WO2009106834A1 - Procédé pour surveiller la déshybridation d’acide nucléique bicaténaire - Google Patents

Procédé pour surveiller la déshybridation d’acide nucléique bicaténaire Download PDF

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Publication number
WO2009106834A1
WO2009106834A1 PCT/GB2009/000541 GB2009000541W WO2009106834A1 WO 2009106834 A1 WO2009106834 A1 WO 2009106834A1 GB 2009000541 W GB2009000541 W GB 2009000541W WO 2009106834 A1 WO2009106834 A1 WO 2009106834A1
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nucleic acid
double stranded
label
probe
potential
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PCT/GB2009/000541
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English (en)
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Philip Niger Bartlett
Tom Brown
James Alistair Richardson
Sumeet Mahajan
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University Of Southampton
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors

Definitions

  • the present invention relates in general to methods for monitoring the dehybridisation of double stranded nucleic acid and for detecting and/or locating mutations in a nucleic acid sample.
  • the invention has useful practical applications in biotechnology, medical diagnostics, forensic science and the like. BACKGROUND
  • SNPs single nucleotide polymorphisms
  • mutations Detection of specific nucleic acid sequences, single nucleotide polymorphisms (SNPs) and mutations has gained huge importance following the sequencing of the human genome.
  • the repertoire of structure-function relationships between genes (and their mutations) and inherited (genetically acquired) diseases continues to expand.
  • a problem with currently existing mutation detection methods is that they only screen for mutations on a single gene at a time. Given that the human genome has 50,000-100,000 genes, this is a severe limitation.
  • the duplexes can be denatured by several methods such as ramping the temperature or washing with decreasing ionic strength solutions (stringency washing). Monitoring this dehybridization process allows mutations to be distinguished as they destabilize the duplex compared to the perfectly complementary target to varying degrees depending on the number of mismatches. Fluorescence is currently the preferred optical technique for detecting mutations using this hybridization/denaturation strategy (/). However, surface-enhanced Raman or resonant Raman scattering (SER(R)S) has been shown to possess significant advantages compared to fluorescence for DNA detection (2-4); these include the ability to multiplex the measurement because of the molecular specificity and narrow line width (ca.
  • SE(R)S surface-enhanced Raman or resonant Raman scattering
  • SERS S-plasmon resonance
  • SPR surface-plasmon resonance
  • electrochemical detection methods 9
  • SERS surface- enhanced Raman scattering
  • Their optical properties can be tuned for SERS (11, 12) through the choice of sphere diameter and film thickness so that typical enhancements of the order of 10 have been demonstrated for structured gold substrates (13).
  • the sensitivity of detection can be further increased by working with molecules which have an electronic transition in resonance with the laser excitation so that the surface enhancement is accompanied by a further resonant enhancement of ⁇ 10 3 to give surface-enhanced resonance Raman scattering (SERRS) (15).
  • SERRS surface-enhanced resonance Raman scattering
  • the use of electrochemical scanning dehybridization using fluorescence monitoring for SNP recognition has been recently demonstrated on silicon substrates with surface- bound hairpin (molecular beacons) probes (25, 26).
  • the authors state that a probe without a stem structure could not generate enough difference to distinguish SNP targets from the perfect match.
  • the present invention seeks to provide improved methods for the detection of one or more mutations in a nucleic acid.
  • a method for monitoring the dehybridisation of double stranded nucleic acid comprising the steps of: (a) providing a nucleic acid probe and a target nucleic acid; (b) forming a double stranded nucleic acid of which at least one of the strands of the nucleic acid is in contact with a solid substrate; (c) applying a potential (and/or temperature) ramp to the solid substrate; and (d) monitoring the dehybridisation of the double stranded nucleic acid across the potential (and/or temperature) ramp, wherein the nucleic probe is a linear nucleic acid probe and/or the target nucleic acid comprises a label.
  • a method for determining the presence of a mutation in a nucleic acid sample comprising the steps of: (a) providing a nucleic acid probe and a target nucleic acid; (b) forming a double stranded nucleic acid of which at least one of the strands of nucleic acid is in contact with a solid substrate; (c) applying a potential (and/or temperature) ramp to the solid substrate; and (d) monitoring the dehybridisation of the double stranded nucleic acid across the potential (and/or temperature) ramp, wherein a difference in the dehybridisation of the double stranded nucleic acid across the potential (and/or temperature) ramp as compared to a control double stranded nucleic acid is indicative of the presence of a mutation and wherein the nucleic probe is a linear nucleic acid probe and/or the target nucleic acid comprises a label.
  • a method for determining the presence of one or more mutations in two or more nucleic acid samples comprising the steps of: (a) optionally amplifying target nucleic acids from two or more different samples; (b) optionally labelling each of the target nucleic acids with a different label; (c) optionally pooling the target nucleic acids; (d) contacting the solid surface of an array with the same or one or more different nucleic acid probes; (e) hybridising the amplified target nucleic acids and the linear nucleic acid probe(s); (f) applying a potential (and/or temperature) ramp to the solid surface of the array; (g) monitoring the dehybridisation of the double stranded nucleic acid(s) across the potential (and/or temperature) ramp; and (h) determining if the two or more samples comprise one or more mutations, wherein a difference in the dehybridisation of the double stranded nucleic acid across the potential (and/or temperature
  • a method for screening or testing a sample for the presence of a mutation comprising the steps of: (a) providing a sample of nucleic acid; and (b) using that nucleic acid as the target nucleic acid in carrying out in vitro the method described herein.
  • a method for analysing a nucleic acid sample eg.
  • DNA fingerprinting comprising the steps of: (a) amplifying one or more short tandem repeat loci from a sample; (b) hybridising the amplified short tandem repeat loci to one or more nucleic acid probes to form a double stranded nucleic acid of which at least one of , the single strands of nucleic acid is in contact with a solid substrate; (c) applying a potential (and/or temperature) ramp to the solid substrate; (d) monitoring the dehybridisation of the double stranded nucleic acid across the potential (and/or temperature) ramp; and (e) determining the melting profile of the one or more short tandem repeat loci, wherein the nucleic probe is a linear nucleic acid probe and/or the target nucleic acid comprises a label.
  • a method for determining the identity or genotype of an organism comprising the steps of: (a) optionally amplifying one or more target nucleic acid sequences from the target organism; (b) hybridising the target nucleic acid(s) to one or more linear nucleic acid probes; (c) applying a potential (and/or temperature) ramp to the solid substrate; (d) monitoring the dehybridisation of the double stranded nucleic acid across the potential (and/or temperature) ramp; (e) determining which nucleic acid probes hybridise to the amplified target nucleic acid; and (f) determining the identity or genotype of said organism, wherein the nucleic probe is a linear nucleic acid probe and/or the target nucleic acid comprises a label.
  • an assay method for identifying one or more agents that modulate the dehybridisation of double stranded nucleic acid comprising the steps of: (a) providing a linear nucleic acid probe, a target nucleic acid and one or more agents; (b) forming a double stranded nucleic acid of which at least one of the strands of the nucleic acid is in contact with a solid substrate in the presence and absence of said agent; (c) applying a potential (and/or temperature) ramp to the solid substrate; (d) monitoring the dehybridisation of the double stranded nucleic acid across the potential (and/or temperature) ramp in the presence and absence of said agent(s); wherein a difference between the dehybridisation in the presence and absence of said agent(s) is indicative that said agent(s) modulates dehybridisation, wherein the nucleic probe is a linear nucleic acid probe and/or the target nucleic acid comprises a label,
  • the solid substrate following the contact of the at least one single strand of nucleic acid with the solid substrate, said solid substrate is treated to reduce non-specific binding.
  • the solid substrate is a solid metallic substrate which shows surface enhancement properties.
  • the substrate is an ordered sphere segment cavity fabricated by templated electrodeposition.
  • the substrate is an ordered sphere segment cavity gold electrode.
  • SERS Surface Enhanced Raman Scattering
  • SERS Surface Enhanced Raman Scattering
  • SERRS Resonance Raman Scattering
  • the method comprises the further step of: (e) removing the target nucleic acid sequence to regenerate the nucleic acid probe.
  • said at least one of the single strands of nucleic acid that is in contact with the solid substrate is bound directly or indirectly thereto without any intervening matrix.
  • said target nucleic acid sequence is labelled.
  • said label is or is not electroactive. In one embodiment, said label is a SERS or a SERRS active label.
  • said label is a redox active SERS or SERRS label.
  • said method comprises the use of an intercalating, groove-binding or DNA backbone binding Raman label which binds to the double stranded nucleic acid.
  • said solid substrate forms part of or is an array.
  • each of the target nucleic acids - such as two or more nucleic acids - is labelled with a detectably different label.
  • each area or well in use on the array comprises at least one nucleic acid probe for detecting a different mutation. In one embodiment, each area or well in use on the array comprises a set of different probes each comprising a different mutation that is known to be associated with at least one disease.
  • the sample is derived or is derivable from a mammal, a human, a plant, a virus, a bacterium, a protozoan or a fungus.
  • said mutation is a suspected genetic defect, a genetic defect or a mutated gene related to a disease.
  • said mutation is a mutation, preferably a single nucleotide polymorphism, that is to be mapped. In one embodiment, the mutation is a single nucleotide polymorphism.
  • the probes have the same number of repeats as the largest commonly encountered short tandem repeat for each locus.
  • more than one short tandem repeat locus is amplified from the sample in the presence of a label, and wherein each label for each short tandem repeat locus is detectably different.
  • the amplified short tandem repeats are hybridised to an array.
  • the melting potential for the one or more short tandem repeat loci is determined.
  • each array may comprise a control nucleic acid for each different short tandem repeat.
  • the present invention has a number of advantages. These advantages will be apparent in the following description.
  • the present invention is advantageous because it is generic in that it may be implemented on any solid substrate - such as a solid metallic substrate - that shows surface enhancement.
  • the present invention is advantageous because it is not limited in applicability either by the design or use of stem loop or hairpin structures.
  • stem loops have an inherent structure that must be destroyed before they can bind to target nucleic acid, whereas linear probes do not require any secondary structure so their hybridization properties to the target are favorable and predictable. Also when a nucleic acid sequence that can form, a hairpin loop actually binds to its complement there is a tendency for the sequence to come off its target and re-form the hairpin loop.
  • the present invention is advantageous because it does not rely on an immobilisation matrix and/or concentration step.
  • the fact that solid substrates are used is exploited to carry out both electrochemical and/or temperature assisted dehybridisation analyses.
  • the present invention is advantageous because the applied potential can be used to speed up, slow down or stop hybridization and/or dehybridisation.
  • the applied potential may be used to speed up, slow down or stop the hybridization and/or dehybridisation at a choice of electrodes whilst allowing the hybridization and/or dehybridisation to proceed as normal at other electrodes.
  • the present invention is advantageous because the potential may be used as an additional way to monitor hybridisation/dehybridisation using methods - such as impedance changes, binding or redox probes - which may be used in parallel with SERS measurements.
  • the present invention is advantageous because the electrochemistry may be used to bring about a local change in pH which may affect hybridisation/dehybridisation.
  • the present invention is advantageous because the methodology can easily be used to detect and characterize mutations with advantages over and above that in fluorescence or other such spectroscopic techniques.
  • the use of sensing platforms such as ordered spherical cavity substrates with surface-enhancing properties provides a greater flexibility for the choice of denaturation technique as well as labels for detection.
  • SERS monitoring can also be carried out under chemical modulation (stringency washing) or at various temperatures to modulate the melting potential or at controlled potentials to modulate the melting temperature. This could be very useful for mutation analysis of shorter or longer than usual DNA sequences which either spontaneously melt at room temperatures or do not denature within the temperature window available (limited by the boiling point of water).
  • the present invention is advantageous because the methodology can be successfully employed for screening for diseases in clinical settings. Further, detecting mutations is important in the field of genomics and gene sequencing.
  • the present invention is advantageous because with the advent of gene chips, arrays and microarrays, the methodology presents itself as a more powerful alternative to fluorescence based techniques and is ideal for integration with chips and micro-arrays. Hence, for example in a microarray each reaction area or well can be individually addressed electrically, if so desired, and single readout is possible.
  • the present invention is advantageous since it can be employed with RNA and other such biomolecules, as described herein.
  • the present invention is advantageous because using a potential and/or temperature ramp to monitor dehybridisation results in sharp melting curves. This means that the results obtained can be readily used to monitor differences in dehybridistion between sequences.
  • the methodology is even sensitive enough to readily detect a single base pair difference and has a sensitivity of at least about 0.1 amol.
  • the present invention is advantageous because using a potential ramp to monitor dehybridisation allows the closer packing of the array elements in nucleic acids on arrays. This means that nucleic acid arrays can be developed which provide more information than previous arrays.
  • the present invention is advantageous because using a potential ramp to monitor dehybridisation can be cheaper than using temperature cycling since this tends to require the use of sophisticated and more costly equipment.
  • the present invention is advantageous because it provides a reliable, high-speed, high-throughput method to detect genetic variations, for applications in, for example, nucleic acid-based diagnostics.
  • DESCRIPTION OF THE FIGURES Figure 1 (A) SEM images and a 3-D model of the ordered sphere segment cavity gold substrates (templated with 600 nm spheres and electroplated to 480 nm) at different magnifications.
  • the scale bar is 2 ⁇ m for the first SEM image and 100 nm for the other two
  • B The probe and the target sequences for the synthetic oligos.
  • C The scheme of the entire process of detection and characterization of DNA sequences beginning with a) and b) preparation of the sensing surface followed by c) passivation with mercaptohexanol to prevent non-specific binding, d) detection using SERS labelled targets, e) dehybridization with either potential or temperature and ending with f) regeneration of the surface for re-use starting again at step d).
  • the dehybridization process is the key step and yields the information which characterizes mutations.
  • Figure 2 The scheme of the entire process of detection and characterization of DNA sequences beginning with a) and b) preparation of the sensing surface followed by c) passivation with mercaptohexanol to prevent non-specific binding, d) detection using SERS labelled targets, e) dehybridization with either potential
  • the SERS intensities of the 1500 cm "1 band of the dye label (Texas Red) for the three targets, ( ⁇ ) wild type (no mutation), the (•) 1653C/T mutation (single point mutation) and the ( ⁇ ) ⁇ F508 mutation (triple deletion) are plotted against the applied potential.
  • the first derivatives of the sigmoidal fits of the intensity curves are shown in the inset with the melting potentials shown above each curve.
  • the SERS intensity of the 1500 cm "1 band of the dye label for the same three targets is plotted against temperature.
  • the first derivatives of the sigmoidal fits of the intensity curves are shown in the inset.
  • Negative PCR control S ⁇ RS spectra recorded at different potentials for the negative control experiment. The exact protocol for hybridization and S ⁇ RS-Emelting was followed with the solution (primarily containing the Cy5 labelled primer and dNTPs) as with the other PCR products. No peaks corresponding to the S ⁇ RS marker could be observed.
  • Figure 13 S ⁇ RS-Emelting in phosphate buffer (pH 8.1 containing 100 mM NaCl).
  • hybridisation has the conventional meaning as used in the art and refers to the process by which double-stranded nucleic acid unwinds and separates into single strands through the breaking of hydrogen bonding between nucleotides. This term is synonymous with “melting” and “denaturation”.
  • hybridisation also has the conventional meaning as used in the art and refers to the process by which a strand of nucleic acid joins with a complementary strand through base pairing.
  • the methods described herein monitor or measure the dehybridisation of a double stranded nucleic acid across a potential and/or temperature ramp. This offers a number of advantages as described herein.
  • the term "ramp" means a change in potential and/or temperature with time such that the starting potential and/or temperature at the beginning of the ramp is different to the potential and/or temperature at the end of the ramp.
  • the change is a stepwise or an incremental change in potential and/or temperature with time.
  • the change in potential and/or temperature from the beginning of the ramp to the end of the ramp may be an increase or a decrease. Increments within the ramp may increase, decrease or be constant.
  • the change in potential and/or temperature may be a potential and/or temperature gradient in which the potential and/or temperature changes with time.
  • the dehybridisation of the double stranded nucleic acid is monitored across the potential and/or temperature ramp at least at 2 different intervals across the ramp.
  • the dehybridisation of the double stranded nucleic acid is monitored across the potential and/or temperature ramp at a plurality of different intervals across the ramp - such as at least at about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or at least at 20 intervals.
  • the potential ramp is monitored at least at about 8 to 14 intervals — such as about 10 to 12 intervals.
  • the temperature ramp is monitored at least at about 16 to 24 intervals — such as about 18 to 22 intervals.
  • the dehybridisation of the double stranded nucleic acid may be monitored at intervals across the entire potential and/or temperature ramp. Each of the increments may be at a different potential and the same temperature. Each of the increments may be at a different temperature and the same potential.
  • the dehybridisation of the double stranded nucleic acid over the ramp is monitored in steps or increments of potential and/or temperature.
  • the steps or increments may increase and/or decrease.
  • the temperature increases across the ramp.
  • the potential decreases across the ramp.
  • the steps or increments are at least at about 0.0001V, 0.001V, 0.01, or 0.1V 3 preferably, -0.001V, -0.01, or -0.1V - such as -0.2V, -0.3V or -0.4V.
  • the potential may be scanned at scan rates of about O.OOOlV/s, O.OOlV/s, 0.01/s, or 0.1V/s, preferably, -O.OOlV/s, -0.01V/s, or -0.1V/s - such as -0.2V/s, -0.3V/s or -0.4V/s. Variable steps or increments may be used.
  • the potential ramp is monitored at about +1.0V, +0.9V, +0.8V, +0.7V, +0.6V, +0.5 V, +0.4V 5 +0.3 V, +0.2V, +0.1V 5 OV, -0.1V, -0.2V, -0.3V, -0.4V, -0.5V, - 0.6V, -0.7V, -0.8V, -0.9V -LOV 5 -1.1V, -1.2V 3 -1.3V, -1.4V, -1.5V or -1.6V or any suitable combination of start and end points — such as from at least about OV to -1.2V, -O.lV to -1.1V, -0.2V to -1.0V, -0.3V to -0.9V 5 -0.4V to -0.8V.
  • the steps or increments are at least about +1 °C - such as at least at about +1.5 0 C 3 +2 0 C 3 +2.5 °C or +3 °C. Variable steps or increments may be used.
  • the temperature ramp is monitored at about +20 0 C 5 +22.5°C 3 +25°C, +27.5°C, +30 0 C 3 +32.5°C, +35 0 C 3 +37.5°C, +40 0 C 3 +42.5 0 C 3 +45°C, +47.5 0 C 3 +50°C, +52.5, +55°C, +57.5°C, +60 0 C 3 +62.5 0 C 5 +65 0 C 5 +67.5°C or +70 0 C or any suitable combination of start and end points — such as from at least about +25°C to at least about +70 0 C.
  • the potential and/or temperature is held at each step or increment for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10O 5 150, 20O 3 250 or 300 or more seconds.
  • variable increments of potential and/or temperate may be used - such as an increment of 10 seconds, followed by an increment of 100 seconds. This may maximise the differentiation between wild type and mutant sequence, for example.
  • the (SER(R)S) intensity of the labelled nucleic acid target is recorded at each step or increment after a fixed time of, for example, about 5, 1O 5 15, 2O 3 25, 50, 100, 150, 200, 250 or 300 seconds.
  • the label intensity increases as the potential becomes more negative from about -0.2 to about -0.8V before then decreasing. In another embodiment, the label intensity decreases to 0 at potentials more negative than -0.8V. In another embodiment, the label intensity remains constant. In another embodiment, the label intensity may decrease slightly and then fall rapidly at the melting potential.
  • the potential and/or temperature ramp may be a predefined (eg. programmed) sequence. The temperature ramp may be monitored in the presence or the absence of an applied electropotential. If the temperature ramp is monitored in the presence of an applied electropotential then the electropotential may or may not remain constant during the monitoring of the temperature ramp. If the electropotential does not remain constant during the monitoring of the temperature ramp then the electropotential may be altered in steps or increments across the temperature ramp, as described above.
  • the potential ramp may be monitored at the same or a different temperature. If the potential ramp is monitored at a different temperature then the temperature may be altered in steps or increments across the potential ramp, as described above.
  • a method for monitoring the dehybridisation of double stranded nucleic acid comprising the steps of: (a) immobilising a nucleic acid probe on a solid substrate; (b) treating the surface of the solid substrate to reduce nonspecific binding; (c) hybridising a target nucleic acid to the nucleic acid probe; (d) applying a potential and/or temperature ramp to the solid substrate; (e) monitoring the dehybridisation of the double stranded nucleic acid across the potential and/or temperature ramp; and (f) optionally washing the surface of the solid substrate to remove the target nucleic acid.
  • the methods described herein comprise the steps of: (a) providing a nucleic acid probe and a target nucleic acid; (b) forming a double stranded nucleic acid of which at least one of the strands of the nucleic acid is in contact with a solid substrate; (c) applying a potential and/or temperature ramp to the solid substrate; and (d) monitoring the dehybridisation of the double stranded nucleic acid across the potential and/or temperature ramp.
  • POTENTIAL Electrochemically induced melting experiments may be carried out in, for example, electrochemical micro-Raman cells. Each of the cells may comprise a thin liquid film on the substrate while the reference (eg. Ag/AgCl) and counter (eg.
  • Platinum wire electrodes are placed on the side.
  • a heating element may be provided in the body of the cell and it may be controlled with a microcontroller circuit.
  • Two thermocouple probes may be incorporated, with digital display, for controlling and monitoring the heating temperature and the temperature of the substrate, respectively.
  • Buffer - such as 10 mM TRIS buffer, pH 7 - is flowed over the solid substrate several times and equilibrated at open circuit potential. Thereafter, a potential sequence is applied or scanned; in one embodiment, the potential is decreased in steps from about 0 V to about -1.4 V vs. the Ag/AgCl pseudo-reference electrode. Spectra may be recorded at each potential step.
  • the profile for the wild type and the mutation with the melting potential may show a difference of about 110 mV and about 60 mV, respectively.
  • the potential and optionally the temperature is measured.
  • TEMPERATURE In one embodiment, the experimental methodology is similar to measuring the potential.
  • the same or a different buffer may be used.
  • the buffer may be a phosphate buffer (pH 8.1) with 0.1 M salt is used instead of TRIS. Using the temperature ramp, it is possible to distinguish between mutations and the wild type DNA sequences.
  • the melting temperature may show a difference of at least about 1 0 C for a mutation — such as a single point mutation or a triple mutation. MUTATION
  • Mutations can arise in sections of nucleotide strands of nucleic acid either by deletion or insertion of part or all of a nucleotide base sequence or by an alteration (eg. substitution) of one or more nucleotide bases.
  • the mutation may or may not have a phenotypic impact.
  • Transcription from mutated nucleic acids may lead to defective protein products which can be a cause of many genetic diseases or disorders and thereby have a phenotypic impact - such as cancers, genetic diseases or increased predisposition to a disease - such as thalassemia, cardiovascular diseases, myopathy, cancer, and more generally genetically inheritable diseases.
  • defective protein products that arise in fermentation processes in biotechnology may also be dysfunctional or harmful.
  • the ability to detect mutations in coding and non-coding nucleic acid is important in, for example, the diagnosis of disease. A single base pair change or small insertion or deletion can result in a frame shift, a stop codon, or a non-conservative amino acid substitution.
  • Mutation in non-coding DNA can also lead to disease, as in, for example, mutations in non-coding splice sites (found in certain cases of cystic fibrosis disease for instance) or mutations in transcriptional regulatory elements (found in certain defects of beta-globin genes).
  • the mutation may be useful in mapping analysis, linkage analysis or mutation profiling and the like and may therefore not have a phenotypic impact.
  • the mutation may be a point mutation - such as a single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • the point mutation may be a single SNP or a plurality of SNPs that may be either contiguous with each other or separated by one or more nucleotides.
  • Detectable point mutations include deletion mutation(s), insertion mutation(s), and substitution mutation(s). It is possible to form four sets of nucleotide mismatches when a mutant and normal DNA segment are annealed together.
  • Each nucleotide pair represents eight possible single base pair mismatches which could be found in a DNA heteroduplex.
  • DNA:RNA and RNA:RNA heteroduplexes can also be formed. Where a heteroduplex includes RNA, 9 single base pair mismatch sets are possible.
  • DNA:DNA, DNA:RNA and RNArRNA heteroduplexes can also be created by insertion, deletion or substitution of nucleotides in the mutant nucleic acid strand.
  • the mutation may even be a larger scale alteration - such as larger mutations than point mutation that affect two or more base pairs - such as three or more, four or more, five or more or six or more base pairs.
  • the alteration may be a deletion or a duplication.
  • a method for determining the presence of a mutation in a nucleic acid sample comprising the steps of: (a) immobilising a nucleic acid probe on a solid substrate; (b) treating the surface of the solid substrate to reduce non-specific binding; (c) hybridising a target nucleic acid (which may be labelled with a SERS label) to the nucleic acid probe; (d) applying a potential and/or temperature ramp to the solid substrate; (e) monitoring the dehybridisation of the double stranded nucleic acid across the potential and/or temperature ramp; and (f) optionally washing the surface of the solid substrate to remove the target nucleic acid, wherein a difference in the dehybridisation of the double stranded nucleic acid across the potential and/or temperature ramp as compared to a control double stranded nucleic acid is indicative of the presence of a mutation.
  • the methods described herein are particularly amenable to determining the presence of one or more mutations.
  • the methods described herein are particularly amenable to determining the presence of one or more SNPs.
  • the process of determining which nucleotide(s) is/are present at one or more positions is referred to by such terms as SNP genotyping, SNP haplotyping, SNP typing, SNP scoring, SNP assaying, SNP profiling, SNP identification, and SNP screening.
  • Methods for detecting which nucleotide is present at a particular position are provided, such as for use in screening for or treating a human disease condition, susceptibility to a human disease condition or hi genome mapping.
  • SNP genotyping includes the steps of collecting a sample of cells, isolating nucleic acid (eg., genomic, mRNA or both) from the cells of the sample and determining the nucleotide present at the SNP position using the methods described herein.
  • nucleic acid eg., genomic, mRNA or both
  • Association or correlation of particular SNP genotypes with a particular phenotype may be performed for a population of individuals who have been tested for the presence or absence of a phenotypic trait of interest and for SNP marker sets.
  • the presence or absence of a set of polymorphisms may be determined for a set of the individuals, some of whom exhibit a particular trait, and some of whom exhibit a lack of the trait.
  • the alleles of each polymorphism of the set are then reviewed to determine whether the presence or absence of a particular allele is associated with the trait of interest. Correlation may be performed by standard statistical methods - such as a chi-squared test and statistically significant correlations between polymorphic form(s). Such correlation can be exploited in several ways.
  • Detection of a polymorphisms correlated with disease in a couple contemplating a family may also be valuable to the couple in their reproductive decisions.
  • the female partner might elect to undergo in vitro fertilization to avoid the possibility of transmitting such polymorphisms from her husband to her offspring.
  • immediate therapeutic intervention or monitoring may not be justified. Nevertheless, the patient can be motivated to begin simple life-style changes (eg., diet, exercise) to confer potential benefits.
  • this information may be used in a number of different methods. Examples of such methods are disease susceptibility screening, disease diagnosis, drug therapy based on an individual's genotype ("pharmacogenomics"), developing drugs based on SNPs associated with a disease, human identification applications - such as forensics - and grouping SNPs into haplotype groups.
  • the contribution or association of particular SNPs with disease phenotypes - such as human disease - enables the SNPs to be used to develop superior diagnostic tests capable of identifying individuals who express a detectable trait, such as human disease, as the result of a specific genotype or individuals whose genotype places them at risk of developing a detectable trait at a subsequent time.
  • the diagnostics may be based on a single SNP or a group of SNPs. Combined detection of a plurality of SNPs (for example, 2, 5, 10, 20, 30, 40, 50, 100, 200, 400, 600, 800 or 1000 or more SNPs) typically increases the probability of an accurate diagnosis.
  • Another aspect relates to a method of determining whether an individual is at risk of developing a trait or whether an individual expresses a trait as a consequence of possessing a particular trait-causing allele.
  • a method of determining whether an individual is at risk of developing a plurality of traits or whether an individual expresses a plurality of traits as a result of possessing a particular trait causing allele is also described. These methods may comprise determining whether the nucleic acid sample contains one or more alleles of one or more SNPs indicative of a risk of developing the trait or indicative that the individual expresses the trait as a result of possessing a particular trait-causing allele.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism.
  • SNP genotype of an individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes affects both the intensity and duration of drug action.
  • the method typically includes assaying the ability of the compound to modulate the activity and/or expression of the SNP-containing nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired activity or expression of the SNP-containing nucleic acid.
  • Modulation includes both up-regulation — such as activation or agonization - or down-regulation - such as suppression or antagonization - of nucleic acid expression.
  • SNPs are also valuable as human identification markers for such applications as forensics and paternity testing. Genetic variations in the nucleic acid sequences between individuals can be used as genetic markers to identify individuals and to associate a biological sample with an individual. Determination of which nucleotides occupy a set of SNP positions in an individual identifies a set of SNP markers that distinguishes the individual. The more SNP positions that are analyzed, the lower the probability that the set of SNPs in one individual is the same as that in an unrelated individual. Suitably, if multiple sites are analyzed, the sites are unlinked. Thus, the SNPs may be used in conjunction with polymorphisms in distal genomic regions.
  • SNPs have advantages over other types of polymorphic markers - such as short tandem repeats (STRs), and therefore SNPs arc the preferred markers for forensic and human identification applications for some applications. SNPs can be easily scored and are amenable to automation, making SNPs the markers of choice for large-scale forensic databases. SNPs are found in much greater abundance throughout the genome than repeat polymorphisms. Population frequencies of two polymorphic forms can usually be determined with greater accuracy than those of multiple polymorphic forms at multi-allelic loci.
  • STRs short tandem repeats
  • Cell-based assays include, but are not limited to, cells naturally expressing the SNP- containing nucleic acid molecule or recombinant cells genetically engineered to express the SNP-containing nucleic acid sequences.
  • nucleic acid encompasses DNA (deoxyribonucleic acid), RNA (ribonucleic acid) and RNAi.
  • the nucleic acid may be single-stranded, double- stranded or triple stranded.
  • the nucleic acid may be chemically modified.
  • the term also encompasses any known nucleic acid analogue, including but not limited to peptide nucleic acid (PNA), nucleic acid analog peptide (NAAP) and locked nucleic acid (LNA), as described below.
  • PNA peptide nucleic acid
  • NAAP nucleic acid analog peptide
  • LNA locked nucleic acid
  • the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc. Nucleic acids may utilize isocytosine and isoguanine to reduce non-specific hybridization.
  • the nucleic acid may be of almost any length, from oligonucleotides of two or more bases up to a full-length chromosomal DNA molecule.
  • the nucleic acid may be an oligonucleotide or a polynucleotide.
  • nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al, Tetrahedron 49(10): 1925 (1993); phosphorothioate (Mag et al, Nucleic Acids Res. 19:1437 (1991); phosphorodithioate (Briu et al, J. Am. Chem. Soc.
  • nucleic acid backbones and linkages see Egholm, J Am. Chem. Soc. 114:1895 (1992).
  • Other analog nucleic acids include those with bicyclic structures including locked nucleic acids (Koshkin et al, J. Am. Chem. Soc. 120:13252-3 (1998)); positive backbones (Denpcy et al, Proc. Natl. Acad. ScL USA 92:6097 (1995); non-ionic backbones (Kiedrowshi et al, Angew. Chem.
  • the double stranded (heteroduplex) nucleic acid described herein is typically composed of a nucleic acid probe and a target nucleic acid.
  • the degree of complementarity between the single nucleic acid strands need not be perfect and there may be base pair mismatches (eg. one or more mutations) which will interfere with the hybridization between the target sequence and the probe. If the degree of complementarity between the two single stranded nucleic acids is not high enough then a double stranded nucleic acid will not form.
  • the degree of stringency can be altered to control the extent to which double stranded nucleic acids form.
  • double stranded nucleic acid will form when the nucleic acid probe and the target nucleic acid are sufficiently complementary to hybridize under the reaction conditions.
  • the general steps of double stranded nucleic acid formation are to denature briefly the nucleic acid probe and/or the target nucleic acid to create single-stranded nucleic acid(s). These are then allowed to renature to regenerate duplex DNA in which the two strands reassociate. Denaturation/renaturation is most conveniently performed by heating the mixture to a temperature between about 90 °C and 100 °C, holding the mixture at the higher temperature for only a few seconds (to minimize potential damage to the nucleic acid), and then returning the temperature to a value of between about 4-25 0 C.
  • a time- and temperature-programmable thermal cycler - such as is used to control PCR - may be used for completing the double stranded nucleic acid formation.
  • target nucleic acid refers to a nucleic acid sequence - such as a gene (or a fragment or portion thereof), a regulatory sequence, genomic DNA 5 cDNA, RNA including mRNA and rRNA, or other nucleic acids as described herein.
  • the target sequence may be a target sequence from a sample, or a it may be a product of an amplification reaction. Nucleic acid probes may be made to hybridize to target sequences to determine the presence or absence of one or more mutations in the target nucleic acid.
  • the target nucleic acid may be derived or derivable from an individual or a tissue suspected of carrying one or more mutations.
  • the amplified target nucleic acid may be about 15 to about 10,000 nucleotides in length — such as about 15 to about 5,000 nucleotides in length, about 15 to about 2,500 nucleotides in length, about 15 to about 1,000 nucleotides in length, about 15 to about 500 nucleotides in length, about 15 to about 250 nucleotides in length, about 15 to about 100 nucleotides in length, about 15 to about 50 nucleotides in length, about 15 to about 25 nucleotides in length or about 20 nucleotides in length — such as 21, 22, 23, 24 or 25 nucleotides in length.
  • the target nucleic acid may be or may be derived from genomic nucleic acid - such as genomic nucleic acid from a eukaryote, a prokaryote.
  • the target nucleic acid may be or may be derived from genomic nucleic acid - such as genomic nucleic acid from a mammal, a human, a plant, a virus, a bacterium, a protozoan or a fungus.
  • the target nucleic acid is linear such that it does not fold back on itself to form a complementary double strand.
  • the target nucleic acid remains linear during the application of the potential and/or temperature ramp to the solid substrate.
  • the target nucleic acid does not form a hairpin or a stem-loop structure.
  • the amplification reaction may be applied to genomic DNA or, in the case of RT- PCR, to cDNA generated by applying a reverse transcription reaction to RNA, usually mRNA.
  • RNA usually mRNA.
  • RT-PCR is somewhat preferred over amplification of genomic targets simply because amplicon size can be maximized without concern about exon size.
  • intronic target sequence except possibly that close to the splice junctions, because the nitrons are often polymorphic in ways which have no known biological consequence.
  • Such "silent" polymorphisms can act as a background signal thereby reducing sensitivity to clinically important mutations which influence the amount and/or structure of mRNA and therefore of expressed protein.
  • each amplified target nucleic acid may be labelled with a detectably different label and so the origin and identify of the amplified target nucleic acid can be readily determined.
  • each of the amplified target nucleic acids are labelled with a detectably different label the origin and identify of the amplified target nucleic acid can be readily determined even though the amplified target nucleic acids have been pooled.
  • the term "detectably different label” means that each of the labels produces a different signal (eg. spectra) such that the identity of each individual label can be determined.
  • the identity of each individual label should be easily distinguishable using the detection methods that are used to detect the signal from the label.
  • the target nucleic acid is in contact with or attached to a solid substrate — such as an array.
  • the target nucleic acid may be labelled.
  • a label as defined herein, is a molecule or atom which, by its chemical nature, provides an identifiable (SER(R)S) signal intensity thereby allowing sensitive detection. Any label that is a SER(R)S active label is contemplated and may be any one of a number of molecules with distinctive Raman scattering patterns. A variety of such Raman labels are known in the art (see, for example, US 5,306,403; US 6,002,471; and US 6,174,677).
  • Non-limiting examples of labels that may be used for Raman spectroscopy include TRIT (tetramethyl rhodamine isothiol), NBD (7-nitrobenz-2-oxa- 1,3-diazole), Texas Red dye, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine, biotin, digoxigenin, 5-carboxy-4',5'-d ⁇ chloro-2',7'-dimethoxy fluorescein, 5-carboxy-2',4 l ,5',7 l -tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxy rhodamine, 6-carboxyrhodamine, 6-carboxytetramethyl amino phthalocyanines, azomethines, cyanines, xanthines, succinylfluoresc
  • Raman labels may be obtained from commercial sources (e.g., Molecular Probes, Eugene, Oreg.). Polycyclic aromatic compounds may function as Raman labels, as is known in the art. Other labels that may be of use include cyanide, thiol, chlorine, bromine, methyl, phosphorus and sulfur groups. In certain embodiments of the invention, carbon nanotubes may be of use as Raman labels. In one embodiment, the labels is a redox active (SERS/SERRS) label. By changing the potential, the oxidation state of the label will also change which will also alter the Raman spectrum. This is a particular advantage of the combined SERS and electrochemical approach described herein and may have significant benefits in terms of detection limit or quantification of the DNA.
  • SERS/SERRS redox active
  • Labels may be attached directly to the nucleic acids or may be attached via various linker compounds.
  • nucleotide precursors that are covalently attached to Raman labels may be purchased from standard commercial sources (eg., Roche Molecular Biochemicals, Indianapolis, Ind.; Promega Corp., Madison, Wis.; Ambion, Inc., Austin, Tex.; Amersham Pharmacia Biotech, Piscataway, NJ.).
  • Raman labels that contain reactive groups designed to covalently react with nucleic acids are commercially available (e.g., Molecular Probes, Eugene, Oreg.).
  • the target nucleic acid sequence is labelled at its 3' and/or 5' end.
  • the label is Texas Red or Cy5. Detection can be either qualitiative or quantitiative.
  • the label may be chemically modified.
  • the label eg. the SERS active label
  • the labelled target nucleic acid can only be detected by SERS when it is close to the solid surface. When it dehybridises it moves away form the surface and the signal will be substantially eliminated. Typically, (unlike fluorescence) the SERS effect is localised to molecules close (-100 nm) to the solid surface.
  • the target nucleic acid is detected by direct detection of incorporated nucleotides.
  • an intercalating, Raman label which binds to the double stranded nucleic acid may be used.
  • the label may be a groove-binding label or it may (weakly) bind to the DNA backbone.
  • purine nucleotides containing adenine or guanine moieties exhibit unique signals that can be detected by SERS or SERRS.
  • nucleoside triphosphate precursors may be incorporated into amplification product and tagged with one or more Raman labels to facilitate their detection, identification and/or quantification
  • nucleoside triphosphate precursors may be covalently modified with a reactive group, such as sulfhydryl, amino, carboxyl, maleimide, biotin and/or other reactive moieties known in the art.
  • the modified nucleotides may be tagged with one or more Raman labels, designed to covalently or non-covalently bind to the reactive groups.
  • Raman labels Another method to incorporate Raman labels is to conjugate the labels with oligonucleotide probes that have sequences complementary to the amplification product and hybridize the oligonucleotide to the amplification product.
  • multiple target nucleic acids such as multiple genes or segments or fragments thereof - may be analysed.
  • the multiple target nucleic acids may be analysed using multiple different labels — such as multiple different labels for different mutations.
  • the probes may be combined rather than arrayed with each of the multiple target nucleic acids distinguished by label and not position.
  • nucleic acid probe refers to a sequence of nucleic acids of known sequence which hybridises to (some portion of) the target nucleic acid sequence. Depending on whether the probe is complementary to the wild type or the mutant target sequence, the probe may sometimes be referred to as the "wild type probe” or the "mutant probe”.
  • the probes are designed to be complementary to a target sequence or an amplicon of the target sequence such that hybridization of the target sequence and the probes of the present invention occurs. This complementarity need not be perfect; that is, there may be any number of base pair mismatches which will interfere with hybridization between the target nucleic acid and the probe.
  • the nucleic acid probe is a linear nucleic acid probe.
  • the nucleic acid probe is linear such that it does not fold back on itself to form a complementary double strand.
  • the nucleic acid probe remains linear during the application of the potential and/or temperature ramp to the solid substrate.
  • the nucleic acid probe does not form a hairpin or a stem-loop structure.
  • SERS/SERRS SERS/SERRS
  • the nucleic acid probe may be labelled if the orientation of the label changes on binding of the target nucleic acid or if some other effect alters its SERS spectrum.
  • the nucleic acid probe may comprise a label - such as a SER(R)S label.
  • the nucleic acid probe is in contact with or attached to a solid substrate — such as an array. No particular limitation on the length of the probe is imposed provided that it hybridizes to the target nucleic acid.
  • the number of bases in the nucleic acid probe may be from 10 to 100 - such as from 10 to 75, 10 to 50, 10 to 25 or notably from 15 to 20 - such as 15, 16, 17, 18, 19 or 20 bases. For highest resolution on a solid substrate - such as an array - smaller oligonucleotides will result in the highest resolution.
  • the probe may comprise additional bases (eg. one additional base), for example, to prevent extension.
  • the nucleic acid probe is a control nucleic acid probe
  • the nucleic acid probe is a probe that can detect a mutant sequence. Accordingly, there is contemplated the use of at least two nucleic acid probes in accordance with the methods described herein.
  • control nucleic acid probe can be any probe which will result in a discernable difference between the signal obtained from the control probe and the signal obtained from the mutant probe
  • control nucleic acid probe is a probe that can detect a wild type sequence.
  • the nucleic acid probe may be in the form of a set or a combination of two or more probes.
  • the set of probes may be used to test for a plurality of associated mutations — such as a plurality of mutations that are associated with a specific disease; a plurality of mutations that are associated with the severity or prognosis of a disease; a plurality of mutations that are associated with a specific treatment regime; or a plurality of mutations that are associated with the identity of a specific genus, strain or species of an organism.
  • a plurality of associated mutations such as a plurality of mutations that are associated with a specific disease; a plurality of mutations that are associated with the severity or prognosis of a disease; a plurality of mutations that are associated with a specific treatment regime; or a plurality of mutations that are associated with the identity of a specific genus, strain or species of an organism.
  • a plurality of associated mutations such as a plurality of mutations that are associated with a specific disease; a plurality of mutations that are associated with the severity or prognosis of a
  • the same target nucleic acid may be amplified from a plurality of sources (eg. patient samples), with each target nucleic acid being amplified with a detectably different label. Accordingly, the origin of the source can be determined by the specific label that is used during amplification.
  • Each of the amplified target nucleic acids may optionally be pooled and contacted with an array. The results from the array can then be readily interpreted by correlating the result obtained with each of the different labels, which can then be used to interpret the results for each source.
  • the probe is synthesised with a dithiol linkage at the 5' end for contact to a solid substrate.
  • a sample containing the target nucleic acid may be a synthetic or natural sample.
  • the sample is derived or derivable from a patient.
  • reference herein to "a sample” excludes the step of physically obtaining said sample from a subject.
  • the sample may be a biological sample.
  • a “biological sample” may be any sample obtained or obtainable from a biological source and/or including material from a biological source.
  • the biological sample may originate or be derived from a cell, a tissue, an organ, a surgical or a biopsy specimen fixed or non-fixed such as bone marrow aspirates, or a biological fluid including body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genito-urinary tracts, tears, saliva, milk, white blood cells, and cell culture supernatants.
  • body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genito-urinary tracts, tears, saliva, milk, white blood cells, and cell culture supernatants.
  • the origin of the sample can be from an animal — such as a mammal or a human, a plant, a virus, a bacterium, a protozoan or a fungus.
  • the sample may be eukaryotic, prokaryotic, or acellular.
  • Cells comprised in the biological sample for example when coming from a tissue, organ, biological fluid or biopsy, may be cultivated in order to increase the number of available cells.
  • the sample may contain cells from a single type or of mixed cell type.
  • the cells, tissues and specimens may originate from normal individuals or from patient suspected to be suffering from or suffering from a disease or a disorder.
  • SERS Surface Enhanced Raman Scattering
  • SERRS Surface Enhanced Resonance Raman Scattering
  • the increase in intensity can be on the order of several million-fold or more.
  • Van Duyne J. Electroanal. Chem. 1977, 84, 1 was the first to recognize this effect as a unique phenomenon and coined the term "surface enhanced Raman scattering".
  • the SERS effect can be enhanced through combination with the resonance Raman effect.
  • the surface-enhanced Raman scattering effect is even more intense if the frequency of the excitation light is in resonance with a major absorption band of the molecule being illuminated.
  • the resultant Surface Enhanced Resonance Raman Scattering (SERRS) effect can result in an enhancement in the intensity of the Raman scattering signal of seven orders of magnitude or more.
  • SERRS Surface Enhanced Resonance Raman Scattering
  • nucleic acid is in contact or is attached to a solid substrate.
  • nucleic acid probe is in contact with or is attached to the solid substrate.
  • target nucleic acid is in contact with or is attached to the solid substrate.
  • the substrate can be modified to contain discrete individual sites appropriate for the attachment or association of nucleic acids.
  • the nucleic acid may be bound directly or indirectly.
  • the nucleic acid may be bound indirectly using a short linker (eg. an ethylene oxide chain) to the thiol which is attached to the surface
  • a short linker eg. an ethylene oxide chain
  • Many different metallic materials and configurations may be used for the solid substrate — such as the SERS active surface. These materials (for example, silver, gold, copper, platinum etc.) may take the form of flat surfaces (electrodes, strips, slides, etc.) or dispersed colloids, particles, droplets, ie.
  • the solid substrate is an electronic conductor for potential melting.
  • An attractive surface for SERRS based assays is metal colloid.
  • a metal colloid combines a very strong SERS activity with the advantage of a liquid medium that can readily be handled.
  • the combination of a SERS readout and a colloidal reagent would allow assays to be run in a manner similar to that used for present clinical chemistry analysis.
  • Another surface that may be used is a glass, impregnated with (conducting) metal particles. This surface may be a silver-impregnated glass, used as a substrate upon which an improved surface enhancement of Raman scattering can be achieved.
  • Certain glasses are commercially available which have a percentage of particulate silver embedded into their formulations.
  • One glass in particular has been developed by Corning Glassworks which may be particularly applicable to SERRS.
  • This product Corning No. 8612 Polarcor, has elongated crystalline silver embedded into its surface to a depth of approximately 35 micrometers.
  • the crystals are oriented in such a fashion so as to capitalize on the plasmon or resonant absorption effects of the silver conduction band electrons.
  • This distribution and orientation of the silver in this product is intended to behave as a polarizer.
  • Light of random polarization whose waves are aligned parallel to the long axes of the particles will be absorbed by the particles.
  • Light waves whose polar orientation is perpendicular to the long axes will be transmitted unattenuated.
  • the former case is the same condition that must exist to produce the pronounced enhancement as seen with SERRS.
  • the SERRS enhancement is considerably greater on a microscopically roughened surface than on a polished surface. This roughening provides for a certain percentage of the total surface area to have proper angular and distance components to absorb the correspondingly polarized component of the impinging light waves.
  • the aforementioned filled glass product satisfies the required conditions without the need for secondary processes like roughening. Side reactions such as oxidation, sulfide formation and photodegradation, which are known to occur on conventional pure silver surfaces, such as electrodes, are also avoided.
  • the product is produced with particle dimensions and spacial distributions to accommodate a relatively broad band of wavelengths. Several discrete band passes throughout the red and near infrared portion of the optical radiation spectrum are available.
  • the substrate used is formed by the electrodeposition of about 450 nm of gold through a close packed monolayer array of uniform spherical polystyrene particles (typically about 600 nm diameter) followed by dissolution of the template. This leaves a close packed hexagonal array of sphere segment voids which interact strongly with visible light.
  • a specific binding member coupled to a Raman active label and bound near the surface of this glass can potentially exhibit an even more pronounced SERRS effect than in conventionally used surfaces for the following reasons: (a) the spacings between the silver surface and the label are more uniform (since the particle orientation is far less random); (b) the orientation of the silver particles with respect to the polarization of the light waves can be made optimal by physically moving the glass and (c) the incident light and the Rayleigh scattered light should be almost totally absorbed into the filled glass, simplifying the removal of reflected or Rayleigh scattered light from the Raman signal.
  • the substrate is a metallic material in which are arranged voids that confine surface plasmons as described in WO02/42836.
  • the substrate is a metallic material in which are arranged voids that have the shape of truncated spheres and in one embodiment, have a diameter from about 50nm to 10,000nm as described in WO2006/027581.
  • nucleic acids can be attached or immobilized to a solid support in a wide variety of ways.
  • the nucleic acid/substrate interaction may be non-covalent — such as electrostatic, hydrophilic, or hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as, streptavidin to the support and the non-covalent binding of the biotinylated probe to the streptavidin.
  • the nucleic acid/substrate interaction may be "covalent" such that the two moieties are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds.
  • Covalent bonds can be formed directly between the nucleic and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the nucleic acid or both molecules.
  • Immobilization may also involve a combination of covalent and non-covalent interactions.
  • the nucleic acid may be attached to an array in a wide variety of ways.
  • the nucleic acids can either be synthesized first, with subsequent attachment to the array, or can be directly synthesized on the array.
  • the solid substrate can be reused by washing the surface to remove the target nucleic acids.
  • spectra may be collected from the same area of the substrate and for each spectrum the laser spot for detection may be moved on the substrate by about 1-2 ⁇ M.
  • data from one or more areas of the array may be captured using a camera.
  • nucleic acid probes are designed to be "substantially complementary" to a nucleic acid target sequence, such that hybridization of the target sequence and the probe(s) occurs.
  • This complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the probe. If the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence.
  • substantially complementary herein is meant that probes are sufficiently complementary to the target sequences to hybridize under normal reaction conditions, particularly high stringency conditions.
  • hybridisation conditions in the context of a nucleic acid sequence, means a nucleic acid sequence having a sequence relationship to a second nucleic acid sequence such that there is perfect alignment of Watson-Crick base pairs along the entire length of both nucleic acid sequences.
  • a variety of hybridisation conditions may be used in accoridnace with the methods described herein in order to control the degree of hybridisation. Typically, the hybridisation conditions are classified by the degree of "stringency”, including high, moderate and low stringency conditions. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringency may be controlled by altering one or more parameters — such as temperature, formamide concentration, salt concentration, chaotropic salt concentration, pH and/or organic solvent concentration, etc.
  • the Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes axe occupied at equilibrium).
  • “maximum stringency” typically occurs at about Tm -5 0 C; "high stringency” at about 5-10 °C below the Tm; “intermediate stringency” at about 10-20 °C below the Tm of the probe; and “low stringency” at about 20-25 °C below the Tm.
  • hybridization conditions are carried out under high ionic strength conditions, for example, using 6 x SSC or 6 x SSPE.
  • high stringency conditions hybridization may be followed by two washes with low salt solution, for example 0.5 x SSC, at the calculated temperature.
  • medium stringency conditions hybridization may be followed by two washes with medium salt solution, for example 2 x SSC.
  • low stringency conditions hybridization may be followed by two washes with high salt solution, for example 6 x SSC.
  • maximum stringency conditions may be used to identify nucleic acid sequences having strict identity or near-strict identity with the hybridization probe; while high stringency conditions may be used to identify nucleic acid sequences having about 80% or more sequence identity with the probe.
  • stringent conditions will typically be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 0 C for short probes (eg. 10 to 50 nucleotides) and at least about 60 °C for long probes (eg. greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • the hybridization conditions may also vary when a non-ionic backbone, i.e. PNA is used, as is known in the art.
  • cross-linking agents may be added after target binding to cross-link, i.e. covalently attach, the two strands of the hybridization complex.
  • Preferred embodiments of the present invention may use "competimers" on arrays, to reduce non-specific binding.
  • the methods described herein may be performed under desired stringency conditions.
  • the target nucleic acid may be an amplified nucleic acid.
  • the amplification method relies on an enzymatic chain reaction.
  • the amplification is an exponential amplification.
  • Many amplification methods have been described hi the literature, for example, general reviews of these methods can be found in Landegren, U., et al., Science 242:229-237 (1988) and Lewis, R., Genetic Engineering News 10:1, 54-55 (1990).
  • amplification methods include polymerase chain reaction (PCR), PCR in situ, ligase amplification reaction (LAR), ligase hybridisation, Q-beta bacteriophage replicase, transcription-based amplification system (TAS) and nucleic acid sequence- based amplification (NASBA) and in situ hybridisation.
  • Primers suitable for use in various amplification techniques can be prepared according to methods known hi the art.
  • Polymerase Chain Reaction (PCR) PCR is a nucleic acid amplification method described inter alia in U.S. Pat. Nos. 4,683,195 and 4,683,202. PCR consists of repeated cycles of DNA polymerase generated primer extension reactions.
  • the target DNA is heat denatured and two oligonucleotides, which bracket the target sequence on opposite strands of the DNA to be amplified, are hybridised. These oligonucleotides become primers for use with DNA polymerase.
  • the DNA is copied by primer extension to make a second copy of both strands.
  • PCR is a molecular biology tool, which must be used in conjunction with a detection technique to determine the results of amplification.
  • An advantage of PCR is that it increases sensitivity by amplifying the amount of target DNA by 1 million to 1 billion fold in approximately 4 hours.
  • PCR can be used to amplify any known nucleic acid hi a diagnostic context (Mok et al., (1994), Gynaecologic Oncology, 52: 247- 252).
  • Self-Sustained Sequence Replication (3SR) 3SR is a variation of TAS, which involves the isothermal amplification of a nucleic acid template via sequential rounds of reverse transcriptase (RT), polymerase and nuclease activities that are mediated by an enzyme cocktail and appropriate oligonucleotide primers (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874). Enzymatic degradation of the RNA of the RNA/DNA heteroduplex is used instead of heat denaturation. RNase H and all other enzymes are added to the reaction and all steps occur at the same temperature and without further reagent additions. Following this process, amplifications of 10 to 10 have been achieved in one hour at 42 0 C. Ligation Amplification (LAR/LAS)
  • This method uses DNA ligase and four oligonucleotides, two per target strand. This technique is described by Wu 3 D. Y. and Wallace, R. B. (1989) Genomics 4:560. The oligonucleotides hybridise to adjacent sequences on the target DNA and are joined by the ligase. The reaction is heat denatured and the cycle repeated.
  • RNA replicase for the bacteriophage Q ⁇ which replicates single- stranded RNA, is used to amplify the target DNA, as described by Lizardi et al. (1988) Bio/Technology 6:1197.
  • the target DNA is hybridised to a primer including a T7 promoter and a Q ⁇ 5' sequence region.
  • reverse transcriptase generates a cDNA connecting the primer to its 5' end in the process.
  • the resulting heteroduplex is heat denatured.
  • a second primer containing a Q ⁇ 3' sequence region is used to initiate a second round of cDNA synthesis.
  • T7 RNA polymerase then transcribes the double-stranded DNA into new RNA, which mimics the Q ⁇ . After extensive washing to remove any unhybridised probe, the new RNA is eluted from the target and replicated by Q ⁇ replicase. The latter reaction creates 10 fold amplification in approximately 20 minutes.
  • strand displacement amplification (SDA; Walker et al, (1992) PNAS (USA) 80:392) may be used.
  • the amplification steps are repeated for a period of time to allow a number of cycles, depending on the number of copies of the original target sequence and the sensitivity of detection, with cycles ranging from 1 to thousands, with from 10 to 100 cycles being preferred and from 20 to 50 cycles being especially preferred.
  • ARRAYS As used herein, the term "array” refers to plurality of probes or nucleic acid target sequences in an array format; the size of the array will depend on the composition and end use of the array. Arrays containing from about 2 different probes or nucleic acid target sequences to many thousands can be made. Generally, the array will comprise from two to as many as 100,000 or more. In addition, each array may comprise a control probe. In some arrays, multiple substrates may be used, either of different or identical composition. Thus for example, large arrays may comprise a plurality of smaller substrates.
  • an array of nucleic acid probes including the wild type and the mutant probe to a queried base is described that are attached or immobilized to a solid support.
  • an array of target nucleic acid sequences is described that are attached or immobilized to a solid support.
  • the spatial location of the array element that shares a complementary sequence with the target nucleic acid that is, whether the wild type or the mutant base occurs in the nucleic acid target may be determined. If both the wild type and the mutant probes give a signal on detection, it indicates the presence of a heterozygote in the nucleic acid sample.
  • an "array" of nucleic acid target sequences is described that are attached or immobilized to a solid support. Methods for the manufacture and use of arrays are known in the art and any such known method may be used. Different methods may be used to create arrays, including synthesis of nucleic acids directly on a chip surface.
  • nucleic acids may be synthesized or purified and then attached to a chip surface.
  • chip substrates that may be used to create arrays are commercially available (e.g., NanoChip.TM. System, Nanogen, San Diego, Calif.; GeneChip.RTM., Affymetrix, Santa Clara, Calif.).
  • Array technology and the various techniques and applications associated with it is described generally in numerous textbooks and documents. These include Lemieux et al., 1998, Molecular Breeding 4, 277-289, Schena and Davis. Parallel Analysis with Biological Chips, in PCR Methods Manual (eds. M. Tnm ' s, D. Gelfand, J.
  • a major application for array technology in the context of the present invention is the identification of gene mutations - such as the identification of a gene mutation in a plurality of (pooled) samples; the identification of a plurality of different gene mutations in a sample; or the identification of a plurality of different gene mutations in a plurality of (pooled) samples.
  • any library may be arranged in an orderly manner into an array, by spatially separating the members of the library.
  • a suitable library for arraying is a nucleic acid library (including DNA, cDNA and oligonucleotide libraries) - such as a nucleic acid probe library.
  • the members of the library are generally fixed or immobilised onto a solid phase, preferably a solid substrate as described herein.
  • the samples may be arranged in such a way that indexing (ie., reference or access to a particular sample) is facilitated.
  • indexing ie., reference or access to a particular sample
  • this not essential since reference to a particular sample may be achieved through the use of different labels on the target nucleic acid.
  • the members of the library are applied as spots in a grid formation. Common methods may be adapted for this purpose.
  • an array may be immobilised on the surface of a microplate, either with multiple samples in an area or well, or with a single sample in each area or well.
  • Various means of arranging and fixing are known in the art and include, for example, pipetting, drop-touch, piezoelectric means, ink-jet and bubblejet technology and electrostatic application.
  • photolithography may be utilised to arrange and fix the samples on the chip.
  • the samples may be arranged by being "spotted" onto the solid substrate; this may be done by hand or by making use of robotics to deposit the sample.
  • arrays may be described as macroarrays or microarrays, the difference being the size of the sample spots.
  • Macroarrays typically contain sample spot sizes of about 300 microns or larger and may be easily imaged by existing gel and blot scanners.
  • the sample spot sizes in microarrays are typically less than 200 microns hi diameter and these arrays usually contain thousands of spots.
  • microarrays may require specialized robotics and imaging equipment, which may need to be custom made. Instrumentation is described generally in a review by Cortese, 2000, The Engineer 14[11]:26. Techniques for producing immobilised libraries have been described in the art. Generally, most prior art methods described how to synthesise single-stranded nucleic acid molecule libraries, using for example masking techniques to build up various permutations of sequences at the various discrete positions on the solid substrate.
  • Format I cDNA probe (500-5,000 bases long) is immobilized to a solid surface using robot spotting and exposed to a set of targets either separately or in a mixture. This method is described hi Ekins and Chu (1999) Trends in Biotechnology 17, 217-218.
  • Format II an array of oligonucleotides (20 ⁇ 25-mer oligos) or peptide nucleic acid (PNA) probes is synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization. The array is exposed to target nucleic acid which is hybridized thereto.
  • PNA peptide nucleic acid
  • the array is an array of electrodes that can apply a potential to and/or pass electrons to or from species in solution.
  • Electrodes are known in the art and include, but are not limited to, certain metals and their oxides, including gold; copper; silver; chromium; titanium; platinum; palladium; silicon; aluminum; metal oxide electrodes including platinum oxide, titanium oxide, tin oxide, indium tin oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenum oxide, ruthenium oxides, and zinc oxide and tungsten oxide; conductive plastics (such as polymers like polythiophenes, polyacrylamide, polyanilines, polypyrroles, and metal impregnated polymers); and carbon (including glassy carbon electrodes, graphite and carbon paste).
  • a preferred electrode is one in which the reference is Ag and/or AgCl and the counter electrode is platinum (wire).
  • the electrodes may be present on a flat surface, which is only one of the possible conformations of the electrode.
  • the conformation of the electrode will vary with the detection method used. For example, flat planar electrodes may be preferred for optical detection methods or when arrays of nucleic acids are made, thus requiring addressable locations for detection. That is, each electrode has an interconnection attached to the electrode at one end and to a device that can control the electrode, on the other end thereby making each electrode independently addressable.
  • the nucleic acid probe(s) is contacted with the solid surface of the array and the target nucleic acid hybridised thereto. Accordingly, in one aspect there is provided a method for determining the presence of one or more mutations in a nucleic acid sample using an array, comprising the steps of: (a) providing target nucleic acid; (b) contacting one or more of the same or different nucleic acid probes with the solid surface of an array; (c) hybridising the target nucleic acid and the linear nucleic acid probe(s); (d) applying a potential and/or temperature ramp to the solid surface of the array; (e) monitoring the dehybridisation of the double stranded nucleic acid(s) across the potential and/or temperature ramp; and (f) determining if said sample comprises one or more mutations, wherein a difference in the dehybridisation of the double stranded nucleic acid across the potential and/or temperature ramp as compared to a control is indicative of the presence of a mutation
  • the target nucleic acid is contacted with the solid surface of the array and the nucleic acid probe(s) hybridised thereto.
  • a method for deteraiining the presence of one or more mutations in a nucleic acid sample using an array comprising the steps of: (a) providing a target nucleic acid; (b) contacting the target nucleic acid with the solid surface of an array; (c) hybridising one or more of the same or different nucleic acid probes to the target nucleic acid; (d) applying a potential and/or temperature ramp to the solid surface of the array; (e) monitoring the dehybridisation of the double stranded nucleic acid(s) across the potential and/or temperature ramp; and (f) determining if said sample comprises one or more mutations, wherein a difference in the dehybridisation of the double stranded nucleic acid across the potential and/or temperature ramp as compared to a control is indicative of the
  • an assay method for identifying an agent that modulates the dehybridisation of double stranded nucleic acid a difference between the dehybridisation in the presence and absence of said agent(s) is indicative that said agent(s) modulates dehybridisation and differences in the presence of different agents may be indicative of relative strengths of binding of the agent(s) with the nucleic acids.
  • DNA-binding molecules may be identified which have desirable drug properties.
  • the term "agent" may be a single entity or it may be a combination of entities.
  • the agent may be an organic compound or other chemical.
  • the agent may be a compound, which is obtainable from or produced by any suitable source, whether natural or artificial.
  • the agent may be an amino acid molecule, a polypeptide, or a chemical derivative thereof, or a combination thereof.
  • the agent may even be a polynucleotide molecule - which may be a sense or an anti-sense molecule.
  • the agent may even be an antibody.
  • the agent may be designed or obtained from a library of compounds, which may comprise peptides, as well as other compounds, such as small organic molecules.
  • the agent may be a natural substance, a biological macromolecule, or an extract made from biological materials such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic agent, a semi-synthetic agent, a structural or functional mimetic, a peptide, a peptidomimetics, a derivatised agent, a peptide cleaved from a whole protein, or a peptides synthesised synthetically (such as, by way of example, either using a peptide synthesizer or by recombinant techniques or combinations thereof, a recombinant agent, an antibody, a natural or a non-natural agent, a fusion protein or equivalent thereof and mutants, derivatives or combinations thereof.
  • the agent will be an organic compound.
  • the organic compounds will comprise two or more hydrocarbyl groups.
  • hydrocarbyl group means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. hi addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group.
  • the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen.
  • the agent comprises at least one cyclic group.
  • the cyclic group may be a polycyclic group, such as a non-fused polycyclic group.
  • the agent comprises at least the one of said cyclic groups linked to another hydrocarbyl group.
  • the agent may contain halo groups.
  • halo includes fluoro, chloro, bromo or iodo.
  • the agent may contain one or more of alkyl, alkoxy, alkenyl, alkylene and alkenylene groups — which may be unbranched- or branched-chain.
  • the agent may be capable of displaying other therapeutic properties.
  • STRs short tandem repeats
  • CAG CAG
  • GATA d(GATA)
  • TCTTA d(TCTTA) n .
  • Many of these loci are polymorphic, with the number of repeats varying between individuals and populations. This allows STRs to be used for human identification, forensic science, disease-related linkage analysis, and paternity/kinship testing.
  • the existing technique for STR analysis is based on fluorescence detection. It involves DNA extraction and amplification using 5'-fluorescently labelled PCR primers followed by electrophoretic separation and analysis.
  • the analytical procedure is carried out in a laboratory environment and requires specialized equipment and skilled technicians. As a result it is a time-consuming process. There is a parallel requirement for a simple and robust method of STR analysis for use at the scene-of-crime and custody suites where portable analysis is required.
  • the methods described herein may be used for the detection of PCR products from STR loci.
  • This method has several advantages over the conventional STR analysis.
  • several samples can be taken from a crime scene, and each amplified separately using primers with different SERS labels.
  • sample 1 blood sample
  • sample 2 hair sample
  • SERS label B primers labelled with SERS label B
  • the PCR products can then be combined, applied to the array, and the STR melting analysis carried out on the mixture of samples. Because of the unique nature of each SERS label, this will simultaneously generate several unique profiles on a single array. This could be particularly beneficial when analysing multiple samples at scenes of violent crime.
  • melting curve analysis by array based E-melting could also form the basis of a bespoke high-throughput system for STR analysis in non- forensic applications, for example in genetic/linkage analysis.
  • the STR method described above will be easily adaptable to the analysis of SNPs and mutations.
  • a method for analysing a nucleic acid sample comprising the steps of: (a) amplifying one or more short tandem repeat loci from a sample; (b) hybridising the amplified short tandem repeat loci to one or more nucleic acid probes to form a double stranded nucleic acid of which at least one of the single strands of nucleic acid is hi contact with a solid substrate; (c) applying a potential and/or temperature ramp to the solid substrate; (d) monitoring the dehybridisation of the double stranded nucleic acid across the potential and/or temperature ramp; and (e) determining the melting profile for the one or more short tandem repeat loci.
  • a method for DNA fingerprinting comprising the steps of: (a) amplifying one or more short tandem repeat loci from a sample; (b) hybridising the amplified (labelled) short tandem repeat loci to one or more nucleic acid probes to form a double stranded nucleic acid of which at least one of the single strands of nucleic acid is hi contact with a solid substrate; (c) applying a potential and/or temperature ramp to the solid substrate; (d) monitoring the dehybridisation of the double stranded nucleic acid across the potential and/or temperature ramp; and (e) obtaining a profile of the short tandem repeat loci. From country to country different STR based DNA profiling systems are hi use. For example, in North America systems which amplify the CODIS 13 core loci are almost universal, while hi the UK the SGM+ system is in use. Whichever system is used, many of the STR regions under test are the same.
  • the system is based around multiplex reactions, whereby many STR regions will be under test at the same time.
  • Organism identification is based around multiplex reactions, whereby many STR regions will be under test at the same time.
  • nucleic acid probes may be designed to determine the genus, species or strain of the bacterium, hi this context, an array may be designed which comprises nucleic acid probes ⁇ eg. 16S or 23 S rRNA probes) that are specific to different genera, species or strains of bacteria.
  • the target nucleic acid such as genomic or amplified nucleic acid - may be contacted with the array and the methods described herein carried out. By analysing the output of the results on the array, the skilled person will be able to determine the identity of the one or more organisms by determining which sequences are and are not present in the organism.
  • an array may be designed with multiple different probes to test for the presence of multiple different organisms — such as multiple different genera, species or strains of organism, hi one specific example, the methods described herein may be used to identify a strain of organism - such as methycillin resistant Staphylococcus aureus (MRSA).
  • MRSA methycillin resistant Staphylococcus aureus
  • the most common strains of MRSA at present are EMRSAl 5 and EMRSAl 6.
  • EMRSAl 6 carries the SCCmec type II, enterotoxin A and toxic shock syndrome toxin 1 genes. Under the new international typing system, this strain is now called MRSA252.
  • MRSA MRSA-associated CC8 strain
  • ST8:USA300 which carries mec type IV, Panton- Valentine leukocidin, PSM-alpha and enterotoxins Q and K and ST8:USA400.
  • Other community-associated strains of MRSA are ST8:USA500 and ST59:USA1000. Accordingly, arrays of nucleic acid probes which are specific to the specific genes and/or gene mutations associated with MRSA may be provided. Following the hybridization of the genomic nucleic acid or an amplified nucleic acid target from the test organism, the methods described herein can be used to determine the genotype of said bacterium. The organism may even be a virus.
  • Viruses can change their genetic information rapidly and typical examples include Influenza and Human Immunodeficiency viruses.
  • the methods described herein can therefore be used to readily type viruses. Such methods may have wide general applicability in the diagnostic setting where it is desirable to test a subject for the presence of organisms and bacteria - such as MRSA. hi particular, the methods described herein readily lend themselves to diagnostic testing on a large scale and at high throughput using multiple patient samples and multiple different probes in order to obtain an accurate and detailed result in a short period of time and at low cost.
  • Identification of oncogenes Many oncogenes have been characterised which are mutated. The methods described herein may allow a rapid assessment as to whether one or more oncogenes have a base change relative to another. Such indications may be of use in the clinical setting since determining a subject's genotype may also implicate which course and/or type of treatment - such as chemotherapy - is to be followed. Confirmation o/in vitro mutagenesis
  • the present invention employs, unless otherwise indicated, conventional techniques of chemistry, electrochemistry, molecular biology, microbiology and recombinant DNA technology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritscb, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N. Y.); B. Roe, J. Crabtree, and A.
  • a method for monitoring the dehybridisation of double stranded nucleic acid comprising the steps of: (a) providing a nucleic acid probe and a target nucleic acid; (b) forming a double stranded nucleic acid of which at least one of the strands of the nucleic acid is in contact with a solid substrate; (c) applying a potential and/or temperature ramp to the solid substrate; and (d) monitoring the dehybridisation of the double stranded nucleic acid across the potential and/or temperature ramp,
  • a method for determining the presence of one or more mutations in two or more nucleic acid samples comprising the steps of: (a) optionally amplifying target nucleic acids from two or more different samples; (b) optionally labelling each of the target nucleic acids with a different label; (c) optionally pooling the target nucleic acids; (d) contacting the solid surface of an array with the same or one or more different nucleic acid
  • a method for screening or testing a sample for the presence of a mutation comprising the steps of: (a) providing a sample of nucleic acid; and (b) using that nucleic acid as the target nucleic acid in carrying out in vitro the method according to the above-mentioned aspects.
  • a method for analysing a nucleic acid sample comprising the steps of: (a) amplifying one or more short tandem repeat loci from a sample; (b) hybridising the amplified short tandem repeat loci to one or more nucleic acid probes to form a double stranded nucleic acid of which at least one of the single strands of nucleic acid is in contact with a solid substrate; (c) applying a potential and/or temperature ramp to the solid substrate; (d) monitoring the dehybridisation of the double stranded nucleic acid across the potential and/or temperature ramp; and (e) determining the melting profile for the one or more short tandem repeat loci.
  • a method for deterrnining the identity or genotype of an organism comprising the steps of: (a) optionally amplifying one or more target nucleic acid sequences from the target organism; (b) hybridising the target nucleic acid(s) to one or more nucleic acid probes; (c) applying a potential and/or temperature ramp to the solid substrate; (d) monitoring the dehybridisation of the double stranded nucleic acid across the potential and/or temperature ramp; (e) determining which nucleic acid probes hybridise to the amplified target nucleic acid; and (f) determining the identity or genotype of said organism.
  • an assay method for identifying an agent that modulates the dehybridisation of double stranded nucleic acid comprising the steps of: (a) providing a nucleic acid probe, a target nucleic acid and at least agent; (b)forming a double stranded nucleic acid of which at least one of the strands of the nucleic acid is in contact with a solid substrate in the presence and absence of said agent; (c) applying a potential and/or temperature ramp to the solid substrate; (d) monitoring the dehybridisation of the double stranded nucleic acid across the potential and/or temperature ramp in the presence and absence of said agent; wherein a difference between the dehybridisation in the presence and absence of said agent is indicative that said agent modulates dehybridisation.
  • Example 1 Materials & Methods Fabrication of substrates (illustrated in Figure 5)
  • glass microscope slides were coated with a 10 nm chromium adhesion layer followed by 200 nm of gold using standard thermal evaporation technique. Thereafter, pieces were cut out from the gold coated slide, typically, measuring 2 cm x 1.5 cm. These were then cleaned by sonicating in isopropanol for 90 minutes, washed and dipped in 10 mM cysteamine in ethanol for at least 48 hours. Subsequently, these treated surfaces are washed with water and assembly of polystyrene spheres (obtained from Duke Scientific) was carried out.
  • the method is a modification of the convective self-assembly method of Nagayama et al. and was carried out to result in a monolayer of hexagonally close packed spheres (27, 28). This served as the template for the formation of ordered spherical cavities.
  • Electrodeposition was carried out using a commercial gold electroplating bath (Tech. Gold 25, Technic Inc.) to which an additive (ECF 60, Technic Inc.) was added for a bright and smooth finish. A fixed area was masked and the charge required for a particular height was calculated. Electrodeposition was carried under potentiostatic control at -0.73 V vs. a home-made saturated calomel electrode (SCE) with a platinum counter electrode.
  • SCE saturated calomel electrode
  • the deposition was terminated when the requisite amount of charge had been passed. Thereafter, the polystyrene spheres were removed by dissolution in DMF to obtain ordered spherical cavity gold substrates, hi the present work 600 nm diameter spheres were employed and the gold film was electrodeposited to 450 nm.
  • Oligonucleotides synthesis Standard DNA phosphorarnidites, solid supports and additional reagents were purchased from Link Technologies Ltd, Sigma and Applied Biosy stems Ltd.
  • C7-aminoalkyl coupling and C7-fluorescein (FAM) synthesis columns were obtained from Link Technologies Ltd and Cy5 and dithiol phosphorarnidites from Glen Research Inc.
  • oligonucleotides were synthesized on an Applied Biosystems 394 automated DNA/RNA synthesizer using a standard 0.2 or 1.0 ⁇ mole phosphoramidite cycles of acid-catalyzed detritylation, coupling, capping and iodine oxidation. Stepwise coupling efficiencies and overall yields were determined by the automated trityl cation conductivity monitoring facility and in all cases were >98.0%. All ⁇ -cyanoethyl phosphoramidite monomers were dissolved in anhydrous acetonitrile to a concentration of 0.1 M immediately prior to use.
  • the coupling time for normal (A 5 G 5 C 3 T) monomers was 25 s and the coupling time for the cy5 and dithiol monomers was extended to 360 s.
  • 3 '-Dye-labelled oligonucleotides were synthesized by postsynthetic labeling of 3'-aminoalkyl oligonucleotides which were synthesized using C7- aminolink cpg. These oligonucleotides were labelled with the NHS esters of Cy3, Cy 5 or Texas Red as described below.
  • the Cy5 chromophore is unstable to prolonged exposure to ammonia so 5 '-Cy 5 oligonucleotides were synthesized using fast deprotecting dmf-G and Ac-dC phosphoramidite monomers, cleaved from the resin by treatment with concentrated aqueous ammonia for 60 min at room temperature then deprotected for 1 h at 55 0 C. 3 '-Oligonucleotide labeling
  • oligonucleotides Purification of oligonucleotides was carried out by reversed-phase HPLC on a Gilson system using an ABI Aquapore column (C8), 8 mm x 250 mm, pore size 300 A. The system was controlled by Gilson 7.12 software and the following protocols were used: Run time 30 mins, flow rate 4 mL per min, binary system, ramp: Time in mins (% buffer B);0 (0); 3 (0); 5 (10); 21 (40); 21(60)* 25 (100); 27 (0); 30 (0). Elution buffer A: 0.1 M ammonium acetate, pH 7.0, buffer B: 0.1 M ammonium acetate with 50% acetonitrile pH 7.0.
  • % buffer B at 21 min was 40% for all oligonucleotides except those containing the hydrophobic Cy3, Cy 5 and Texas Red dyes which required 60% buffer B). Elution of oligonucleotides was monitored by ultraviolet absorption at 295 nm. Texas Red oligonucleotides gave two product peaks corresponding to the 5- and 6- regioisomers of Texas Red. The first peak (5-isomer) was collected and used in the subsequent SERS experiments and the second peak was discarded. After HPLC purification oligonucleotides were desalted using NAP-10 Sephadex columns (GE Healthcare), aliquoted into eppendorf tubes and stored at -20 0 C. All oligonucleotides were characterized by MALDI-TOF mass spectrometry. PCR amplification
  • a synthetic DNA template (50 ng) containing the wild type sequence or the ⁇ F508 mutation was amplified in a sample volume of 20 ⁇ L, containing 0.2 ⁇ M 5' Cy5 labelled forward primer, 0.05 ⁇ M reverse primer, 1 mM dNTPs (Promega, UK), 2.5 mM MgCl 2 and 1 unit hot start DNA polymerase (Eppendorf HotMaster ® ) in 1 x PCR buffer.
  • the PCR reactions were performed using a thermocycler (Eppendorf Mastercycler Gradient) with a denaturation step of 95 0 C for 2 min, followed by 15 cycles of 94 0 C for 20 s, 54 0 C for 45 s and 70 °C for 45 s.
  • the sequences of the oligonucleotides are shown in Table 1. Immobilization of probe oligonucleotides on the substrate
  • 1 ⁇ M solutions of the dithiol modified 25-mer oligonucleotides were prepared in pH 8.1 phosphate buffer containing 0.1 M NaCl. It was extremely important that the substrates were clean. This was ensured by thoroughly cleaning them by sonicating in DMF for at least 3 h. Thereafter, they were immediately transferred to de-ionized water and sonicated for another 15 min. The substrates were preserved in water until used. For immobilizing the probe oligonucleotides, the substrates were dipped in the 1 ⁇ M solutions and kept in a refrigerator maintained at 6°C for 48 h.
  • the substrates were taken out and washed with pH 8.1 phosphate (0.1 M NaCl) buffer several times and dipped in 0.01 M mercapto-hexanol solution prepared in pH 8.1 phosphate (1 M NaCl) buffer for 20 min. This ensured that any non-specific binding of the target oligonucleotides with the substrate was prevented.
  • the substrates were taken out and rinsed with pH 8.1 phosphate (0.1 M NaCl) buffer several times and employed for detection or mutation analysis.
  • Surface coverage determination of probe on gold surface The well known procedure of Steel et al. based on coloumetry was employed to determine the coverage of immobilized probes on gold surfaces using [Ru(NH 3 ) 6 ] 3+ as the redox marker (29).
  • the [Ru(NH 3 ) 6 ] 3+ cation is expected to localize on the surface, bound to the phosphate groups of the single strand.
  • a 500 ms pulse from an initial potential of 0.1 V vs. SCE to -0.4 V vs. SCE was employed for reducing [Ru(NH 3 ) 6 ] 3+ .
  • Coulometric curves and the adsorbtion isotherm for [Ru(N ⁇ 3 ) 6 ] 3+ is presented in Figure 6. The intercepts of the coulometric curves without and in presence of [Ru(NH 3 ) 6 ] 3+ yield the double layer charge (g d i) and the surface excess related charge (nFATo), respectively (refer equation 1).
  • the electrochemically induced melting experiments were carried out in custom designed electrochemical micro-Raman cell. It utilizes a horizontal geometry for viewing under the Raman microscope maintaining a thin 150 micron thin liquid film on the substrate while the reference (Ag/ AgCl) and counter (Platinum wire) electrodes are placed on the side. It is possible to flow solutions over the substrate during an experiment without disturbing the position of the substrate.
  • a heating element is provided in the body of the cell and can be controlled with a microcontroller circuit. Two thermocouple probes are incorporated, with digital display, for controlling and monitoring the heating temperature and the temperature of the substrate, respectively.
  • the substrates were dipped in pH 7, 10 mM TRIS buffer and the buffer was flown over the substrates several times in the cell at open circuit potential.
  • the experiments were carried out at room temperature.
  • a potential sequence was applied, typically, starting at -0.2 V followed by -0.1 V decrement until - 1.3 V or less.
  • the potential was held at each step for 300 s.
  • All electrochemical studies were carried out employing an ⁇ coChemie ⁇ Autolab potentiostat/galvanostat. Raman spectra were also recorded every 300 s with the first spectrum recorded 250 s after the beginning of the potential pulse.
  • Raman spectra were acquired using a 5Ox objective (NA: 0.75) on a Renishaw 2000 Raman microscope instrument equipped with a 632.8 nm He-Ne laser.
  • the Raman microscope system has a motorized stage with a precision XYZ stage controller.
  • Raman spectra were acquired from a 4 ⁇ m x 4 ⁇ m or larger area on the substrate with the laser being moved 1-2 ⁇ m each time using the stage controller. This was done to avoid any bleaching effects of the dye and was aided by the fact that signals on our substrates are reproducible. Nevertheless, suitable control experiments were however, carried out to check this aspect.
  • spectra for the targets with the Texas Red label spectra were acquired for 10-30 s and for Cy5 labelled oligonucleotides it was 1-10 s, under static mode centred at 1400 cm “1 or 1450 cm “1 with the laser power measured at the sample being 3 mW.
  • the entire procedure for thermal melting for detecting mutations was essentially similar to that described for Emelting except that no potential sequence was applied while the temperature was ramped up from room temperature at 1 0 C per minute to 6O 0 C or as appropriate in pH 8.1, 0.01 M phosphate buffer with 0.1 M NaCl was used. The temperature of the substrate was monitored and noted down at the instance the acquisition of a Raman spectra was started.
  • a DNA mutation discrimination method (termed SERS-melting) is reported that uses SERS to follow denaturation on an ordered sphere segment cavity gold electrode. Denaturation is driven by either electrochemical or thermal changes and followed by SERS; differences in the melting potential or temperature allow the discrimination of mutations. As 'proof of concept', using this method it was possible to distinguish between the ⁇ F508 (triple deletion), 1653C/T (point) mutation and wild-type human Cystic Fibrosis Transmembrane Regulator (CFTR) gene with differences between the SERS-melting potentials of the mutations of ⁇ 110 and 60 mV and differences between their SERS-melting temperatures of ⁇ 11 and 4 0 C from the perfect wild type match. The practical utility of this method is demonstrated by using it to differentiate between PCR products of a mutated and wild type sequence.
  • CFTR Cystic Fibrosis Transmembrane Regulator
  • the allele specific 25-mer probes for detecting the wild type and mutated sequences were synthesized with dithiol linkages at the 5 '-end for attachment to the gold surface. Similarily, the oligonucleotide targets were synthesized by standard solid-phase techniques and labelled at the 3 '-end with common fluorophores, which served as SERS labels. The probe and target sequences are shown in Figure IB.
  • the targets were detected in a hybridization assay; the entire scheme for detection and mutation discrimination is displayed in Figure 1C.
  • SER(R)S detection was carried out in a custom designed cell with electrochemical and temperature control.
  • Hybridization of the labelled targets was carried out in 10 ⁇ M solutions for 20 min in pH 8.1 phosphate buffer containing 0.1 M NaCl. Thereafter the substrates were rinsed with the buffer at least 3 to 4 times, hi experiments to discriminate mutations the same substrate was used each time with the different targets and spectra were collected from the same area on the substrate although for each spectrum the laser spot was moved on the substrate by 1-2 ⁇ m. This not only reduced any bleaching and photodegradation effects but also serves to demonstrate that the SERS sensitivity, surface modification and melting effects are reproducible across our substrates. In fact the substrates were reusable and no change in melting characteristics was observed on repeated measurements.
  • SERS-Emelting SERS-electrochemical-melting experiment
  • 10 mM TRIS buffer, pH 7 was flowed over the substrate several times and the surface equilibrated at open circuit potential for several min. Thereafter, a potential sequence was applied; in this particular example, the potential was decreased in steps from -0.2 V to -1.5 V.
  • SER spectra were recorded at each potential step after a fixed time interval of 250 s.
  • the label for both the perfect match and the mutations was the dye Texas Red. Texas Red has an absorption maximum at 596 nm therefore it is in pre-resonance with the 633 run laser used in the current work. In principle any molecule with a high Raman cross section could be employed.
  • the intensity of the peak for the SERS marker increases as the potential becomes more negative from -0.2 to -0.8 V before then decreasing and ultimately falling to zero at potentials more negative than -0.8 V.
  • the initial increase is reversible and could be due to a potential dependent change in the orientation of the dsDNA and the 3' Texas Red label. It is also possible to distinguish mutations by following the temperature induced dehybridization, SERS-Tmelting, on these substrates.
  • the experimental methodology is similar to that used for SERS-Emelting except that pH 8.1 phosphate buffer containing 0.1 M salt was used. Instead of changing the potential the temperature was ramped at 1 °C/min to induce dehybridization.
  • Cy5 is a cyanine dye with an absorption maximum of 647 nm it is therefore resonant with the 633 nm laser used to record the spectra.
  • the signals for the SERRS enhanced Cy5 peaks are 4-5 times higher than the partially resonant Texas Red peaks shown in figure 2, which is expected.
  • the SERRS spectra at various potentials are presented in figure 3A and normalized melting profiles for four peaks of Cy5 are plotted in figure 3B.
  • SER(R)S provides vibrational spectroscopic data manifesting in different peaks, each of which can be monitored, thus allowing for multiplexability besides giving more insight on the state of the molecule, and therefore, dehybridization in this case.
  • the intensity of the peaks decreases with potential but we note that the peak positions do not shift significantly with change in potential, which is expected as the dye is not attached to the surface.
  • Cy5 as the label for the complementary target, the electrochemical melting profile shifts to less negative potentials when compared with those obtained with Texas Red labelled oligonucleotides.
  • the choice of the label provides another means of manipulating denaturation on the surface.
  • Cy5 has a lower pKa than Texas Red; hence, Cy5's negatively ionized state prevents it from coming closer to the surface as potential is scanned negative. Therefore, the Cy5 label initiates dehybridization at lower potentials (and temperature) compared to Texas Red labelled oligonucleotides. This is also consistent with the observation that with Cy5 the initial increase in intensity is not observed as with Texas Red and that the melting profiles for Texas Red labelled oligonucleotides are sharper compared to Cy5 labelled sequences. This highlights the destabilizing influence of the 'negatively' charged label, Cy5, compared to the stabilizing influence of Texas Red on duplexes attached to the surface.
  • SERS-melting a new method for distinguishing mutations employing SERS detection (termed SERS-melting) on ordered gold sphere segment cavity substrates and we have demonstrated its potential utility in a real application.
  • Many practical applications are envisaged leveraging the advantages of SER(R)S over fluorescence and other detection methods especially in being able to combine immense molecule-specific information with high sensitivity.
  • the other advantage of our SERS substrate approach is its fundamental suitability for high throughput analysis, miniaturization and the possibility of multidimensional control of the denaturation process by electrochemical potential, temperature and/or chemical parameters.
  • SERS-melting could prove to be an enabling technology in the fields of diagnostics, genomics and forensic science.
  • Example 3 Reproducibility of SERS-melting characteristics and re-usability of substrates
  • Example 4 Current profiles obtained during Emelting ( Figure 10). Amperograms obtained during the electrochemical denaturation of hybrids of the PCR products, both the ⁇ F508 mutation and the wild type, on our sphere segment cavity substrates are presented. Current densities are very low and mostly indicating capacitive currents, though after -0.7 V some steady state current is observed possibly due to some hydrogen evolution or oxygen reduction etc.
  • Example 5 - PCR control experiment
  • the STR analysis method involves already optimized PCR amplification of the standard panel of human forensic STR loci using the primer sequences used in the SGM+ kit ⁇ Forensic Science International (2000), 112, 151), substituting SERS labels for fluorescent dyes.
  • This multiplex will then be applied to an array of oligonucleotide probes immobilized on a structured gold surface which is complementary to the STR amplicons, and also contains the mixed sequence regions adjacent to the STR' s to act as anchors.
  • These probes will have the same number of repeats as the largest commonly encountered STR for each locus.
  • the common D16S539 alleles comprise 8 to 14 repeats of d(GATA), with reported allele frequencies exceeding 0.01.
  • the SERS-labelled PCR products will be hybridized to probes on the array and this will be greatly accelerated by applying a potential to the gold surface. Upon completion of hybridization the potential at the surface will be reversed to promote controlled de-hybridisation (melting) of the STR amplicons. During this process, prominent features of the SERS spectrum of the label will be monitored during irradiation with a laser light source. This data will be converted into a melting curve, and then to its first derivative which will define the melting potential (E m ) of each of the STRs. The position on the array will define the STR locus. The E m of each STR will vary according to the number of tandem repeats, and the combination of melting temperatures for each locus will provide a unique profile for an individual. REFERENCES 1. R. T. Ranasinghe, T. Brown, Chem. Commun., 5487 (2005).
  • the present invention provides methods as defined in the following numbered paragraphs:
  • a method for monitoring the dehybridisation of double stranded nucleic acid comprising the steps of: (a) providing a nucleic acid probe and a target nucleic acid;
  • nucleic probe is a linear nucleic acid probe and/or the target nucleic acid comprises a label.
  • a method for determining the presence of a mutation in a nucleic acid sample comprising the steps of: (a) providing a nucleic acid probe and a target nucleic acid;
  • nucleic probe is a linear nucleic acid probe and/or the target nucleic acid comprises a label.
  • the solid substrate is a solid metallic substrate which shows surface enhancement properties. 5.
  • the substrate is an ordered sphere segment cavity fabricated by templated electrodeposition.
  • a method for determining the presence of one or more mutations in two or more nucleic acid samples comprising the steps of: (a) optionally amplifying target nucleic acids from two or more different samples;
  • nucleic probe is a linear nucleic acid probe and/or the target nucleic acid comprises a label.
  • each of the target nucleic acids is labelled with a detectably different label.
  • each area or well in use on the array comprises at least one nucleic acid probe for detecting a different mutation.
  • each area or well in use on the array comprises different probes for detecting a different mutation that is known to be associated with at least one disease.
  • a method for screening or testing a sample for the presence of a mutation comprising the steps of:
  • a method for analysing a nucleic acid sample comprising the steps of:
  • nucleic probe is a linear nucleic acid probe and/or the short tandem repeat loci comprises a label.
  • a method for determining the identity or genotype of an organism comprising the steps of: (a) optionally amplifying one or more target nucleic acid sequences from the target organism;
  • nucleic probe is a linear nucleic acid probe and/or the target nucleic acid comprises a label.
  • An assay method for identifying an agent that modulates the dehybridisation of double stranded nucleic acid comprising the steps of:
  • nucleic probe is a linear nucleic acid probe and/or the target nucleic acid comprises a label.

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Abstract

La présente invention concerne, dans un aspect, un procédé pour surveiller la déshybridation d’acide nucléique bicaténaire, comprenant les étapes consistant à : (a) fournir une sonde d’acide nucléique et un acide nucléique cible; (b) former un acide nucléique bicaténaire dont au moins un des brins de l’acide nucléique est en contact avec un substrat solide; (c) appliquer une rampe de potentiel au substrat solide; et (d) surveiller la déshybridation de l’acide nucléique bicaténaire le long de la rampe de potentiel, caractérisé en ce que la sonde nucléique est une sonde d’acide nucléique linéaire et/ou l’acide nucléique cible comprend un marqueur.
PCT/GB2009/000541 2008-02-27 2009-02-27 Procédé pour surveiller la déshybridation d’acide nucléique bicaténaire WO2009106834A1 (fr)

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Citations (3)

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US6245508B1 (en) * 1993-11-01 2001-06-12 Nanogen, Inc. Method for fingerprinting utilizing an electronically addressable array
US20050147976A1 (en) * 2003-12-29 2005-07-07 Xing Su Methods for determining nucleotide sequence information

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US6245508B1 (en) * 1993-11-01 2001-06-12 Nanogen, Inc. Method for fingerprinting utilizing an electronically addressable array
US5721102A (en) * 1995-10-13 1998-02-24 Lockheed Martin Energy Systems, Inc. Surface enhanced Raman gene probe and methods thereof
US20050147976A1 (en) * 2003-12-29 2005-07-07 Xing Su Methods for determining nucleotide sequence information

Non-Patent Citations (3)

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CHUMANOV G ET AL: "SURFACE ENHANCED RAMAN SCATTERING FOR DISCOVERING AND SCORING SINGLE BASE DIFFERENCES IN DNA", PROCEEDINGS OF THE SPIE, SPIE, BELLINGHAM, VA, US, vol. 3608, 25 January 1999 (1999-01-25), pages 204 - 210, XP001183203, ISSN: 0277-786X *
GREEN MINO ET AL: "SERS PLATFORMS FOR HIGH DENSITY DNA ARRAYS", FARADAY DISCUSSIONS, ROYAL SOCIETY OF CHEMISTRY, CAMBRIDGE, GB, vol. 132, 1 January 2006 (2006-01-01), pages 269 - 280,DISCU, XP009072997, ISSN: 0301-7249 *
STOKES ROBERT J ET AL: "Quantitative enhanced Raman scattering of labeled DNA from gold and silver nanoparticles", SMALL, WILEY VCH, WEINHEIM, DE, vol. 3, no. 9, 1 September 2007 (2007-09-01), pages 1593 - 1601, XP002479365, ISSN: 1613-6810 *

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