WO2016142687A1 - Analyse d'acide nucléique impliquant des sondes d'acide nucléique ancrées dans la surface - Google Patents

Analyse d'acide nucléique impliquant des sondes d'acide nucléique ancrées dans la surface Download PDF

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Publication number
WO2016142687A1
WO2016142687A1 PCT/GB2016/050617 GB2016050617W WO2016142687A1 WO 2016142687 A1 WO2016142687 A1 WO 2016142687A1 GB 2016050617 W GB2016050617 W GB 2016050617W WO 2016142687 A1 WO2016142687 A1 WO 2016142687A1
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Prior art keywords
nucleic acid
acid probe
length
probe
nucleotides
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PCT/GB2016/050617
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English (en)
Inventor
Philip Nigel Bartlett
Tom Brown
Evanthia Papadopoulou
Nittaya Gale
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University Of Southampton
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Publication of WO2016142687A1 publication Critical patent/WO2016142687A1/fr

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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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/60—Detection means characterised by use of a special device
    • C12Q2565/607—Detection means characterised by use of a special device being a sensor, e.g. electrode
    • 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
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/60—Detection means characterised by use of a special device
    • C12Q2565/632—Detection means characterised by use of a special device being a surface enhanced, e.g. resonance, Raman spectrometer

Definitions

  • the present invention relates to a method of nucleic acid analysis, in particular using surface- anchored nucleic acid probes, and associated uses thereof.
  • nucleic acid arrays on solid surfaces have been extensively used in gene expression profiling and the detection of microorganisms using a range of optical (e.g. fluorescence, surface plasmon resonance (SP ), surface enhanced Raman spectroscopy) 1 and electronic sensing platforms.
  • optical e.g. fluorescence, surface plasmon resonance (SP ), surface enhanced Raman spectroscopy
  • a short single-stranded nucleic acid is immobilised on an assay surface via one end, adopting vertical orientation, to promote target binding.
  • end-tethered DNA doesn't necessarily adopt a fixed and upright orientation at the surface, a variety of triggers such as surface potential, 3,4 temperature, pH, ionic strength, 4 the length of the DNA and the nature of the DNA-end that is tethered 5 can influence the orientation and tilting angles on the surface.
  • the high probe density of end-tethered DNA molecules on the surface can lead to slow hybridization kinetics and low hybridization efficiencies, 6 this problem is more intense when real DNA fragments are used as targets (PCR products are usually >100 base pairs).
  • Optimization of the probe density 7 and the use of hybridization rate accelerators (e.g. dextran sulphate, CTAB) 8,9 have been employed to improve the hybridization efficiencies.
  • an aim of the present invention is to improve nucleic acid analysis requiring the use of a nucleic acid probe.
  • a method of analysing nucleic acid in a sample comprising:
  • nucleic acid probe which is anchored to a substrate surface from a point located in a mid-region of the nucleic acid probe
  • a method of analysing nucleic acid in a sample comprising: -providing a nucleic acid probe, which is anchored to a substrate surface from a nonterminal residue of the nucleic acid probe; and
  • the invention allows the specific immobilization of the nucleic acid in a horizontal orientation relative to the substrate surface.
  • Horizontally-immobilised nucleic acid on the substrate surface has several advantages such that it can lead to inherent lower probe density on the surface with increased hybridization efficiency; it can locate the nucleic acid backbone closer to a sensor surface allowing increased sensitivity; and it can allow the nucleic acid to adopt a more fixed orientation on the surface that is less susceptible to external parameters (i.e. pH, surface potential, temperature).
  • the invention provides a simple, straightforward methodology for specific tethering of DNA probes which can use a single anchor, and wherein the anchor can be placed approximately in the middle of the probe to ensure horizontal orientation of the attached nucleic acid.
  • a successful hybridisation of the nucleic acid probe to the target nucleic acid sequence can indicate the presence of the target nucleic acid in the sample.
  • the failure to hybridise the nucleic acid probe to the target nucleic acid sequence may indicate the absence of the target nucleic acid in the sample, or at least the absence of detectable quantities thereof.
  • the method may further comprise detecting the amount or concentration of the target nucleic acid in the sample.
  • Providing a nucleic acid probe molecule anchored to a substrate may comprise
  • nucleic acid probe may comprise or consist of nucleic acid selected from any one of the group comprising DNA, NA, and a nucleic acid analogue, such as PNA or LNA; or combinations thereof.
  • the nucleic acid probe may comprise or consist of DNA.
  • the nucleic acid probe may comprise or consist of PNA.
  • the nucleic acid probe may be at least about 8 nucleotides in length.
  • the nucleic acid probe may be at least about 10 nucleotides in length.
  • the nucleic acid probe may be at least about 12 nucleotides in length.
  • the nucleic acid probe may be at least about 15 nucleotides in length.
  • the nucleic acid probe may be about 20 nucleotides in length.
  • the nucleic acid probe may be no more than about 15 nucleotides in length.
  • the nucleic acid probe may be no more than about 20 nucleotides in length.
  • the nucleic acid probe may be no more than about 30 nucleotides in length.
  • the nucleic acid probe may be no more than about 40 nucleotides in length.
  • the nucleic acid probe may be no more than about 100 nucleotides in length.
  • the nucleic acid probe may be no more than about 150 nucleotides in length.
  • the nucleic acid probe may be between about 8 and about 150 nucleotides in length.
  • the nucleic acid probe may be between about 8 and about 100 nucleotides in length.
  • the nucleic acid probe may be between about 8 and about 80 nucleotides in length.
  • the nucleic acid probe may be between about 8 and about 50 nucleotides in length.
  • the nucleic acid probe may be between about 8 and about 35 nucleotides in length.
  • the nucleic acid probe may be 30 between about 8 and about 30 nucleotides in length.
  • the nucleic acid probe may be between about 8 and about 25 nucleotides in length.
  • the nucleic acid probe may be about 30 nucleotides in length.
  • a plurality of probe nucleic acid molecules may be provided.
  • the nucleic acid probes in a plurality of probes may vary in length.
  • a plurality of nucleic acid probes may have an average length of about 30 nucleotides.
  • a plurality of nucleic acid probes may have an average length of between about 8 and about 50 nucleotides.
  • a plurality of nucleic acid probes may have an average length of between about 8 and about 60 nucleotides.
  • a plurality of nucleic acid probes may have an average length of between about 8 and about 80 nucleotides.
  • a plurality of nucleic acid probes may have an average length of between about 8 and about 100 nucleotides.
  • a plurality of nucleic acid probes may have an average length of between about 8 and about 150 nucleotides.
  • a plurality of nucleic acid probes may have an average length of between about 8 and about 35 nucleotides.
  • a plurality of nucleic acid probes may have an average length of between about 8 and about 25 nucleotides.
  • the nucleic acid probe may comprise a known/pre-determined sequence.
  • the nucleic acid probe may be complementary to the target nucleic acid sequence.
  • the nucleic acid probe may be 100% complementary to the target nucleic acid sequence.
  • the nucleic acid probe may be at least about 95%, or at least about 90% complementary to the target nucleic acid sequence.
  • the nucleic acid probe may be at least about 80% complementary to the target nucleic acid sequence.
  • the nucleic acid probe may be complementary to the target nucleic acid sequence along the whole length of the probe.
  • the nucleic acid probe may be complementary to the target nucleic acid sequence along a length of at least about 8 consecutive nucleotides of the probe.
  • the nucleic acid probe may be complementary to the target nucleic acid sequence along a length of at least about 10 consecutive nucleotides of the probe.
  • the nucleic acid probe may be complementary to the target nucleic acid sequence along a length of at least about 15 consecutive nucleotides of the probe.
  • the nucleic acid probe may be complementary to the target nucleic acid sequence along a length of at least about 18 consecutive nucleotides of the probe.
  • the nucleic acid probe may be complementary to the target nucleic acid sequence along a length of at least about 25 consecutive nucleotides of the probe.
  • the nucleic acid probe may be sufficiently complementary to the target nucleic acid sequence to be able to selectively hybridise under stringent conditions.
  • the nucleic acid probe may hybridise to target nucleic acid, such as under stringent conditions.
  • the nucleic acid probe may be anchored to a substrate surface only from one or more point(s) located in a mid-region of the nucleic acid probe.
  • the nucleic acid probe may be anchored to a substrate surface only from a point located in a mid-region of the nucleic acid probe.
  • the nucleic acid probe may comprise a single anchor point.
  • the nucleic acid probe may not comprise two or more anchor points.
  • the nucleic acid probe may not comprise three or more anchor points.
  • the nucleic acid probe may not be anchored to the substrate from a hairpin loop region of the nucleic acid probe.
  • the anchor points may be immediately adjacent to each other, e.g. on neighbouring nucleotides or the anchors may be attached within the same nucleotide.
  • the anchor points may be attached no more than 4 nucleotides apart on the same strand.
  • the anchor points may be attached no more than 3 nucleotides apart on the same strand.
  • the anchor points may be attached no more than 2 nucleotides apart on the same strand.
  • the anchor points may be attached no more than 1 nucleotide apart on the same strand.
  • the anchor points may not be separated by a major or minor groove when the nucleic acid is duplexed.
  • the anchor points may all be on the same region of the strand where it faces the anchor surface (i.e. not on another region of the strand that would be a helical turn away in the context of duplexed nucleic acid).
  • the provision of only a single mid-region anchor point (or closely positioned anchor points) when anchoring a nucleic acid to a substrate advantageously allows a double helical structure to form and unwind during hybridisation events and during denaturing-hybridisation events, and other nucleic acid manipulation events.
  • two or more anchor points where provided along the strand of nucleic acid, they may interfere with unwinding and hybridisation if they span across the major or minor grooves when the nucleic acid is duplexed.
  • the second strand would be entangled between the nucleic acid probe, the anchors, and the surface.
  • the mid-region of the nucleic acid probe may not comprise a terminal nucleotide of the nucleic acid probe.
  • the mid-region of the nucleic acid probe may comprise a series of nucleotides that are positioned at, or near, to the middle residue of the nucleic acid probe.
  • the mid-region of the nucleic acid probe may be at least 3 nucleotides away from either end of the nucleic acid probe.
  • the mid-region of the nucleic acid probe may be at least 4 nucleotides away from either end of the nucleic acid probe.
  • the mid-region of the nucleic acid probe may be at least 5 nucleotides away from either end of the nucleic acid probe.
  • the mid-region of the nucleic acid probe may not comprise a hairpin loop (otherwise known as a stem loop).
  • the nucleic acid probe comprises a hairpin loop
  • the hairpin loop may be considered equivalent to a terminal nucleotide in position on the probe, for example because it effectively forms an end of the duplexed nucleic acid. Therefore, in an embodiment wherein the nucleic acid comprises a stem loop, the anchor point may be located in the stem of the stem loop, such as in a mid-region of the stem.
  • the mid-region of a stem loop structured nucleic acid may not comprise part of the loop.
  • terminal nucleotide is understood to be the most 5' nucleotide or the most 3' nucleotide, for example the end nucleotide of any particular nucleic acid strand.
  • near-terminal is understood to include the terminal nucleotide, the immediate upstream/downstream nucleotide from the terminal nucleotide. In some embodiments "near- terminal” may also include nucleotides within 1, 2, 3, or 4 nucleotides of the terminal nucleotide.
  • the mid-region of the nucleic acid probe may comprise a series of nucleotides that are distanced at least 10% from either of the terminal nucleotides relative to the entire nucleic acid probe length.
  • the mid-region of the nucleic acid probe may comprise a series of nucleotides that are distanced at least 20% from either of the terminal nucleotides relative to the entire nucleic acid probe length.
  • the mid-region of the nucleic acid probe may comprise a series of nucleotides that are distanced at least 30% from either of the terminal nucleotides relative to the entire nucleic acid probe length.
  • the mid- region of the nucleic acid probe may comprise a series of nucleotides that are distanced at least 40% from either of the terminal nucleotides relative to the entire nucleic acid probe length.
  • the mid-region of the nucleic acid probe may comprise a series of nucleotides that are distanced at least 45% from either of the terminal nucleotides relative to the entire nucleic acid probe length.
  • the mid-region of the nucleic acid probe may be at least within 10% of the total nucleic acid length away from the central residue or central pair of residues of the nucleic acid probe.
  • the mid-region of the nucleic acid probe may be at least within 20% of the total nucleic acid length away from the central residue or central pair of residues of the nucleic acid probe.
  • the mid- region of the nucleic acid probe may be at least within 30% of the total nucleic acid length away from the central residue or central pair of residues of the nucleic acid probe.
  • the mid- region of the nucleic acid probe may be at least within 40% of the total nucleic acid length away from the central residue or central pair of residues of the nucleic acid probe.
  • the mid- region of the nucleic acid probe may be at least within 50% of the total nucleic acid length away from the central residue or central pair of residues of the nucleic acid probe.
  • the mid- region of the nucleic acid probe may be at least within 60% of the total nucleic acid length away from the central residue or central pair of residues of the nucleic acid probe.
  • the mid- region of the nucleic acid probe may be at least within 70% of the total nucleic acid length away from the central residue or central pair of residues of the nucleic acid probe.
  • the mid- region of the nucleic acid probe may be at least within 80% of the total nucleic acid length away from the central residue or central pair of residues of the nucleic acid probe.
  • the mid-region of the nucleic acid probe may be at least 5 nucleotides away from either end of the nucleic acid probe. In an embodiment wherein the nucleic acid probe is between about 18 and about 25 nucleotides in length, the mid-region of the nucleic acid probe may be at least 6 nucleotides away from either end of the nucleic acid probe. In an embodiment wherein the nucleic acid probe is between about 18 and about 25 nucleotides in length, the mid-region of the nucleic acid probe may be at least 7 nucleotides away from either end of the nucleic acid probe.
  • the mid-region of the nucleic acid probe may be at least 8 nucleotides away from either end of the nucleic acid probe. In an embodiment wherein the nucleic acid probe is between about 20 and about 35 nucleotides in length, the mid-region of the nucleic acid probe may be at least 7 nucleotides away from either end of the nucleic acid probe. In an embodiment wherein the nucleic acid probe is between about 20 and about 35 nucleotides in length, the mid-region of the nucleic acid probe may be at least 6 nucleotides away from either end of the nucleic acid probe.
  • the mid-region of the nucleic acid probe may be at least 7, 8, 9 or 10 nucleotides away from either end of the nucleic acid probe. In an embodiment wherein the nucleic acid probe is between about 30 and about 40 nucleotides in length, the mid-region of the nucleic acid probe may be at least 8 nucleotides away from either end of the nucleic acid probe. In an embodiment wherein the nucleic acid probe is between about 30 and about 40 nucleotides in length, the mid-region of the nucleic acid probe may be at least 10 nucleotides away from either end of the nucleic acid probe.
  • the mid-region of the nucleic acid probe may be at least 12 nucleotides away from either end of the nucleic acid probe. In an embodiment wherein the nucleic acid probe is between about 30 and about 40 nucleotides in length, the mid-region of the nucleic acid probe may be at least 13 or 14 nucleotides away from either end of the nucleic acid probe.
  • the anchor point of the nucleic acid probe may be located in a series of nucleotides that are positioned at, or near, to the middle residue of the nucleic acid probe.
  • the anchor point of the nucleic acid probe may be located 3 nucleotides away from either end of the nucleic acid probe.
  • the anchor point of the nucleic acid probe may be located at least 4 nucleotides away from either end of the nucleic acid probe.
  • the anchor point of the nucleic acid probe may be located at least 5 nucleotides away from either end of the nucleic acid probe.
  • the anchor point of the nucleic acid probe may be located at a nucleotide that is distanced at least 10% from either of the terminal nucleotides relative to the entire nucleic acid probe length.
  • the anchor point of the nucleic acid probe may be located at a nucleotide that is distanced at least 20% from either of the terminal nucleotides relative to the entire nucleic acid probe length.
  • the anchor point of the nucleic acid probe may be located at a nucleotide that is distanced at least 30% from either of the terminal nucleotides relative to the entire nucleic acid probe length.
  • the anchor point of the nucleic acid probe may be located at a nucleotide that is distanced at least 40% from either of the terminal nucleotides relative to the entire nucleic acid probe length.
  • the anchor point of the nucleic acid probe may be located at a nucleotide that is distanced at least 45% from either of the terminal nucleotides relative to the entire nucleic acid probe length.
  • the anchor point of the nucleic acid probe may be at least within 10% of the total nucleic acid length away from the central residue or central pair of residues of the nucleic acid probe.
  • the anchor point of the nucleic acid probe may be at least within 20% of the total nucleic acid length away from the central residue or central pair of residues of the nucleic acid probe.
  • the anchor point of the nucleic acid probe may be at least within 30% of the total nucleic acid length away from the central residue or central pair of residues of the nucleic acid probe.
  • the anchor point of the nucleic acid probe may be at least within 40% of the total nucleic acid length away from the central residue or central pair of residues of the nucleic acid probe.
  • the anchor point of the nucleic acid probe may be at least within 50% of the total nucleic acid length away from the central residue or central pair of residues of the nucleic acid probe.
  • the anchor point of the nucleic acid probe may be at least within 60% of the total nucleic acid length away from the central residue or central pair of residues of the nucleic acid probe.
  • the anchor point of the nucleic acid probe may be at least within 70% of the total nucleic acid length away from the central residue or central pair of residues of the nucleic acid probe.
  • the anchor point of the nucleic acid probe may be at least within 80% of the total nucleic acid length away from the central residue or central pair of residues of the nucleic acid probe.
  • the anchor point may refer to all of the anchor points provided on the nucleic acid probe, for example where multiple anchor points are provided, all of the anchor points are intended to be included in this term. Alternatively, only a single anchor point may be provided, whereby “the anchor point” refers directly to that single anchor point.
  • the anchor point of the nucleic acid probe may be at least 5 nucleotides away from either end of the nucleic acid probe. In an embodiment wherein the nucleic acid probe is between about 18 and about 25 nucleotides in length, the anchor point of the nucleic acid probe may be at least 6 nucleotides away from either end of the nucleic acid probe. In an embodiment wherein the nucleic acid probe is between about 18 and about 25 nucleotides in length, the anchor point of the nucleic acid probe may be at least 7 nucleotides away from either end of the nucleic acid probe.
  • the anchor point of the nucleic acid probe may be at least 8 nucleotides away from either end of the nucleic acid probe. In an embodiment wherein the nucleic acid probe is between about 20 and about 35 nucleotides in length, the anchor point of the nucleic acid probe may be at least 7 nucleotides away from either end of the nucleic acid probe. In an embodiment wherein the nucleic acid probe is between about 20 and about 35 nucleotides in length, the anchor point of the nucleic acid probe may be at least 6 nucleotides away from either end of the nucleic acid probe.
  • the anchor point of the nucleic acid probe may be at least 7, 8, 9 or 10 nucleotides away from either end of the nucleic acid probe. In an embodiment wherein the nucleic acid probe is between about 30 and about 40 nucleotides in length, the anchor point of the nucleic acid probe may be at least 8 nucleotides away from either end of the nucleic acid probe. In an embodiment wherein the nucleic acid probe is between about 30 and about 40 nucleotides in length, the anchor point of the nucleic acid probe may be at least 10 nucleotides away from either end of the nucleic acid probe.
  • the anchor point of the nucleic acid probe may be at least 12 nucleotides away from either end of the nucleic acid probe. In an embodiment wherein the nucleic acid probe is between about 30 and about 40 nucleotides in length, the anchor point of the nucleic acid probe may be at least 13 or 14 nucleotides away from either end of the nucleic acid probe.
  • the nucleic acid probe may be double stranded or single stranded. In one embodiment, the nucleic acid probe is double stranded nucleic acid. In another embodiment, the nucleic acid probe is single stranded nucleic acid. In one embodiment, the nucleic acid may not comprise a hairpin loop (also known as a stem loop). In one embodiment, the nucleic acid may not be capable of forming a hairpin loop structure. In an embodiment, wherein the nucleic acid is double stranded, the sense and anti-sense strand (i.e. the complementary strands) may be separate molecular entities, for example not joined by a hairpin loop.
  • the sense and anti-sense strand i.e. the complementary strands
  • the sense and anti-sense strand may be separate molecular entities, for example not joined by a covalent bond.
  • the anchor point of the nucleic acid probe may be not at an end residue of the nucleic acid probe.
  • the anchor point may not be within 3 nucleotides from end of the nucleic acid probe.
  • the anchor point may not be within 4 nucleotides from end of the nucleic acid probe.
  • the anchor may comprise any compound capable of tethering a nucleic acid to a substrate surface.
  • the anchor may be a small molecule, or a polymer. Any appropriate chemistry may be used to anchor the nucleic acid probe to the substrate surface, for example click-chemistry may be used to anchor the nucleic acid probe to the substrate surface by reaction of a chemical group on the nucleic acid probe with an opposing/complementary reactive group on the substrate.
  • the substrate surface and/or the nucleic acid probe may comprise reactive or charged groups for anchoring the nucleic acid probe to the substrate surface.
  • the anchor may comprise a thiol anchor. In one embodiment a thiol anchor may be attached to a thymine base on the nucleic acid probe.
  • the anchor may comprise a phosphoramidate bond.
  • the anchor may comprise a triazole.
  • the nucleic acid may be anchored by immobilisation of the nucleic acid using a carbodiimide crosslinker, such as EDC (also called EDAC; l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, or DCC (dicyclohexyl carbodiimide).
  • EDC also called EDAC; l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, or DCC (dicyclohexyl carbodiimide).
  • the nucleic acid may be anchored by immobilisation of the nucleic acid using the carbodiimide linker upon a surface modified with stearic acid or octadecylamine.
  • the nucleic acid may be anchored by immobilisation of the nucleic acid using a carbodiimide crosslinker, such as EDC, upon a surface modified with primary amino groups or aminoethanethiol.
  • the nucleic acid may be anchored through attachment of nucleic acid, such as ssDNA, onto a phosphoric acid-terminated surface.
  • the phosphoric acid may comprise MBPA (mercaptobutylphosphoric acid).
  • the nucleic acid may be anchored through attachment of nucleic acid onto a film of aluminum alkenebisphosphonate on the surface of the substrate.
  • the nucleic acid may be anchored onto a mercaptosilane coating of the surface via the amino groups of the nucleic acid bases.
  • the nucleic acid may be anchored using functionalised polypyrrole.
  • the nucleic acid may be anchored using any one of the covalent cross-linking reactions discussed in Pividori et al., (2000. Biosensors & Bioelectronics 15; pp. 191-303 - incorporated herein by reference), and as illustrated in Figure 8.
  • the nucleic acid probe may comprise a modified nucleotide, comprising a linker or reactive group to form an anchor.
  • the linker group for attachment to the surface may be termed an anchor unit.
  • the nucleic acid probe may comprise a modified thymine for use as an anchor.
  • the anchor may comprise a modified thymine.
  • the modified thymine may comprise a deoxythymidine (dT) modified with a linker.
  • the modified thymine may comprise a deoxythymidine (dT) modified with an anchor unit.
  • the anchor unit may comprise a linker comprising thiol groups, such as dithiols.
  • the linker may comprise at least two or three dithiols as a surface anchor.
  • the linker may comprise a propagylamidopentanol linker attached to the thymine, such as at the C5 position of the thymine.
  • the nucleic acid probe may comprise modified thymine comprising a deoxythymidine (dT) modified with a linker comprising three dithiols as a surface anchor and a propagylamidopentanol linker attached to the C5 position of the thymine.
  • the linker or reactive group to form an anchor may comprise biotin for linking with streptavidin, or comprise streptavidin for linking with biotin.
  • the nucleic acid probe may be anchored to a modified surface by the use of silane coupling agents to introduce functional groups to the surface (such as thiols, amines, or aldehydes) for linking to a nucleic acid probe modified with an appropriate reactive group, which would form an anchoring bond.
  • silane coupling agents to introduce functional groups to the surface (such as thiols, amines, or aldehydes) for linking to a nucleic acid probe modified with an appropriate reactive group, which would form an anchoring bond.
  • the anchor may be between about 0.2nm and about lOOnm in length. In another embodiment, the anchor may be between about 0.2nm and about 50nm in length. In another embodiment, the anchor may be between about 0.2nm and about 15nm in length. In another embodiment, the anchor may be between about 0.2nm and about lOnm in length. Alternatively, the anchor may be between about 0.3nm and about 5nm in length. Alternatively, the anchor may be between about 0.5nm and about 4nm in length. Alternatively, the anchor may be between about 0.5nm and about 3nm in length. Alternatively, the anchor may be between about 0.8nm and about 4nm in length. The anchor may be between about 0.8nm and about 2.3nm in length.
  • the anchor may be between about 0.8nm and about 2.3nm in length, for example when the nucleic acid probe is at least 4.5nm in length.
  • the anchor may be between about 0.2nm and about 2.3nm in length.
  • the anchor may be between about 0.5nm and about 2.3nm in length.
  • the anchor may be between about 0.2nm and about 3nm in length.
  • the anchor may be between about 0.2nm and about 2.5nm in length.
  • the anchor may be between about 0.5nm and about 2.5nm in length.
  • the anchor may be at least 0.2nm in length.
  • the anchor may be at least 0.3nm in length.
  • the anchor may be at least 0.5nm in length.
  • the anchor may be no more than 50nm in length. In another embodiment, the anchor may be no more than 20nm in length. In another embodiment, the anchor may be no more than lOnm in length. Alternatively, the anchor may be no more than 5nm in length.
  • the length of the anchor may be less than 51% of the length of the nucleic acid probe.
  • the length of the anchor may be less than 50% of the length of the nucleic acid probe.
  • the length of the anchor may be less than 49% of the length of the nucleic acid probe.
  • the length of the anchor may be less than 48% of the length of the nucleic acid probe.
  • the length of the anchor may be less than 40% of the length of the nucleic acid probe.
  • the length of the anchor may be less than 30% of the length of the nucleic acid probe.
  • the length of the anchor may be less than 25% of the length of the nucleic acid probe.
  • the length of the anchor may be less than 20% of the length of the nucleic acid probe.
  • the length of the anchor may be less than 15% of the length of the nucleic acid probe.
  • the length of the anchor may be less than 10% of the length of the nucleic acid probe.
  • the length of the anchor may be less than 5% of the length of the nucleic acid probe.
  • the length of the anchor may be more than 5% of the length of the nucleic acid probe.
  • the length of the anchor may be more than 10% of the length of the nucleic acid probe.
  • the length of the anchor may be more than 20% of the length of the nucleic acid probe.
  • the length of the anchor may be between about 10% and 50% of the length of the nucleic acid probe.
  • the length of the anchor may be between about 1% and 49% of the length of the nucleic acid probe.
  • the length of the anchor may be between about 5% and 49% of the length of the nucleic acid probe.
  • the length of the anchor may be between about 1% and 30% of the length of the nucleic acid probe.
  • the length of the anchor may be between about 0.5% and 49% of the length of the nucleic acid probe.
  • the anchor linking the nucleic acid probe to the surface is small enough to hold the hybridized nucleic acid probe in a substantially horizontal orientation (e.g. substantially parallel to the substrate surface) and prevent the hybridized nucleic acid probe from adopting a substantially vertical orientation (e.g. substantially perpendicular relative to the substrate surface).
  • the substrate may comprise a nanoparticle, a nanotube or rod.
  • the substrate may comprise a wafer consisting of, or layered with, a metal.
  • the substrate surface may be porous or roughened, for example by etching.
  • the substrate may comprise a metallic substrate, such as gold.
  • the metallic substrate may comprise or consist of gold, silver, platinum, copper or aluminium.
  • the substrate comprises or consists of a metallic nanoparticle, such as a gold nanoparticle.
  • the substrate may comprise an electrode, for example as discussed in Lucarelli et al. (2004. Biosensors and Bioelectronics 19; pp. 515-530 - incorporated herein by reference).
  • the substrate may comprise a carbon electrode.
  • the substrate may comprise a glass surface or a semiconductor surface.
  • the substrate surface and/or the nucleic acid probe may comprise modifications to enable the nucleic acid probe attachment.
  • the surface may comprise, or may be modified to comprise, any one of the following group comprising polylysine; amine; epoxy diazonium ion; SU-8; unmodified glass; agarose film; membrane, such as nitrocellulose; gold; mercaptosilanes; maleimide; iodoacetyl; aldehyde; isothiocyanate; aminated surfaces; and avidin; or combinations thereof.
  • the surface may comprise, or may be modified to comprise, any one of the following group comprising polylysine; amine; epoxy diazonium ion; SU-8; unmodified glass; agarose film; membrane, such as nitrocellulose; or combinations thereof.
  • the surface may comprise, or may be modified to comprise, unmodified glass.
  • the nucleic acid is thiol modified for attachment, the surface may comprise, or may be modified to comprise, any one of the following group comprising gold; mercaptosilanes; maleimide; and iodoacetyl; or combinations thereof.
  • the surface may comprise, or may be modified to comprise, any one of the following group comprising aldehyde; epoxy; isothiocyanate; active esters; and carboxylic groups or combinations thereof.
  • the surface may comprise, or may be modified to comprise, an aminated surface.
  • the nucleic acid is biotin modified, the surface may comprise, or may be modified to comprise, avidin; or vice versa.
  • the sample may comprise a bodily fluid sample.
  • the sample may comprise a tissue sample.
  • the sample may comprise an environmental sample, such as a water, air, or soil sample.
  • the sample may comprise a food or beverage sample.
  • the sample may comprise a cell culture sample.
  • the sample may comprise a sample of pre-extracted nucleic acid.
  • the sample may consist of nucleic acid and a solute.
  • the sample may be from a mammal.
  • the mammal may be human.
  • the sample may comprise a blood or blood plasma sample.
  • the sample may be selected from any of the group comprising blood; blood plasma; mucous; urine; faeces; cerebrospinal fluid; tissue, such as organ tissue; lung aspirate; or combinations thereof.
  • the target nucleic acid sequence may be varied in length, for example as provided by a restriction enzyme digests of longer nucleic acid strands.
  • the target nucleic acid sequence may be at least 8 nucleotides in length.
  • the target nucleic acid sequence may be at least 12 nucleotides in length.
  • the target nucleic acid sequence may be at least 15 nucleotides in length.
  • the target nucleic acid sequence may be at least 18 nucleotides in length.
  • the target nucleic acid sequence may be at least 25 nucleotides in length.
  • the target nucleic acid sequence may be no more than about 200 nucleotides in length.
  • the target nucleic acid sequence may be no more than about 180 nucleotides in length.
  • the target nucleic acid sequence may be no more than about 150 nucleotides in length.
  • the target nucleic acid sequence may be no more than about 100 nucleotides in length.
  • the target nucleic acid sequence may be no more than about 80 nucleotides in length.
  • the target nucleic acid sequence may be no more than about 60 nucleotides in length.
  • the target nucleic acid sequence may be no more than about 40 nucleotides in length.
  • the target nucleic acid sequence may be no more than about 35 nucleotides in length.
  • the target nucleic acid sequence may be between about 8 and about 200 nucleotides in length.
  • the target nucleic acid sequence may be between about 12 and about 150 nucleotides in length.
  • the target nucleic acid sequence may be between about 18 and about 150 nucleotides in length.
  • the target nucleic acid sequence may be about 30 nucleotides in length.
  • reference to the length of the target nucleic acid may refer to the average length of the target nucleic acid in a pool of nucleic acid.
  • the target nucleic acid sequence may comprise or consist of DNA or RNA.
  • the target nucleic acid sequence may comprise a mixture of DNA and RNA.
  • the target nucleic acid sequence may comprise genomic nucleic acid.
  • the target nucleic acid sequence may comprise viral RNA; mRNA; ncRNA; small RNA; and siRNA; or combinations thereof.
  • the target nucleic acid sequence may comprise mitochondrial nucleic acid.
  • the target nucleic acid sequence may comprise or consist of chromosomal and/or non-chromosomal DNA.
  • the target nucleic acid sequence in the sample may comprise a mixture of mammalian and non-mammalian nucleic acid.
  • the target nucleic acid sequence in the sample may comprise a mixture of mammalian and microbial nucleic acid.
  • the target nucleic acid sequence in the sample may comprise a mixture of mammalian and bacterial and/or viral nucleic acid.
  • the target nucleic acid sequence in the sample may comprise a mixture of mammalian and fungal nucleic acid.
  • the target nucleic acid sequence in the sample may comprise a mixture of mammalian and pathogen nucleic acid.
  • the target nucleic acid sequence the sample may comprise a mixture of species and/or strains.
  • the target nucleic acid sequence may comprise a species and/or strain specific sequence.
  • the target nucleic acid sequence may comprise a pathogen's nucleic acid sequence.
  • the target nucleic acid sequence may comprise microbial nucleic acid sequence.
  • the target nucleic acid sequence may comprise fungal nucleic acid sequence.
  • the target nucleic acid sequence may comprise nucleic acid sequence selected from any of the group comprising bacterial nucleic acid sequence; viral nucleic acid sequence; parasitic nucleic acid sequence; protozoan nucleic acid sequence; and fungal nucleic acid sequence; or combinations thereof.
  • the target nucleic acid sequence may comprise a cell type and/or cell state specific sequence.
  • the target nucleic acid sequence may be extracted from the sample. For example, the target nucleic acid sequence in the sample may be purified or partially purified prior to hybridisation and/or amplification. The extraction of target nucleic acid sequence may be carried out by the skilled person by standard laboratory techniques.
  • the target nucleic acid sequence may be amplified prior to hybridising the nucleic acid probe.
  • the sample may be provided with pre-amplified target nucleic acid sequence.
  • the amplification of target nucleic acid sequence may comprise or consist of polymerase amplification.
  • the amplification of target nucleic acid sequence may comprise or consist of PCR.
  • the PCR may use a pair of primers. One or both primer of the primer pair may be affinity tagged, thereby providing affinity tagged PCR product.
  • the target nucleic acid sequence may be affinity tagged.
  • the affinity tag may comprise biotin.
  • the hybridisation of the nucleic acid probe with the target nucleic acid sequence may be detected by an electrochemical genosensor.
  • the hybridisation of the nucleic acid probe with the target nucleic acid sequence may be detected by surface enhanced resonance Raman scattering (SERRS) or surface enhanced Raman scattering (SERS) assay, for example for detecting dye labelled nucleic acid.
  • the dye may be a Raman-active dye, such as an azo dye.
  • the hybridisation may be detected via an increase in a current signal of an electroactive indicator (e.g. that preferentially binds the double-stranded DNA).
  • the electroactive indicator may be used in conjunction with, or alternatively to, enzyme or redox labels, or from other hybridisation-induced changes in electrochemical parameters.
  • Detecting hybridisation may comprise the use of differential pulse voltammetry (DPV), square wave voltammetry (SWV) and/or potentiometric stripping analysis (PSA).
  • Detecting hybridisation may comprise the use of an intercalative compound/groove binder and/or enzyme label.
  • DPV differential pulse voltammetry
  • SWV square wave voltammetry
  • PSA potentiometric stripping analysis
  • Detecting hybridisation may comprise the use of an intercalative compound/groove binder and/or enzyme label.
  • the probe nucleic acid and/or target nucleic acid may be labelled.
  • the label may comprise an organic dye, organic fluorophore, fluorescent dye, IR absorbing dye, UV absorbing dye, metachromatic dye, photochromic dye, thermochromic dye, or sulphonephthalein dye.
  • the label may comprise an azo-dye.
  • the label may comprise enzyme labels, nanoparticle labels, or radio- labels.
  • the label may comprise fluorescence dye (cyanine dye (Cy3, Cy5), Texas Red, Rhodamine Red, Alexa Dye, Bodipy dye, Atto dye, FAM, TET, HEX, JOE, Cy3, TAM RA, or ROX,).
  • Nanoparticles labels may be used to enhance the signal, and may be further modified with DNA or an appropriate dye.
  • the label may comprise electrochemical dyes (e.g. methylene blue, Co(phen)3(CI04)3) or HRP enzyme.
  • the nucleic acid probe may be anchored to, and arranged in, an array (i.e. a microarray).
  • a plurality of different nucleic acid probes may be arranged in an array in order to detect a range of different target nucleic acid sequences.
  • a plurality of the same nucleic acid probes may be anchored in an array for detecting hybridisation with target nucleic acid sequence under varying conditions. Varying conditions could include physical parameters such as temperature, and/or detecting hybridisation in the presence of additional components, such as potential therapeutic molecules, for example, arranged to target a nucleic acid sequence.
  • nucleic acid probe molecule which is anchored to a substrate
  • anchor point is located in a mid-region of the nucleic acid.
  • anchor points may only be located in the mid-region of the nucleic acid probe.
  • nucleic acid probe molecule which is anchored to a substrate
  • anchor point is not located at a terminal residue of the nucleic acid.
  • a plurality of nucleic acid probes may be anchored to the substrate.
  • a nucleic acid probe comprising an anchor group arranged to anchor the nucleic acid to a substrate, wherein the anchor group is located in a mid-region of the nucleic acid probe.
  • the nucleic acid does not comprise terminal or near-terminal anchor groups; or b) the anchor group is the only anchor group arranged to anchor the nucleic acid to a substrate.
  • a nucleic acid probe comprising an anchor group arranged to anchor the nucleic acid to a substrate, wherein the anchor group is not located at a terminal residue of the nucleic acid probe.
  • a plurality of nucleic acid probes may be provided. In one embodiment, a plurality of nucleic acid probes may be between 2 and 100 nucleic acid probes. In another embodiment, a plurality of nucleic acid probes may be between 2 and 50 nucleic acid probes. In another embodiment, a plurality of nucleic acid probes may be between 2 and 25 nucleic acid probes. In another embodiment, a plurality of nucleic acid probes may be between 2 and 10 nucleic acid probes. In another embodiment, a plurality of nucleic acid probes may be between 2 and 7 nucleic acid probes. In another embodiment, a plurality of nucleic acid probes may be between 2 and 6 nucleic acid probes.
  • a plurality of nucleic acid probes may be between 3 and 100 nucleic acid probes. In another embodiment, a plurality of nucleic acid probes may be between 10 and 100 nucleic acid probes. In another embodiment, a plurality of nucleic acid probes may be at least 2 nucleic acid probes. In another embodiment, a plurality of nucleic acid probes may be at least 3 nucleic acid probes. In another embodiment, a plurality of nucleic acid probes may be at least 4, 5, 10, 20, or 30 nucleic acid probes.
  • a microarray comprising a plurality of nucleic acid probes anchored to a substrate, wherein the anchor point is located in a mid-region of the nucleic acid probe.
  • the anchor points may only be located in the mid-region of the nucleic acid probe.
  • the nucleic acid probes may be separated, for example into groups of the same nucleic acid probe, into compartments in the microarray. Such compartmentalisation may facilitate the testing of different samples and/or conditions.
  • the surface density of the nucleic acid probe (once anchored) may be about 10 12 per cm 2 .
  • the surface density of the nucleic acid probe (once anchored) may be about 4.2 x 10 11 per cm 2 .
  • the surface density of the nucleic acid probe (once anchored) may be between about 4.2 x 10 11 per cm 2 and about 10 12 per cm 2 .
  • the surface density of the nucleic acid probe (once anchored) may be between about 1 x 10 11 per cm 2 and about 10.5 12 per cm 2 .
  • the surface density of the nucleic acid probe (once anchored) may be between about 1 x 10 10 per cm 2 and about 10 13 per cm 2 .
  • the surface density of the nucleic acid probe may be less than about 10 13 per cm 2 . In another embodiment, the surface density of the nucleic acid probe (once anchored) may be less than about 10 12 per cm 2 . In another embodiment, the surface density of the nucleic acid probe (once anchored) may be less than about 5 x 10 11 per cm 2 .
  • the surface density of the nucleic acid probe may be calculated using a chronoamperometric method. The probe may be hybridised to its target prior to anchoring to the substrate surface, or hybridised to its target after anchoring to the substrate surface. A lower density of nucleic acid probe may be provided on the substrate surface by hybridising the nucleic acid probe to its target prior to anchoring.
  • Figure 1 schematic diagram to show A) an end tethering approach where the DNA adopts a vertical orientation, and B) a middle tethering approach where the DNA adopts a horizontal and more fixed orientation.
  • Figure 2 Structure of the dithiol linker attached on the thymine base.
  • Figure 3 (a- b) SE S spectra of the DNA probe-1 (short probe with a thiol linker appox. in the middle) that was immobilised at 40 °C and was hybridised with (a) fully- complementary short target DNA(Target 1) and (b) non-complementary short target DNA (Target 3).
  • the DNA target was labelled with Texas Red and Cy3B at the 5' and 3' ends respectively.
  • Figure 4 SERS spectra of the DNA probe-1 (short probe with a thiol linker approx. in the middle) hybridised with (a) fully-complementary long target DNA (Target 2) and (c) non-complementary long target DNA (Target 4), (b) SERS spectra of DNA probe-4
  • Figure 5 SERS spectra of the long DNA probe (77 bases long, Probe 3) with a thiol in the middle and hybridised (a) to a fully complementary long DNA target (76 bases long, Target 2) and (b) non complementary long DNA target (79 bases long, Target 4).
  • Figure 6 SERS spectra of the DNA probe 2 hybridised to the long complementary target (target 2) and the long non complementary target (target 4).
  • Figure 7 SERS spectra of the (a) DNA probe 1 and (b) DNA probe 2, hybridised to the long complementary target (target 2). The spectra are all displayed with the same intensity but have been offset for clarity.
  • Figure 8 Some common reactions used for the covalent binding of DNA on different electrochemical transducer surfaces, (a) Immobilisation of ssDNA on glassy carbon electrodes (through deoxyguanosine group (dG)n-DNA) using carbodiimide method (EDC: l-3(-dimethylaminopropyl)-3-ethyl-carbodiimide;NHS:N- hydroxysulfosuccinimide).
  • Figure 9 (a) - (b) Structure of dithiol and hexaethylene glycol linker attached at the (a) 3'and (b) 5' end of the DNA probes, (c) Structure of the Cy3B modification at the 3' end and (d) the Texas Red modification at the 5' end.
  • FIG. 10 Binding isotherm for [Ru(NH 3 ) 6 ] + with DNA duplex immobilised horizontally on an Au SSV substrate.
  • the duplex was hybridized in solution and then the duplex strand (0.5 ⁇ of dsDNA in 0.05 M Na 2 S0 4 ) was immobilised on the SSV Au surface overnight. The surface was then passivated with mercaptohexanol (1 mM).
  • a method of the present invention is to specifically immobilize a DNA probe horizontally on the surface. This orientation leads to lower probe density which can increase the hybridization efficiency as well as locating the DNA backbone closer to the sensor surface thereby allowing increased sensitivity.
  • DNA sequences that were aligned horizontally to the surface have been shown to be effective for sensitive electronic 10 and SERS 11 label free detection of DNA.
  • the DNA was physically adsorbed on the surfaces, whereas specific attachment via a linker would be more efficient and more controlled for the design of molecular assays.
  • a recent approach described the specific horizontal immobilization of a 15-mer PNA probe on a silicon dioxide surface via three linker molecules attached at three locations ( ⁇ points) along the PNA backbone.
  • the invention provides a simple, straightforward methodology for specific tethering of DNA probes using a single linker, by placing the linker approximately in the middle of the probe to ensure horizontal orientation of the attached dsDNA.
  • the thiol linker is attached to a thymine base.
  • Previously vertically tethered DNA was used on Au sphere segment void (SSV) surfaces which give large SE S enhancements, 13 14 to develop assays for sensitive DNA discrimination by targeting the detection of single nucleotide polymorphism 15 and tandem repeats.
  • SSV Au sphere segment void
  • 13 14 to develop assays for sensitive DNA discrimination by targeting the detection of single nucleotide polymorphism 15 and tandem repeats.
  • 16 Specifically, a negative potential is applied on the Au SSV surface to melt the DNA and the melting is monitored by recording the SERS signal of the labelled DNA target as a function of applied potential. When the DNA target diffuses away from the surface after dsDNA dissociation, the signal of the SERS label decreases significantly.
  • a modified 30bp oligonucleotide probe ( 5' -h exy n o I - ATATC ATCTTTG GTGT*TTCCTC A TGCTTTA- 3') (SEQ ID NO: 1) was synthesized.
  • the T* indicates a deoxythymidine (dT) modified with a linker consisting of three dithiols as a surface anchor and a propagylamidopentanol linker attached at the C5 position of the thymine, as shown in Figure 2.
  • the modified dT was the 16 th base along the probe starting from the 5' end.
  • the length of the DNA probe is ⁇ 10.2 nm and the length of the linker between the thymine and the Au surface (from propagylamidopentanol, phosphate and the first dithiol) can range from 0.8 nm to 2.3 nm depending on whether the alkyl chain is curled or extended. No spacers have been used between the three dithiol units and thymine base to minimize the bending of the DNA duplex and facilitate a fixed and rigid orientation on the surface. Immobilization of the DNA probe at room temperature prior to hybridization was avoided since the DNA might coil up on the surface in an unsuitable manner.
  • the DNA probe was hybridized to its target in solution to guarantee that the desired rigid duplex was formed before surface immobilization.
  • the Au SSV surface was then incubated in the 0.5 ⁇ DNA solution at room temperature, overnight, following passivation with mercaptohexanol.
  • the DNA probe was first immobilized on the surface at 40 Q C for 6 hours in order to allow the DNA to bind in its uncoiled form. 4
  • the Au surface was then passivated with mercaptohexanol before hybridized to its target DNA at room temperature for 2 hours. It should be noted that in both cases the DNA was immobilized at low ionic strength (0.05 M Na 2 S0 4 ) since there was no need to screen the duplex charge in these inherently low probe density surfaces.
  • Figure 3 (a)-(d) shows SERS spectra of the DNA probe with the thiol linker attached on the thymine base assembled on the gold SSV under both sets of conditions with both the complementary and non-complementary target DNA. Significantly, bands for both Texas Red and Cy3B are visible when the DNA probe with the thiol linker located on the thymine base is hybridized to the complementary target, Figure 3 (a).
  • Texas Red has a stronger resonance contribution (A max , 589 nm) with the 633 nm laser used here than the Cy3B dye (A max , 558 nm).
  • a similar difference in SERS intensity has been observed when two different DNA strands individually labelled with the two dyes were immobilized vertically on the surface.
  • the horizontally tethered dsDNA was denatured either electrochemically (by application of -1.2 V vs Ag/AgCI), or thermally (by heating to 80 °C).
  • the target DNA was labelled with a single Texas Red fluorophore at the 5' end. After DNA denaturation by either method, the Texas Red SERS signal was lost due to the labelled DNA target being released and diffusing away from the surface.
  • the surface density of the DNA was calculated using a chronoamperometric method. The surface coverage was found slightly different, depending on the immobilization methods:
  • This study demonstrates a new simple and effective methodology for the covalent attachment of DNA probes on gold surfaces that promotes horizontal orientation of the dsDNA.
  • the method is sensitive for the discrimination of complementary and non-complementary DNA.
  • the DNA duplexes can also be melted on the surface and their melting profile can be monitored using either a labelled DNA target or unmodified target with an added binding agent specific for double stranded DNA (i.e. methylene blue).
  • This new immobilization strategy can be applied to a wide range of optical or electronic biosensors and has the potential to improve the hybridization efficiency of long DNA targets on the surface as well as the overall sensitivity of the sensors.
  • the same methodology can be applied to successfully induce horizontal orientation of the DNA on surfaces up to the persistence length of dsDNA (> 150 bp).
  • a methodology that allows the specific tethering of dsDNA horizontally on the surface is provided. This can be achieved using a linker on the DNA probe, which will be placed approximately in the middle of the DNA sequence.
  • the DNA probe modified with a surface-linker is first immobilised on a gold nanostructured surface by incubation of the surface in a DNA solution at 40°C for 6 hours in order to allow the DNA to bind in its uncoiled form.
  • the surface is then passivated mercaptohexanol in order to prevent unspecific binding of the DNA to the surface.
  • the surface tethered DNA probe is then hybridised to its target DNA at room temperature for 2 hours. SERS spectroscopy was utilised to show that the DNA was hybridised in a horizontal orientation. To monitor the orientation of the DNA duplex, the target DNA was labelled on each end with different fluorophores: Texas Red at the 5' end and Cy3B or Cy3 at the 3' end.
  • Target -1 Comp-short target 30 bases
  • Target 2 Comp long target 78 bases
  • Target 3 Non Comp-short target 30 bases
  • Target 4 Non Comp-long target 79 bases
  • Target 5 Comp long target 76 bases
  • Figure 3(a) and (b) shows the results where a short probe (30 bases long) that had the surface- linker in the middle and was hybridised with a short complementary target (30 bases long) and a short non-complementary target (30 bases long).
  • Figure 4 shows the results of utilizing a short probe (30 bases long) that had a surface-linker in the middle, hybridised to a longer complementary target (76 bases long) and a longer non- complementary target.
  • the figure shows that when the probe was hybridised to a fully complementary long target the SERS spectrum contains bands associated with both dyes. Therefore the dsDNA has been successfully orientated parallel to the Au surface.
  • the probe was hybridised to a non-complementary long DNA target, no SERS signal is observed. Two different probes have been used to show that the result is reproducible and can be applied to different DNA sequences.
  • Figure 5 shows the results of utilising a long probe (77 bases long), with a surface-linker approx. in the middle, hybridised to a fully complementary long target (76 bases long).
  • the SERS spectrum contains bands associated with mainly Texas Red. Small bands of Cy3 can be observed.
  • the figure also shows that when the probe was hybridised to a non-complementary target, there is an insignificantly small signal of Texas Red demonstrating that the hybridization is specific to the complementary sequence.
  • Figure 6 shows the results of utilising a short probe (25 bases long) that had three linkers, one in the middle and one in each end of the DNA (Probe 2). The figure shows that when the probe was hybridised to a fully complementary short target the SERS spectrum contains bands associated with both dyes. When the probe was hybridised to a non-complementary long DNA target, no SERS signal is observed.
  • Figure 7 shows the spectra of dsDNA when (a) short DNA probe (probe 1, one linker in the middle) was used hybridised with a long DNA target (target 2) and (b) short DNA (probe 2, three linkers across the DNA backbone) was used hybridised with a long DNA target (target 2). It obvious that when there is only one linker on the DNA probe the signal from both of the dyes is significantly stronger, demonstrating that the horizontal immobilisation of the DNA is more successful when the probe has only one thiol in the middle.
  • the distance from base to base in the DNA sequence is 0.34 nm.
  • the length of the linker, for this experiment, between the thymine and the Au surface (from propagylamidopentanol, phosphate and the first dithiol) can range from 0.8 nm to 2.3 nm depending on whether the alkyl chain is curled or extended. Therefore, for this linker, the DNA probe is preferably no smaller than 14 bases (the length of 7 bases will be 2.38 nm). If the DNA sequence is smaller than 14 bases then there is a possibility that the DNA can orientate perpendicular on the surface (when the linker is extended which is not very likely).
  • linker For shorter DNA sequences a shorter linker can be employed.
  • the length of the linker should be optimised with regards to the length of the DNA probe. 3. Methods to immobilise DNA on a surface.

Abstract

La présente invention concerne un procédé d'analyse d'acide nucléique dans un échantillon comprenant les étapes consistant à : - utiliser une sonde d'acide nucléique, qui est ancrée dans une surface de substrat uniquement à partir d'au moins un point situé dans une région médiane de la sonde d'acide nucléique; et détecter la présence ou l'absence d'une séquence d'acide nucléique cible dans l'échantillon par l'hybridation de la sonde d'acide nucléique avec la séquence d'acide nucléique cible si elle est présente.
PCT/GB2016/050617 2015-03-06 2016-03-07 Analyse d'acide nucléique impliquant des sondes d'acide nucléique ancrées dans la surface WO2016142687A1 (fr)

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Citations (1)

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WO2005047301A1 (fr) * 2003-11-07 2005-05-26 Solexa Limited Ameliorations apportees a des reseaux polynuceotidiques

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WO2005047301A1 (fr) * 2003-11-07 2005-05-26 Solexa Limited Ameliorations apportees a des reseaux polynuceotidiques

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