Classifications

• • 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
  • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

• the present invention relates to a method and apparatus for characterising a nucleic acid in terms of the number and location of methylated CpG islands present therein.
• CpG islands are regions of the human genome which contain a high density of sites where a cytosine nucleotide is adjacent a guanine nucleotide on the same polynucleotide strand thereby being only separated by a single phosphate group.
• the cytosine is susceptible to facile enzymatic methylation into 5- methylcytosine.
• it has been estimated that up to 80% of such CpG sites are methylated in this way. Once methylated, the 5-methylcytosine exhibits a tendency to undergo deamination to thymine.
• US2011097728 discloses a method for assaying methylated CpG islands by assaying with at least one gene indicative of prostate cancer for example methyltransferase family member 1, glutathione peroxidise 7, paladin and fibroblast growth factor 20.
• WO2010/107716 describes a method for detecting multiple methylated CpG islands in a DNA sample using abortive transcription technology in which DNA signal generators called Abortive Promoter Cassettes (APCs) are generated. This enables RNA polymerase to produce uniform, short RNA molecules from synthetic promoters in the APCs as signals of the presence of methylated CpG islands.
• APCs Abortive Promoter Cassettes
• US2010075331 describes a method for analyzing the methylation status of a genomic DNA sample, comprising the steps of (i) fragmenting said sample and enriching said sample for sequences comprising CpG islands, (ii) generating a single stranded DNA library, (iii) subjecting said sample to bisulfite treatment, (iv) clonally amplifying individual members of said single stranded DNA library by means of emulsion PCR, and (v) sequencing said amplified clonally amplified single stranded DNA library.
• US2010136541 describes various methods for the identification and verification of methylation marker sequences including the use of fluorophores.
• a method of screening a biological sample for biomarkers for colon cancer comprising: (a) determining the expression level of all or substantially all of nucleic acid sequences of the biological sample which comprise at least one methylated CpG site in a promoter-first exon region; (b) determining the expression level of the all or substantially all of nucleic acid sequences that have been demethylated; (c) comparing the expression level from step (a) with the expression level from step (b) and (d) identifying those nucleic acid sequences exhibiting a significant increase in the expression level after demethylation as compared to the expression level of the same nucleic acid sequences in the methylated state.
• WO 2010/080617 discloses methods for detecting, characterizing or separating DNA based on methylation. The methods involve passing a DNA sample through a nanopore, wherein an electric field is established across the membrane comprising the nanopore and generates a threshold voltage across a nanopore such that only methylated DNA or DNA having a particular methylation content or pattern is forced through the nanopore.
• the inventors named in WO 2010/080617 also report in Mirsaidov et al (Biophysical Journal: Biophysical Letters, 96: L32-L34, 2008) that a synthetic nanopore can be used to detect methylation based on the voltage threshold for permeation of methylated DNA through the nanopore.
• WO 2011/109825 relates to the detection of nucleic acid lesions and adducts using nanopores.
• a method of obtaining sequence information from a nucleic acid which involves coupling a current modulating compound to a preselected nucleotide type in a nucleic acid to form a nucleic acid adduct, directing the nucleic acid adduct into a channel and measuring changes in current through the channel in response to the current modulating compound to detect the preselected nucleotide type.
• US 2010/0044211 discloses an apparatus for the detection of one or more target molecules which comprises a membrane that separates a first chamber and a second chamber, wherein the membrane comprises a nanochannel that is configured to allow passage of the target molecule(s), an electrical detection unit configured to detect the passage of the target molecule(s) through the nanochannel and an optical detection unit configured to identify the one or more target molecules passing through the nanochannel. Also disclosed is a method of detecting one or more target molecules which comprises applying an electrical source across such a membrane, detecting an electrical signal change upon passage of the target molecule(s) through the nanochannel, applying an electromagnetic energy source to the target molecule(s) and detecting an optical signal from the target molecule(s) generated by the electromagnetic energy source.
• US 2008/0242556 discloses a method for characterizing one or more macromolecules using a nanofluidic device which involves translocating at least a portion of at least one region of the macromolecule through a fluidic nanochannel segment disposed substantially parallel to the surface of a substrate, wherein the fluidic nanochannel segment is capable of containing and elongating at least a portion of a region of the macromolecule, and has a cross-sectional dimension of less than about 1000 nm and a length of at least about 10 nm; monitoring, through a viewing window capable of permitting optical inspection of at least a portion of the contents of the fluidic nanochannel segment, one or more signals related to the translocation of one or more regions of the macromolecule through the nanochannel; and correlating the monitored signals to one or more characteristics of the macromolecule.
• the macromolecule only passes through the nanopore and not the detection window; the macromolecule is simply viewed through the detection window.
• a method for mapping the number and location of methylated CpG islands in a target nucleic acid which comprises the steps of (1) translocating a target nucleic acid having detectable elements characteristic of the presence of the methylated CpG islands therein through an analysing device comprising a nanopore and a detection window and (2) causing the detectable elements to be detected as they pass though the detection window.
• the detectable elements are suitably detected so as to output data or a signal in the form of a distribution profile of the detectable elements along the length of the target nucleic acid.
• the distribution profiles so obtained can be used as is or added to a database of like profiles so that over time an extensive reference set is built up which constitutes a valuable research tool enabling genetic, biochemical and therapeutic conclusions and insights to be drawn therefrom.
• nucleic acid means a polymer of nucleotides. Nucleotides themselves are sometimes referred to as bases (in single stranded nucleic acid molecules) or as base pairs (in double stranded nucleic acid molecules) in an interchangeable fashion. Nucleic acids suitable for use in the method of the present invention are typically the naturally-occurring nucleic acids DNA or RNA or synthetic versions thereof. However the method can also be applied if desired to analogues such as PNA (peptide nucleic acid), LNA (locked nucleic acid), UNA (unlocked nucleic acid), GNA (glycol nucleic acid) and TNA (threose nucleic acid).
• PNA peptide nucleic acid
• LNA locked nucleic acid
• UNA unlocked nucleic acid
• GNA glycol nucleic acid
• TNA threose nucleic acid
• the nucleic acids themselves in turn suitably comprise a sequence of at least some of the following nucleotides: adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) 4- acetylcytidine, 5- (carboxyhydroxylmethyl)uridine, 2-O-methylcytidine, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylamino-methyluridine, dihydrouridine, 2-O-methylpseudouridine, 2-O-methylguanosine, inosine, N6- isopentyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyl adenosine, 2-methylguanosine, 3- methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine
• the length of the target nucleic acid sequence is expressed in terms of the number of nucleotides it contains.
• the term “kilobase” (kb) means 1000 nucleotides whilst “megabase” (Mb) means 1,000,000 nucleotides.
• the target nucleic acid used in the method of the present invention can in principle contain any number of nucleotides up to an including the number typically found in a human or other mammalian gene. However the method of the present invention is also applicable to smaller oligonucleotide fragments (e.g.
• fragments of a human gene which are at least 10 bases (for single stranded nucleic acids) or base pairs (for double stranded nucleic acids) long, more typically at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 500 or more bases/base pairs long or lkb, 2kb, 5kb, lOkb, 20kb, 50kb, lOOkb, 250kb, 500kb or up to 1Mb or more long.
• the target nucleic acid itself may be derived directly or indirectly from any available biological sample including but not limited to materials such as blood, sputum or urine.
• the target nucleic acid is further comprised of detectable elements characteristic of the former's methylated CpG islands.
• these detectable elements can be any element within or attached to the target nucleic acid at a site of a methylated CpG island which exhibits a detectable characteristic as the latter passes through the detection window of the analysing device.
• the detectable elements will all be of the same type for a given nucleic acid sample, although a given target nucleic acid may be analysed multiple times using different detectable elements if so desired. In fact it may be beneficial in certain circumstances to map the same target nucleic acid in this way to ensure that all the methylated CpG islands are identified.
• the detectable elements are such that they are able to generate, either directly or indirectly, corresponding characteristic data and/or a signal when caused to pass through the detection window.
• this characteristic data and/or signal is generated by the emission of photons characteristic of the detectable element fluorescing or Raman scattering incident light within the detection window.
• the detectable element may form part of the molecular structure of the methylated CpG island itself or may subsist in an element attached thereto which is made identifiable using Raman spectroscopy by the addition of the marker molecule referred to below.
• the detectable elements are generated by attaching a molecule capable of acting as a marker to the site of the methylated CpG island.
• a marker molecules can be attached by either physical or chemical means and, in the case of the latter, by covalent, ionic, dative bonding or ligation.
• One preferred class of such marker molecules are derivatives of proteins which are capable of binding to a methylated CpG site.
• proteins include but are not limited to methyl-CpG-binding domain proteins such as MeCP2, MBD1, MBD2 and MBD4.
• these proteins are chemically modified before use to include conventional fluorophore moieties including but not limited to xanthene moieties e.g.
• fluorescein, rhodamine and their derivatives such as fluorescein isothiocyanate, rhodamine B and the like; coumarin moieties (e.g. hydroxy-, methyl- and aminocoumarin) and cyanine moieties such as Cy2, Cy3, Cy5 and Cy7.
• Preferred fluorophore moieties are those derived from the following commonly used dyes: Alexa dyes, cyanine dyes, Atto Tec dyes, and rhodamine dyes.
• Atto 633 (ATTO-TEC GmbH), Atto 740 (ATTO- TEC GmbH), Rose Bengal, Alexa FluorTM 750 C 5 -maleimide (Invitrogen), Alexa Fluor 532 C 2 -maleimide (Invitrogen) and Rhodamine Red C 2 -maleimide and Rhodamine Green.
• Alexa FluorTM 750 C 5 -maleimide (Invitrogen)
• Alexa Fluor 532 C 2 -maleimide Invitrogen
• Rhodamine Red C 2 -maleimide and Rhodamine Green Rhodamine Green.
• the comparison is performed computationally and can be based on a set of logic decision rules, or on a range of regression and classification methods (linear or not), or on pattern matching and machine learning methods (such as neural networks, kernel methods or graphical models).
• the comparison can be performed by a computer that has a database or reference set of distribution profiles for known nucleic acids and a memory containing instructions which, when executed by the processor, compare the distribution profile of the target nucleic acid to the reference set. In the case where no matching is found the target nucleic acid can be added to the reference set for future reference if so desired.
• the target nucleic acid having the necessary detectable elements is analysed by translocating it through an analysing device comprising a nanopore having a detection window.
• the target nucleic acid is translocated through both the nanopore and the detection window.
• this detection window is defined by a localised electromagnetic field generated by plasmon resonance.
• the interaction between this electromagnetic field, the detectable elements and incident electromagnetic radiation impinging on the detection window is used to generate an increased level of fluorescence or Raman scattering which can be easily detected and analysed.
• an analysing device can be found in our WO 2009/030953 the contents of which are incorporated herein by reference.
• this analysing device comprises a nano-perforated substrate separating sample providing and receiving chambers.
• the nano-perforated substrate may either be fabricated from an inorganic insulator or from organic or biological material.
• the nano-perforated substrate is an inorganic insulator such as a silicon carbide wafer.
• the nanopore is between lnm and lOOnm in diameter preferably lnm to 50nm, 2nm to 30nm, 5nm to 20nm or 5nm to 15nm.
• the target nucleic acid is suitably caused to translocate from the sample to the receiving chambers via the nanopore by electrophoresis.
• Passage through the nanopore ensures that the target nucleic acid translocates in a coherent, linear fashion so that it emerges from the outlet thereof in a nucleotide by nucleotide fashion enabling the detectable elements and therefore the methylated CpG islands to be detected in sequence.
• the analysing device is suitably provided with a detection window juxtaposed either within the nanopore or adjacent its outlet.
• this detection window is defined by one or more metallic moieties fabricated from gold or silver capable of undergoing plasmon resonance under the influence of incident electromagnetic radiation from a coherent source such as a laser. This plasmonic resonance generates the strong localised electromagnetic field through which the target nucleic acid passes.
• the exact geometry of these metallic moieties determines the geometry of the detection window and hence affects the nature of the interaction with the detectable elements.
• the geometry of the detection window can be chosen so as to be optimised for increased photon emission, rather than for lateral localisation.
• the detection window is sized so that the length in the z dimension is from 1 to 100 preferably from 10 to 50 nanometres.
• the signal generated by the interaction of the detectable elements and the electromagnetic field can be detected by a detector such as a photocounter in the case of fluorescence or a spectrometer in the case of Raman scattering.
• the output of such a device will typically be an electrical signal characteristic of the target nucleic acid's distribution profile of methylated CpG islands.
• the method of the present invention may suitably employ multiple detectors and multiple analysing devices.
• an array of pairs of detectors and analysing devices may be used with each detector being arranged to detect photons generated using its paired analysing device.
• Other detectors including other detectors for detecting fluorescence such as a photomultiplier or single photon avalanche diode may be used.
• the characteristic data stream and/or signal is generated by fluctuations in an electrical property of the detection window and/or its contents (e.g. changes in voltage, resistance or current flow occasioned by the detectable element blocking or enabling the flow of ions in the nucleic acid's associated translocation medium between electrodes).
• fluctuations in an electrical property of the detection window and/or its contents e.g. changes in voltage, resistance or current flow occasioned by the detectable element blocking or enabling the flow of ions in the nucleic acid's associated translocation medium between electrodes.
• Preferred translocation media used here are aqueous alkali metal electrolytes such as an aqueous potassium or sodium halide, nitrate or sulphate solution.
• an apparatus for identifying a target nucleic acid comprising detectable elements characteristic of methylated CpG islands
• the apparatus comprising: an analysing device comprising a nanopore having a detection window, wherein the analysing device is capable of plasmon resonance to produce a localised electromagnetic field which defines the detection window; a detector for detecting detectable elements of the target nucleic acid as they pass through the detection window to produce a distribution profile of the detectable elements along the target nucleic acid; and optionally a computer system for comparing the distribution profile to a reference set of distribution profiles for known nucleic acids.
• the computer system typically comprises a memory and a processor. Computer executable instructions can be provided which when executed by the processor compare the distribution profile of the target nucleic acids to a reference set of distribution profiles to identify the target nucleic acid or other relationships between it and the data in the database.
• Figure 1 is a flow diagram showing a method in accordance with an aspect of the present disclosure
• FIG 2 schematically illustrates an apparatus for the method of Figure 1
• Figures 3a-b illustrates the evolution of a schematic distribution profile for the target DNA analysed in the apparatus of Figure 2.
• Figure 1 represents a flow diagram showing a method in accordance with the present invention.
• the method comprises, at step S10, translocating a target nucleic acid of human origin having detectable elements through a nanopore having a detection window.
• the nanopore is part of an analysing device which has a gold plasmonic structure that is capable of plasmon resonance under incident laser light to produce a localised electromagnetic field which defines the detection window.
• the detectable elements are caused to fluoresce and are detected as they pass through the detection window to produce a distribution profile characteristic of the number and location of the methylated CpG islands in the target nucleic acid.
• the distribution profile of the target nucleic acid is compared against a reference set of distribution profiles.
• Figure 2 schematically illustrates an apparatus for performing the method of Figure 1 comprising an analysing device 24, a photodetector 30, a data acquisition card 32 and a computer 34.
• 24 comprises a non-electrically conducting silicon carbide wafer perforated with a plurality of 4nm diameter nanopores 28 and associated gold plasmonic structures 26 (doughnut shaped) juxtaposed over the outlet of 28 to define detection windows 40.
• the methylated CpG islands of a human patient's DNA 20 are labelled with a marker molecule to create detectable elements 22 and the DNA itself is caused to translocate though 28 and 40 by electrophoresis.
• the marker molecule is for example produced from the protein MECP2 which itself is first produced in accordance with the methodology described in Lewis et al, Cell 69(6), 905-14 (1992) before being treated with the blue dye derivative NHS-AMCA (Succinimydyl-7-amino-4-methylcoumarin-3-acetic ester NHS ester (ex for example Thermo Scientific) using known methods to make the protein capable of undergoing fluorescence.
• 26 generate a localised electromagnetic field around the outlets of 28 which interacts with each 22 in turn causing them to fluoresce and emit photons 38 which are captured by 30.
• a laser (not shown) of frequency 350nm and power 12uW is used to induce plasmon resonance in 26.
• 26 comprise one or more pairs of electrodes connected to each other via a battery and an ammeter (not shown) and the detectable elements created at the methylated CPG sites are sized so as to interfere with the flow of ions between these electrodes arising from the sample's associated translocation medium (in this case aqueous potassium chloride).
• a potential difference is continuously applied across the electrodes and the resulting fluctuations in the current flowing between the electrodes (or any equivalent voltage fluctuations or changes in electrical resistance) are continuously monitored as a function of time and/or the progress of the translocation event to generate a data stream analogous to that described in the previous paragraph.
• 34 comprises a processor and memory connected to a central bus structure which is in turn connected to a display via a display adapter and one or more input devices (such as a mouse and/or keyboard). 34 further comprises a communications adapter which is also connected to the central bus. The communications adapter can receive communications, in particular communications containing new distribution profiles for new nucleic acid samples, which can be sent to the computer over a suitable communications link such as the internet.
• the computer 34 contains a database or reference set 36 of distribution profiles of methylated CPG sites for known human DNA samples and the memory of the computer contains instructions which when executed by the processor compare the measured distribution profile of the target DNA sample (shown in Figure 3) to this reference set to characterise the target DNA sample using pattern matching software.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Microbiology (AREA)
• Immunology (AREA)
• Physics & Mathematics (AREA)
• Molecular Biology (AREA)
• Biotechnology (AREA)
• Biophysics (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=46052106

Family Applications (1)

Country Status (2)

Cited By (2)

Citations (4)

• 2012
  • 2012-03-16 GB GB201204727A patent/GB201204727D0/en not_active Ceased
• 2013
  • 2013-03-15 WO PCT/GB2013/050661 patent/WO2013136087A1/fr not_active Ceased

Patent Citations (4)

Non-Patent Citations (4)

Also Published As

Similar Documents

Legal Events