaluation of the thermal stability using a DNA specific electroactive indicator, methylene blue, that facilitated a specific evaluation of the loss of DNA probes from the surface was carried out by heating the sensor surface for 10 minutes in an aliquot of thermostatted buffer. The sensor was subsequently transferred to the measurement solution, maintained at 21 °C, and a DPV measurement recorded. In this work the methylene blue has been chosen to allow a specific evaluation of the loss of DNA probes from the surface. As can be seen in Fig. 2, it was observed that no loss of thiolated DNA probes was recorded between 30 and 65 °C.

Fig. 2 Evaluation of the influence of temperature on the stability of the probe immobilisation on the gold electrode surface. The functionalised electrode was heated in buffer solution (0.5 M NaCl and 20 mM Trizma, pH 7.4) and the electrochemical detection via methylene blue was achieved in constant temperature, room temperature. Measurement solution: 20 µM MB in 0.5 M NaCl and 20 mM Trizma, pH 7.4. Standard deviation is the result of 3 experiments.

The pH of Trizma buffer is known to be influenced by temperature, whilst the melting temperature of DNA duplexes is not affected by a change in pH within the range of pH values from 6.5 to 8.0.⁴⁰ Thus, in order to ensure that the changes in pH of the buffer, as a function of temperature, did not influence duplex stability, a study of the variation of the pH of the buffer with the temperature was performed. The pH of the buffer solution clearly varied with the temperature with a slope of 0.02 pH unit per 1 °C.

UV-based melting temperature studies, at different pH values, between 6.5 and 8, were carried out confirming that these pH values have no effect on the melting temperature.

Effect of ionic strength

The ionic strength is critical for the stabilisation of DNA duplexes. In order to optimise the ionic strength employed for the labelless electrochemical melting analysis, as well as the ionic strength for a maximal differentiation between wild type and mutant targets, a study of the effect of the concentration of NaCl in the hybridisation/washing buffer, on duplex stability and on sequences discrimination, was carried out (the concentration in the measurement buffer was maintained at 0.5 M in order to minimise electrostatic interactions between DNA and MB).

The results of this evaluation are presented as percentage increase in signal as compared to background signal (response due to the presence of probe on the surface) as detailed below:

Signal variation (%) = 100 × [(i_amp − i_reg)/i_reg] (1)

where i_amp peak current recorded with hybridised target and i_reg peak currents after regeneration i.e. denaturation of formed duplex

In Table 2 the signal variations, recorded for the hybridisation of the fully complementary target (Mut) and those recorded for the three mismatch target (WT), as a function of three different salt concentrations, are reported. The increase in NaCl concentration in the hybridisation buffer resulted in an increase of the efficiency of the hybridisation process; for example by increasing the NaCl concentration from 0.05 M to 0.5 M an increase in analytical response (signal variation), from about 30 to 121%, was recorded. Moreover the increase in the NaCl concentration in the hybridisation buffer facilitated a better discrimination between the mutant and wild type synthetic targets difference in signal variation passed from 9 to 36%. From the above studies, it was decided to carry out the melting curve analysis in the presence of 0.5 M NaCl.

Table 2 Effect of ionic strength on discrimination factor between mutant and wild type targets. Standard deviation is the results of 3 experiments

[NaCl]/mM	Mut amplicon, signal variation (%)	WT amplicon, signal variation (%)	Signal variation difference (%)
50	30.5 ± 0.3	21.6 ± 2.5	8.9
200	78.7 ± 0.6	62.0 ± 5.7	16.7
500	121.0 ± 4.9	84.6 ± 2.5	36.4

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Labelless electrochemical melting curve analysis

The effect of temperature on the stability of the fully matched DF508 mutant amplicon and on the three mismatch wild type amplicon duplexes, when mutant probes are immobilised on the sensor surface, is shown in Fig. 3A. The melting behaviour of the two different on-surface confined duplexes shows a clear discrimination between the DF508 mutant amplicon and the corresponding wild type amplicon. To pinpoint the exact melting temperature (i.e. the inflection point of an electrochemical melting curve), the first derivative of the electrochemical melting curve vs. temperature is plotted in Fig. 3B. The melting temperature for the fully complementary duplex is 57 °C, whilst for the three-mismatch case it was observed to be 35 °C.

Fig. 3 Melting curves of the mutant and wild type amplicon duplexes with mutant complementary probe immobilized on the electrode surface. Sensor heating was performed for 10 minutes The methylene blue voltammograms represented at each temperature is a result of the measurement of the pre-heated electrode surface separately in a buffer solution (0.5 M NaCl and 20 mM Trizma, pH 7.4) and then cooled to the room temperature. Measurement solution: 20 µM MB in 0.5 M NaCl and 20 mM Trizma, pH 7.4 (A). First derivative of the electrochemical melting curves (A) vs. temperature (B). Standard deviation is the results of 3 experiments.

Using the melting curve analysis, it was also possible to identify the optimal experimental conditions for specific mutation thermal modulated genosensor. Enhancement in discrimination between DF508 mutant and wild type amplicons was achieved via increasing temperature. By increasing the temperature from 27 °C to 55 °C, the discrimination factor, defined as the ratio between the specific and unspecific response, increased from 1.1 to 4.3, demonstrating the usefulness of temperature modulation for genosensors.

4. Conclusion

Here we reported on a facile and widely applicable method for labelless electrochemical melting curve analysis. Using a model system based on the DF508 cystic fibrosis mutation, the melting curve analysis of synthetic analogues of single stranded PCR amplicons facilitated a clear discrimination between complementary and non-complementary DNA with an immobilised probe. The approach does not require labelling and the melting curve analysis can be carried out at low concentrations of target DNA. The reported approach can be used in array based detection of mutations, where probes against specific target mutants/SNPs would be immobilised on individual electrodes of the array, and the entire array is subject to temperature ramping and the dissociation of the amplicons from the immobilised probes monitored using a multiplex potentiostat. Alternatively, and as also reported in this work, the melting curve analysis can be used to optimise conditions for a thermal modulated genosensor targeting one specific mutation, or group of mutations with similar melting behaviour. Further work is focused on moving to detection of SNPs, as well as array based simultaneous detection of multiple SNPs/mutants using electrochemical melting curve analyses, representing a cost-effective, easy to use approach with far less complicated detection systems than required by i-FRET or fluorescence analysis.

Acknowledgements

This work was carried out with partial financial support from the Commission of the European Communities, specific RTD programme “Coeliac Disease Management Monitoring and Diagnosis using Biosensors and an Integrated Chip System, CD-MEDICs, FP7-2007-ICT-1-216031”. Dr Valerio Beni kindly acknowledges the European Community's, Seventh framework programme (FP7/2007-2013) under grant agreement no PIGF-GA-2008-220928 for the financial support. Hany Nasef wishes to thank Universitat Rovira i Virgili for his BRDI scholarship.

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