WO2009007743A1 - Nucleic acid detection method - Google Patents

Abstract

Nucleic acid detection method The use of single-channel current (nanopore) recordings in combination with chemically modified DNA to retrieve sequence-specific information can be applied for detecting single nucleotide polymorphisms (SNPs), for expression profiling, and for the sizing of highly repetitive sequences of forensic and biomedical importance.

Description

Nucleic acid detection method

Introduction

The invention relates to the use of single-channel current (nanopore) recordings in combination with chemically modified DNA to retrieve sequence-specific information. The approach can be applied for detecting single nucleotide polymorphisms (SNPs), for expression profiling, and for the sizing of highly repetitive sequences of forensic and biomedical importance.

Background

Membrane protein channels and artificial nanopores provide the opportunity to detect analytes via electrical recordings. As a transmembrane potential is applied across a membrane containing the channels or pores, an ionic current passes through the pores which fluctuates if the pores are partially or completely blocked by an analyte. Such fluctuations in the current can be analysed to identify both the concentration and identity of an analyte, the latter from its distinctive current signature. Stochastic sensing, which uses currents from single pores, is an especially attractive prospect because it is highly sensitive and provides a rapid and reversible response which allows real-time monitoring of analytes.

Stochastic sensing has been used to detect ionic molecules, organic molecules and macro molecules such as single-stranded RNA and DNA. In this single molecule approach, a transmembrane potential drives individual DNA and RNA strands through a nanopore and an ionic current is at the same time driven through the pore by the applied potential. As the DNA or RNA strand passes through the pore this current is modulated depending on the extent of blockage of the pore by the strand and characteristic blockades in ionic current are seen. As DNA homopolymers of different composition give rise to different characteristic current blockades and modulations [J. J. Kasianowicz, E. Brandin, D. Branton, D. W. Deamer, Proc Natl Acad Sci U S A 1996, 93, 13770], it has been suggested to use nanopores to sequence individual DNA strands. Progress has been made on the sequence-specific detection of static DNA strands [S. Howorka, L. Movileanu, O. Braha, H. Bayley, Proc Natl Acad Sci U S A 2001, 98, 12996; S. Howorka, S. Cheley, H. Bayley, Nat Biotechnol 2001, 19, 636; N. Ashkenasy, J. Sanchez-Quesada, H. Bayley, M. R. Ghadiri, Angew Chem Int Ed Engl 2005, 44, 1401] and individual nucleotides [Y. Astier, O. Braha, H. Bayley, J Am Chem Soc 2006, 128, 1705].

However, the identification of individual bases in translocating DNA strands has so far not been achieved as the fast passage of the DNA molecule through the pore leads to insufficient discrimination between the four bases [H. Bayley, Current Opinion in Biotechnology 2006, 10, 628]. Specifically, at the transmembrane potentials required for transport of the DNA through the pore, the DNA molecule moves rapidly at around 1 to 5 μs per base, which does not allow single nucleotide resolution with conventional recording procedures as there is too much noise at the required bandwidth to distinguish between G, C, A and T.

US 2005/0053961 relates to the characterisation of polymers, such as DNA, by their interaction with a pore to bring about changes in conductance across the pore which are indicative of the characteristics of the polymer. It is suggested that the nucleotide bases of DNA will influence pore conductance during passage of a DNA molecule through a pore and the sensitivity of the system may be increased by using modified bases, such as methylated bases and biotinylated triphosphates.

It is an aim of the present invention to improve the sensitivity of single-molecule nanopore recordings to retrieve sequence-specific information from DNA.

It is a further aim of the invention to provide a method for detecting single nucleotide polymorphisms (SNPs), for expression profiling, and for the sizing of highly repetitive sequences of forensic and biomedical importance.

Summary of the invention

In a first aspect the invention relates to a method of detecting the presence or absence of one or more bases or the number of repeats of one or more bases in an analyte nucleic acid molecule, comprising providing a membrane having first and second sides and containing a nanoscale pore having a lumen spanning the membrane; providing a fluid comprising the analyte nucleic acid molecule and an ionic salt on the first side of the membrane and a solution comprising an ionic salt on the second side of the membrane; and applying a potential difference across the membrane and measuring the resulting current over time, wherein the analyte nucleic acid molecule is chemically modified to modulate the duration and/or amplitude of the measured current and thereby allow identification of the analyte nucleic acid molecule and detection of the presence or absence of the one or more bases or the number of repeats of the one or more bases.

Preferably the analyte nucleic acid molecule comprises an extended primer oligonucleotide consisting of an original primer portion and an extended oligonucleotide portion, which is chemically modified in the original primer portion and optionally in the extended oligonucleotide portion, the presence or extent of chemical modification in the extended oligonucleotide portion being related to the presence or absence of the one or more bases or the number of repeats of the one or more bases.

In another aspect the invention relates to a method of detecting the presence or absence of one or more bases or the number of repeats of one or more bases in an analyte nucleic acid molecule, comprising: providing a sample of nucleic acid molecules and a chemically modified primer complementary to a portion of the analyte nucleic acid molecule; allowing the primer to bind to analyte nucleic acid molecules in the sample and to extend wherein one or more chemically modified nucleotides are incorporated into the extended primer oligonucleotide when one or more complementary bases are present in the bound analyte nucleic acid molecule; providing a membrane having first and second sides and containing a nanoscale pore having a lumen spanning the membrane; providing a fluid comprising the chemically modified extended primer oligonucleotide and an ionic salt on the first side of the membrane and a solution comprising an ionic salt on the second side of the membrane; and applying a potential difference across the membrane and measuring the resulting current over time, wherein the extent of chemical modification of the extended primer oligonucleotide modulates the duration and/or amplitude of the measured current and thereby allows identification of the analyte nucleic acid molecule and detection of the presence or absence of the one or more bases or the number of repeats of the one or more bases in the analyte nucleic acid molecule.

These methods can be used to detect a single nucleotide polymorphism (SNP) or a mutation in the analyte nucleic acid molecule or to detect the number of repeats of a highly repetitive sequence in the analyte nucleic acid molecule. The primer extension approach can be used for multiplexing to analyse multiple different SNPs in a biological sample. The use of multiple DNA primers which are modified with different sequences of chemical tags which give rise to a characteristic pattern of current modulations is the key to achieve multiplexing. In this way, the sequence of the primer can be inferred from the characteristic pattern of current modulations in the traces. Detecting the signatures in the sample with the extended primer therefore indicates that a specific SNP must have been present in the original sample.

Preferably each type of chemical modification is associated with a specific base. In other words a single base may have one or more chemical modifications, but each chemical modification may be associated with only a single base and not two or more consecutive bases.

Preferably the chemical modification is smaller than 1.5 nm diameter, more preferably smaller than 1 nm diameter, more preferably smaller than 0.5 nm diameter and most preferably about 0.3 nm diameter. The modification may act to increase the diameter of the nucleic acid molecule, thereby slowing its passage through the nanopore.

Preferably the chemical modification is a peptide tag, preferably consisting of from 2 to 6 amino acids. For example, the peptide tag may be selected from hexahistidine, hexaarginine, hexaaspartic acid, histidine(4), histidine(2) and tyrosine(3). The primer may be modified with one or more chemical tags which are different to the one or more chemical tags used to detect the presence or absence of the one or more bases or the number of repeats of the one or more bases. The chemical tag may also allow the extended primer oligonucleotide to be isolated from other nucleic acid molecules.

In another aspect the invention provides that each type of base which is detected is labelled with a different chemical tag which gives rise to a specific current signature. Since it is possible to infer which type of base is present in the extend primer strand by looking at the current signature, this enables part or all of a nucleic acid molecule to be sequenced.

In another aspect the invention provides a method of mRNA profiling comprising: providing a sample comprising a first mRNA species and a second mRNA species, a chemically modified first primer complementary to a portion of said first mRNA species and a chemically modified second primer complementary to a portion of said second mRNA species; allowing the primers to bind to the mRNA species in the sample and to extend wherein one or more chemically modified nucleotides is incorporated into the extended primer oligonucleotides when one or more complementary bases are present in the bound mRNA species; providing a membrane having first and second sides and containing a nanoscale pore having a lumen spanning the membrane; providing a fluid comprising the chemically modified extended primer oligonucleotides and an ionic salt on the first side of the membrane and a solution comprising an ionic salt on the second side of the membrane; and applying a potential difference across the membrane and measuring the resulting current over time, wherein the chemical modification of the extended primer oligonucleotides modulates the duration and/or amplitude of the measured current and thereby allows identification and quantification of the first and second mRNA species.

Preferably the chemical modification of the first primer is different to the chemical modification of the second primer and/or different chemically modified nucleotides are incorporated into the first and second mRNA species. In another aspect the invention provides a method of sequencing a nucleic acid molecule comprising: providing a sample comprising the nucleic acid molecule and a chemically modified primer complementary to a portion of said nucleic acid molecule; allowing the primer to bind to the nucleic acid molecule in the sample and to extend wherein one or more chemically modified nucleotides is incorporated into the extended primer oligonucleotide when one or more complementary bases are present in the bound nucleic acid molecule, wherein each type of chemically modified nucleotide is labelled with a different chemical tag which gives rise to a specific current signature; providing a membrane having first and second sides and containing a nanoscale pore having a lumen spanning the membrane; providing a fluid comprising the chemically modified extended primer oligonucleotides and an ionic salt on the first side of the membrane and a solution comprising an ionic salt on the second side of the membrane; and applying a potential difference across the membrane and measuring the resulting current over time, wherein the chemical modification of the extended primer oligonucleotides modulates the duration and/or amplitude of the measured current and thereby allows identification of one or more nucleotides in the nucleic acid molecule.

Brief description of drawings

The present invention will now be described with reference to the accompanying drawings, in which:

Figure 1 shows the cross-sectional view of the heptameric αHL pore. The model was generated using crystallographic data (Song, L.; Hobaugh, M. R.; Shustak, C; Cheley, S.; Bayley, H.; Gouaux, J. E. Science. 1996, 274, 1859-1866.) and PyMoI. The internal diameters of the channel are: 2.9 nm, cis entrance; 4.1 nm, internal cavity; 1.3 nm, inner constriction; 2 nm, trans entrance of the β-barrel.

Figure 2 shows an individual nanopore embedded in a membrane which separates two reservoirs filled with electrolyte solution. Figure 3 shows schematically a single channel current traces of (A) an unmodified synthetic oligonucleotide, (B) a synthetic oligonucleotide modified with one chemical tag covalently attached to a single base, (C) a synthetic oligonucleotide modified with a different chemical tag covalently attached to a single base, and (D) a synthetic oligonucleotide modified with two different chemical tags, each covalently attached to a single base.

Figure 4 shows the use of chemically modified DNA to detect an SNP using primer extension and representative single channel current traces. 4A. In the case of the presence of an SNP, a chemically modified nucleotide is incorporated resulting in an additional blockade signature. 4B. In the case of the absence of an SNP, no chemical tag is incorporated resulting in no additional blockade signature.

Figure 5 shows the use of chemically modified DNA to size the length of highly repetitive DNA strands. 5 A. In the case of the presence of two repeats, two chemically modified nucleotides are incorporated resulting in an additional blockade signature of length 2xt₂. 5B. In the case of the presence of seven repeats, seven chemically modified nucleotides are incorporated resulting in an additional blockade signature of length 7xt₂.

Figure 6 shows how the incorporation of chemically modified nucleotides can proceed in a two-step procedure. In a first step, a nucleotide with a small linker is incorporated efficiently into DNA. After DNA synthesis, the remaining chemical tag is attached to the linker.

Figure 7 shows single-channel current recordings for (A) a hexa-arginine modified oligonucleotide of 27 bases in length, (B) a hexa-histidine modified oligonucleotide of 27 bases in length, (C) an unmodified oligonucleotide of 27 bases in length, and (D) an oligonucleotide of 27 bases in length modified with two peptide tags, hexa-histine and tri-tyrosine.

Figure 8 shows (A) The α- hemolysin (α HL) pore embedded in a lipid bilayer. (B) The chemical linkage between DNA and peptide within construct H6C1-O1. (C) Schematic representation of the α HL pore and a representative single channel current trace. (D) Events caused by oligonucleotide Ol without a peptide tag. (E) Trace for the translocation of H6C1-O1. (F) Events for H6C 1-01 -terminal. The traces were obtained from recordings at 2 M KCl, 20 mM Tris, pH 8.0, and filtered and sampled at 10 kHz and 50 kHz, respectively.

Figure 9 shows representative nanopore translocation events for (A) R7C1-O1, (B) Y3C1-O1 exhibiting a current step, (C) Y3C1-O1 exhibiting a current slope, and (D) Y3/Y3-O3. The insets display the magnified view of the high-amplitude region of the corresponding events. (E) Amino acid side chains or histidine, arginine and tyrosine. (F) Scheme to account for the current signature of Y3/Y3-O3 events in (D).

Detailed description of the invention

According to the invention, an individual nanopore is embedded in a membrane which separates two reservoirs filled with electrolyte solution (see Fig. 2). The pore can be a protein pore in a lipid bilayer membrane or a solid-state nanopore composed of Si₃N₄. The application of a transmembrane potential leads to the flow of a small ionic current which can be measured using a current amplifier.

Nanopore recordings can be used to detect nucleic acid molecules, e.g. DNA or RNA, at the single molecule level. When added to the reservoir, nucleic acid strands are electrophoretically driven through the pore as shown in Fig. 3A. The temporary blockade of the pore leads to a reversible reduction in the current flowing through the pore. Given that the current is flowing through a single channel, nanopore recordings are capable of detecting individual nucleic acid strands.

The new approach uses chemically modified nucleic acid molecules in order to slow down the passage of the nucleic acid strand through the pore and to specifically detect individual bases. The use of chemically modified DNA strands allows tuning of the cross-sectional diameter of ssDNA to existing pore dimensions rather than matching the pore dimensions to the size of the DNA strand. As shown in Fig. 3B, one chemical tag was covalently attached to a single base in a synthetic oligonucleotide. The chemical tag increases the cross-sectional area of the DNA to over 1 nm which is similar to the 1.2 nm wide nanopore. Recordings show that the chemical tag slows down the passage of the DNA strand by a factor of up to 15 leading to an average blockade duration tl (Fig. 3B). In addition, the tag changed the amplitude of the current blockade (al) (Fig. 3B). Attaching a different chemical tag led to a different blockade signature (different duration t2 and amplitude a2) (Fig. 3C). Attaching two different chemical tags led to a further different blockade signature (Fig. 3D). This is also illustrated in Fig. 7 as described below. Furthermore, decreasing the size of one tag shortened the duration of the corresponding blockade in the recordings. Finally, a chemically modified deoxythymidine nucleotide derivative was prepared and successfully incorporated into DNA strands using template-directed polymerase- catalyzed synthesis. This opens the possibility to use biological templates for the generation of chemically modified DNA.

This invention is not limited to any particular type of nanoscale pore. Any suitable pore may be used. However, in the present invention preferably the pore is an organic pore. The term organic takes its usual meaning in the art, therefore the organic pore substantially comprises carbon and hydrogen, and also other elements, especially nitrogen, oxygen, sulfur, phosphorus and halogens. These pores exhibit very high mechanic stability which makes them ideally suited for rugged electrical sensor devices.

More preferably the pore is a protein pore. A protein pore is a pore which is predominantly protein; however, other types of molecules may also be present. Examples of protein pores suitable for use in the invention include alpha hemolysin, pneumolysin, outer membrane proteins such as porins, and other bacterial pore-forming toxins (Gilbert, R. J. (2002) Cell MoI Life Sci 59, 832-44) (Parker, M. W., and Feil, S. C. (2005) Prog Biophys MoI Biol 88, 91-142) such as streptolysin O (Bhakdi, S., Tranum- Jensen, J., and Sziegoleit, A. (1985) Infect Immun 47, 52-60) or LukF (Olson, R., Nariya, H., Yokota, K., Kamio, Y., and Gouaux, E. (1999) Nat Struct Biol 6, 134- 40). The latter are oligomeric assemblies of protein subunits. The diameter of the lumens of protein pores depends on the type of pore and ranges from 1.2 nm for alpha hemolysin (Song, L., Hobaugh, M. R., Shustak, C, Cheley, S., Bayley, H., and Gouaux, J. E. (1996) Science 274, 1859-66) to 26 nm for pneumolysin (Tilley, S. J., Orlova, E. V., Gilbert, R. J., Andrew, P. W., and Saibil, H. R. (2005) Cell 121, 247-56).

Particularly preferably the protein pore is a α-hemolysin (αHL) polypeptide. αHL is a bacterial toxin which self-assembles to form a heptameric protein pore. The X-ray structure of the αHL pore resembles a mushroom with a wide cap and a narrow stem, which spans the lipid bilayer (Fig. 1) (Song, L.; Hobaugh, M. R.; Shustak, C; Cheley, S.; Bayley, H.; Gouaux, J. E. Science. 1996, 274, 1859-1866). The external dimensions of the heptameric αHL pore are 10 x 10 nm, while the central channel is 2.9 nm in diameter at the cis entrance and widens to 4.1 nm in the internal cavity (Fig. 1). In the transmembrane region, the channel narrows to 1.3 nm at the inner constriction and broadens to 2 nm at the trans entrance of the β-barrel. The defined structure of αHL has facilitated extensive engineering studies and has led to the development of tools for the targeted permeabilization of cells (Eroglu, A.; Russo, M. J.; Bieganski, R.; Fowler, A.; Cheley, S.; Bayley, H.; Toner, M. Nat Biotechnol. 2000, 18, 163-167) as well as new biosensor elements which permit the stochastic sensing of molecules (Bayley, H.; Cremer, P. S. Nature. 2001, 413, 226-230).

Although organic pores are preferred, the invention is not limited to pores of this type. Alternative embodiments of the invention exist wherein the pore is an inorganic pore. Preferably the inorganic pore is composed of silica, silicon nitride, alumina, titanium, gold, platinum, zirconia or a combination thereof. Particularly preferred is a solid-state nanopore composed Of Si₃N₄.

Although the pore is not limited in relation to the material that it comprises, the invention is limited in relation to the size of the pore. The pore must be a nanoscale pore. By nanoscale is meant that the pore is one wherein the lumen has a diameter of less than 1 μm. Preferably the lumen has a diameter of less than 100 nm. More preferably the lumen has a diameter of 10 nm or less. Preferably the pore has a diameter of at least 1 nm. References to the diameter of the pore are to be interpreted as the diameter of the pore at its minimum value. In this regard see fig. 1 which illustrates that the diameter of the lumen may vary at different positions along its length. Engineered nanopores may also be used in accordance with the invention. Engineered pores include pores which have been modified to affect the passage of molecules therethrough. The modification may be at the inner constriction or at either entrance. In the present invention it is preferred to use a pore which has been engineered to be complementary to the oligonucleotide molecule passing through the pore or the chemical tag attached thereto. Such modification could enhance the current modulation and allow greater discrimination between different chemical tags. For example if the oligonucleotide has been modified with a positively charged chemical tag then it may be beneficial to use a protein nanopore which has been engineered to have negative residues in the inner constriction of the pore. As the oligonucleotide passes through the pore there would be electrostatic interactions between the positively charged chemical tag and the negative residues in the lumen, which further slow the passage of the oligonucleotide through the pore and therefore specifically modulate the current. Apart from electrostatic interactions, other types of interactions are possible such as hydrogen bonding, van der Waals, steric bulk pi-pi interactions and hydrophobic interactions, metal chelate complex formation (e.g. NTA Ni HIS), secondary structures which have to be unfolded for their passage (e.g. hairpin structure).

The pores described above provide a route, channel or path across a membrane, from one side to the other side. The membrane, or barrier, itself is not limited in type, and would need to be chosen by consideration of species it is desired to pass through the pore or prevent from passing through the pore, and therefore between the membrane's first and second sides. For example, the membrane must be stable in the presence of this species.

The membranes used in the method of the invention are not limited to biological materials: biological membranes are only one example of membranes according to the invention.

However, preferably the membrane is organic. In one embodiment the organic membrane is an organic polymer, most preferably the organic polymer is a polycarbonate or polyterephtalate polymer. Most preferably the organic membrane is a lipid bilayer. In a particularly preferred embodiment of the invention, the membrane comprising a pore is formed by allowing mutant polypeptides K46C, K8C or S106C to assemble on rabbit erythrocyte membranes to form heptameric pores.

Alternatively the membrane is inorganic, wherein preferably the inorganic membrane is a gold-plated porous membrane prepared by the template synthesis method by depositing gold along the pore walls of a polycarbonate template membrane (Martin, C. R., Nishizawa, M., Jirage, K., and Kang, M. (2001) J Phys Chem B 105, 1925).

In addition, preferably when the membrane is a lipid bilayer the pore is an organic pore, preferably a polymeric organic pore or a protein pore. Preferably when the membrane is an organic polymer the pore is an organic pore. Preferably when the membrane is an inorganic membrane, the pore is an inorganic pore.

In a particularly preferred embodiment of the invention, the membrane is a lipid bilayer and the pore is a protein pore or a solid-state nanopore composed Of Si₃N₄.

According to the invention, the membrane will usually comprise a single pore, the lumen of which provides a single channel between the first side of the membrane and the second side of the membrane. The use of single-molecule detection leads to a higher sensitivity. This could help reduce the number of PCR cycles and errors which are encountered in the course of the preparation of samples for microarray or bead- based detection schemes. The membrane may also comprise more than one but less than ten pores provided that the current modulations caused by the translocation of a DNA strand through a first pore are not compromised by the current modulations caused by the translocation of another DNA strand through a second pore.

It will be apparent to the skilled person that many electrolytes may be used in accordance with the invention. For example suitable electrolytes are KCl or NaCl in the concentration range from 0.5 M to 4 M, or organic electrolytes such as ammonium acetate. The transmembrane potentials can vary from 30 to 200 mV in the case of lipid bilayer membranes but the upper boundary can be up to several V in the case of inorganic pores which are more robust. In accordance with the invention either single stranded or double stranded DNA can permeate through the pore. For example, single stranded DNA may comprise an extension primer which has been extended, while double stranded DNA may comprise an extension primer which has been extended, in addition to the complementary DNA strand. In both cases, chemical modifications on the DNA will give rise to current blockades when passing through a pore. In this case of single stranded DNA it may be preferable to use the αHL pore. In this case of single stranded DNA it may be preferable to use the Si₃N₄ pore. It may be advantageous to use double stranded DNA because this can be easily obtained in pure form by selectively digesting non- hybridised single stranded DNA in a biological sample, while leaving double stranded DNA intact. An additional purification procedure to remove nucleotides from the duplexed analyte would not be necessary. Enyzmes which selectively remove single stranded DNA are well known in art and include Exonuclease I (/E. coli/) and Exonuclease T.

As mentioned above, in the present invention one or more bases of the nucleic acid molecule is modified with a chemical tag. The role of the chemical tag is to affect the ease with which the nucleic acid molecule may pass though the pore and therefore to alter the current signal. By the term "current signal" or "current signature" we refer to the specific alterations in both the duration and the amplitude of the current which result from the chemically modified nucleic acid molecule passing through the pore. In addition, current signature can also include other characteristics such as current noise or characteristic time-dependent changes in the current blockades such as slopes of increasing or decreasing current with in a blockade event.

In order to achieve this goal, the size and type of the chemical tag is selected by consideration of the size of pore, and the degree to which it is desired to alter the ability of the nucleic acid molecule to a pass through the pore. Therefore the present invention is not limited in relation to the size or the type of the chemical tag. However, in some embodiments it may be preferred that the chemical modification is smaller than 1.5 nm diameter, more preferably smaller than 1 nm diameter, more preferably smaller than 0.5 nm diameter and most preferably about 0.3 nm diameter. The modification may act to increase the diameter of the nucleic acid molecule, thereby slowing its passage through the nanopore.

In some embodiments it may be preferable to use more than one type of chemical tag in order that different current signatures may be distinguished. For example it may be desirable for different species of nucleotide to be modified with different chemical tags in order that their current signatures can be clearly distinguished. For example, it may be preferred that each type of chemical modification is associated with a specific base.

In other words a single base may have one or more chemical modifications, but each chemical modification may be associated with only a single base and not two or more consecutive bases.

In one embodiment the primer is chemically modified with a chemical tag which allows identification, and optionally isolation, of the nucleic acid molecule (extended primer). The pattern of chemical tags on a given primer is specific for the sequence of the primer and the pattern of chemical tags encodes for a specific and unique current signature which identifies the sequence of the primer and hence the sequence of the template the primer has bound to. Further, the base or bases which represent the SNP or mutation or are part of a highly repetitive sequence are chemically modified with one or more different chemical tags which have a different current signature. This allows detection and/or identification of the presence or concentration of the base.

For the primer extension only a maximum of four different chemical tags can be used. By contrast, the primer may contain more than four different types of tags. For example, using solid phase oligonucleotide synthesis it is possible to generate an oligonucleotide which contains among other bases three adenines each of which carry a different chemical tags giving rise to a specific current signature. Given the multiple combinations of different chemical tags in the primer portion, it is possible to encode a multitude of different primers of different sequence. This enables multiplexing and the detection of several different SNPs or different mRNA types. The binding of the multiply labelled primers to the template is not affected by the presence of the chemical tags. In the case of undesired interactions, the chemical tags can be positioned at the portion of a primer which does not bind to the template strand. Preferably the chemical tag is a peptide. Such a modular tag, in this case formed of amino acids, enables the skilled person to easily make numerous different tags with different current signatures. Preferably the peptide tag consists of from 2 to 6 amino acids, but any suitable length may be used bearing in mind that the length of the peptide will correspond directly to the duration of the channel blockade and the duration component of the current signature. Examples of suitable peptide tags are hexahistidine, hexaarginine, hexaaspartic acid, tetrahistidine, dihistidine and trityrosine.

Other chemical tags that may be used in accordance with the invention include aromatic systems such as phenyl rings and substituted derivatives thereof, condensed aromatic systems such as naphthalene, phenanthrene, pyrene, and substituted derivatives thereof, saturated organic cyclic compounds such as cycloalkanes including cholesterol and derivatives thereof, spiro-molecule and other bicyclic compounds, tricyclic compounds such as adamantanes, monomeric and oligomeric carbohydrates such as cyclodextrins and substituted derivatives thereof, organometallic compounds including rhodium acetate complexes, metallocenes such as ferrocenes and substituted derivatives thereof, inorganic compounds, porphyrins and substituted organic derivatives thereof.

Methods of synthesis of these types of chemical tags are well known to the person skilled in the art.

Any number of chemical tags may be attached to the DNA molecule. The number of chemical tags attached to the DNA may be altered for example to vary the ease with which molecules may pass through the pore and therefore the current pattern recorded.

A greater number of chemical tags will hinder the passage of molecules through the pore, whereas a smaller number will relatively ease the passage of molecules through the pore. Preferably, however, from one to ten chemical tags are attached to the DNA molecule. Embodiments of the invention are known in which one, two, three, four, five, six, seven, eight, nine or ten chemical tags are attached to the DNA molecule.

More preferably from one to four chemical tags are attached to the DNA molecule. The chemical tag must be bound to the nucleotide in some way. The invention is not limited in respect of how this binding is achieved. Preferably the chemical tag is attached to the nucleotide by various covalent linkages such as amide, disulfide, thioether bonds or linkages generated by Diels-Alder, Click-chemistry, or related pericyclic reactions. The chemical linkages bind the chemical tags to the base. Preferably, purine bases carry a chemical linker at position 7 and pyrimidine bases at position 5. These positions are preferred because nucleotide analogues with linkers at these positions are known to be accepted as substrates by polymerase enzymes. The chemical tags can be attached at various stages to the DNA. For example, synthetic oligonucleotides which are used as primers for the extension reaction can be obtained by linking the tag to a synthetic oligonucleotide carrying a modified base with a linker as shown in Example 1. Alternatively, oligonucleotides with chemical tags can be obtained via solid phase oligonucleotides synthesis using phosphoramidite analogues carrying the chemical tags. Furthermore, the chemical tags can be incorporated into a DNA strand by template-directed DNA polymerisation using triphosphate nucleotide analogues. The polymerisation can be performed using nucleotide analogues carrying the chemical tags, or, in case the polymerase is not promiscuous, using analogues with linkers, which are derivatized with the chemical tag once the base has been incorporated into the DNA strand.

When the tag is incorporated by polymerisation, either only the labelled nucleotide or a mixture of labelled nucleotide and unlabelled nucleotides are used, depending on the specific application. For example, if a primer should be extended by just one base, the labelled base, then the extension reaction is only performed in the presence of the labelled nucleotides (e.g. an 5-ethynyl-dUTP). If longer stretches should be extended, then more than one base can be included depending on the sequence. For the extension of the sequence UAG the bases 5-ethynyl-dUTP, dATP and dGTP would be used. The inclusion and/or exclusion of particular bases, whether labelled or not, is a way to regulate the length of the extended portion. In all cases, the nucleotide mixture is, however, devoid of the unlabelled version of the labelled nucleotides. For example, a nucleotide mix may contains 5-ethynyl-dUTP but not dTTP. As will be apparent to the skilled person, the primer extension is based on complementary base incorporation; misincorporations do not occur. Specifically, the different bases in the template direct the incorporation of one or more complementary labelled bases in the extended primer product. As each type of base may contain a specific chemical tag which gives rise to a specific current signature it is possible to infer which type of base is present in the extend primer strand by looking at the current signature.

Promiscuous polymerases such as Deep Vent exo- can be used for the incorporation of labelled nucleotides (JACS, 2006, 128, 1398-1399).

When the tag is incorporated by polymerisation, the primer may be designed to bind to the oligonucleotide template upstream (5') of the SNP, mutation or highly repetitive sequence. Preferably the primer will bind to the template oligonucleotide less than 20, preferably less than 10 and most preferably 1 to 2 bases upstream of the SNP, mutation or highly repetitive sequence. This ensures that minimal extension of the primer is required in order to achieve labelled extension products. The primer should preferably be designed to assist in interpretation of the current signals measured. For example, if the SNP of interest is an adenine base and so labelled adenine nucleotides are to be incorporated into the extension product when the SNP is present, then it may be preferable to ensure that the primer is designed such that no other adenine residues are encoded prior to the SNP. Alternatively, the current signal should be interpreted to take into account other labelled adenine residues prior to the one of interest.

The incorporation of chemically modified nucleotides can proceed in one step or in a two-step procedure. In the one-step procedure the completely modified nucleotide is being incorporated into the DNA strands via polymerases. Some of the artificial bulky nucleotides might be incorporated efficiently. The problems of low incorporation yield can be overcome by a two-step procedure as shown in Fig. 6. In a first step, a nucleotide with a small linker is incorporated efficiently into DNA. After DNA synthesis, the remaining chemical tag is attached to the linker. The remaining chemical tag can be attached to the base using a variety of chemistries such as Diels-Alder, Click-chemistry, or related pericyclic reactions, or disulfide exchange reactions. The extended primer product may optionally be purified using the chemical tag in one of the extended bases or in the primer. For example, the extended primer product may contain a hexahistidine tag attached to a base in the extended section. As shown in Example 1, a histidine tag facilitates the purification of the modified DNA strand via immobilised metal affinity chromatography. Other equally suitable methods of purification using chemical tags are well known to the person skilled in the art. In the proposed purification scheme, extended DNA will be purified from unextended primers and the biological templates because the latter do not contain histidine-tags.

When the nanopore is the alpha-HL pore or another protein pore, only single stranded nucleic acid molecules can be translocated through because the narrow constriction of this pore is not wide enough for the passage of double stranded DNA. The use of the single stranded DNA requires the isolation of the primer extension product and its separation from the unextended primer and the template sequence. In principle, double stranded DNA could be used in combination with other nanopore such as inorganic pores. This would permit the analysis of an extended primer product which is still hybridized to the template sequence. It is, however, preferable to remove the template strand from the extended primer product due to the ill-defined length of the template. Should a long DNA or RNA strand cause the clogging of a nanopore, this may be cleared by the reversal of the transmembrane potential.

Any kind of nucleic acid sample may be used in accordance with the invention. For example the sample may comprise numerous types of nucleic acid molecule and it will be apparent from the current measurements which are the molecules of interest. Alternatively the nucleic acid molecules of interest can be purified prior to passing them through the nanopore, for example by using one of the chemical modifications to separate the nucleic acid molecules of interest using chromatography or the like as discussed above.

The method of the invention may be used for detecting single nucleotide polymorphisms (SNPs), for expression profiling, and for the sizing of highly repetitive sequences of forensic and biomedical importance. For example, peptide tags can be incorporated into copied DNA strands from biological samples using chemically modified nucleotides and sequence-specific primer extension. This approach would be suitable to sense the presence or absence of single-nucleotide polymorphisms by incorporating and detecting a modified base only if the target mutation is present, or for sizing the highly repetitive DNA regions in trinucleotide expansion disease genes by labelling the same base in all repeats.

The use of chemically modified DNA to detect SNPs using primer extension is illustrated in Fig. 4. An oligonucleotide carrying two chemical tags binds to the template DNA strand. The primer oligonucleotide carries two chemical tags which cause a specific current signature and therefore encode the identity of the primer. Primer extension leads to the synthesis of the complementary DNA strand. In the case of an SNP (Fig. 4A), a chemically modified nucleotide is incorporated, while no chemical tag is added in the absence of the SNP in a different DNA strand (Fig. 4B). The presence or absence of the SNP can be detected by monitoring the presence of absence of the additional blockade signature. This approach can be extended to analyse multiple different SNPs in a biological sample. Multiple DNA primers may be modified with different sequences of chemical tags which give rise to characteristic patterns of current modulations. The sequence of the primer can therefore be inferred from the characteristic pattern of current modulations in the traces. Detecting the signatures in the sample with the extended primer therefore indicates that a specific SNP must have been present in the original sample.

The use of chemically modified DNA to size the length of highly repetitive DNA strands is illustrated in Fig. 5. The expression "highly repetitive sequence" is well known in the art. Repetitive DNA sequences with a length range from a hundred to a few thousand bases are found in non-coding regions of the human genome in the form of microsatellite DNA. These sequences are composed of terra-, tri-, or dinucleotides such as CA repeats and recur 10 to 100 times without interruption. As the number of repeats at a given chromosomal locus is characteristic for each individual, microsatellite DNA is used for genotyping in recombination mapping, studies on population genetic, and paternity tests. Prominent examples of repeat sequences occurring in coding genome regions are genes of trinucleotide expansion diseases such as Huntington's, Myotonic dystrophy and Friedreich's ataxia. The expanded trinucleotide regions arise due to strand slippage during DNA replication and have been identified as molecular reason for pathogenic changes (Bates, G. P. (2005) Nat Rev Genet 6, 766-73, Li, S. H., and Li, X. J. (2004) Trends Genet 20, 146-54). In the case of Huntington and eight other inherited neurodegenerative diseases, expanded CAG repeats encode for polyglutamate tracts, which can lead to misfolding, aggregation and dysfunction of the disease-relevant proteins (Yoon, S. R., Dubeau, L., de Young, M., Wexler, N. S., and Arnheim, N. (2003) Proc Natl Acad Sci U S A 100, 8834-8, Ross, C. A., and Poirier, M. A. (2004) Nat Med 10 Suppl, S 10-7). Importantly, the number of repeats in the tracts strongly influences the proteins' tendency to misfold or aggregate. For example, a stretch of more than 40 glutamate residues in the huntingtin gene causes the onset of Huntington's disease, with the severity of the disorder proportional to the number of additional residues. Similar correlations between repeat number and pathogenicity have also been observed for other trinucleotide expansion disease genes (Timchenko, L. T. (2003) Trinucleotide repeat diseases of the nervous systems, Springer).

As shown in Fig. 5A. in the case of the presence of two repeats of a highly repetitive sequence, two chemically modified nucleotides are incorporated resulting in an additional blockade signature of length 2xt₂. In contrast, Fig. 5B shows that in the case of the presence of seven repeats, seven chemically modified nucleotides are incorporated resulting in an additional blockade signature of length 7xt₂. In this way it is possible to distinguish between the numbers of repeats of the sequence of interest.

In relation to expression profiling, the proportions of two or more types of mRNA from one or two different samples may be identified to obtain information about the physiological or disease state of cells and tissues. For example mRNA from different genes may be compared or mRNA from different cell types or organisms may be compared.

In its simplest embodiment, the relative proportion of two different types of mRNA within one tissue sample is analysed. Two different primers are used, which recognise specifically bind to either one of the mRNA templates. The primers differ not only in their base sequence but also in the pattern of the chemical tags such as arginine or tyrosine (but preferably not histidine) peptides which encode for a specific and distinguishable current signature. After binding to the biological templates, which can be optionally converted into cDNA, the primers are extended by at least one additional nucleotide. The extended nucleotide carries a chemical tag such as the ethynyl group which is then reacted with the azido-derivatized histidine peptide in a Click-reaction as shown in example 3 and 4. The primer extension products may be purified using for example immobilised metal affinity chromatography as illustrated in example 1.

After removal of unextended primer and template, the two extended primers are analysed using single channel current recordings. The translocation of the two DNA strands gives rise to two different current signatures. The current signatures are composed of two segments; the first is caused by the primer-specific tags and the other by the extension specific tag. The extension specific tag and signature is identical for both types of DNA but the primer-specific tags are different and allow us to discriminate between the two different extended primer products. It should be noted that the signature caused by the extension specific tag is not absolutely required as the histidine tag has already been used to separate unextended from extended primers. The presence of the extension signature is nevertheless an additional criterion to positively identify the extended primer.

The current recordings may be performed to sense at least 1000 events for the two types of extended primer products. The proportion of the events is proportional to the ratio of the two mRNA types in the biological sample. Additional calibration with reference standards of known ratios of extended primer products can be conducted as an option to enhance the accuracy of the measurements.

Unlike microarray and bead-based detection technologies, nanopore recordings use electrical rather than optical signals to sense biomolecules. A low-cost miniaturized electrical read-out device which carries out the method according to the invention could be used in point-of-care applications. The products and methods of the invention will now be illustrated by the following examples which are not intended to limiting. The scope of the invention is defined by the appended claims.

Example 1

The coupling of a peptide strand to a DNA oligonucleotide to form a peptide- oligonucleotide conjugate (POCs), its purification and analysis by MS. Preparation of internally modified 27mer oligonucleotide -peptide conjugate

Scheme for the synthesis of a DNA oligonucleotide carrying a single peptide strand attached to an internal base

A) Preparation of 3-(2-Pyridyldithio)-propanoic acid

3-(2-Pyridyldithio)-propanoic acid was synthesized as a precursor for the generation of Λ/-succinimidyl 3-(2-pyridyldithio)-propanoate (SPDP). 2,2'-Dipyridyldisulfide (DPDS, 10.98 g, 0.0498 mol) was dissolved in 80 mL anhydrous ethanol. Acetic acid (1.5 mL) and 3-mercapto-propanoic acid (2.64 g, 2.16 ml, 0.0249 mol) were added and the solution left stirring at room temperature for 2 hours. After this time the solvent was removed under reduced pressure to yield a viscous yellow oil which was placed under high vacuum to remove all traces of acetic acid. The crude product was purified by a basic AI2O3 column using dichloromethane/ethanol (3/2) as the eluent. Once the yellow band corresponding to the thione by-product had eluted from the column 4 ml of acetic acid per 100 ml solvent was added to elute the desired product. Fractions containing this compound were pooled and the solvent removed under reduced pressure. The resulting viscous oil was placed on a high vacuum line to remove all traces of acetic acid and yield the title compound (4.1 g, 0.019 mol, 77% yield). Calculated mass: 215.01; ESI⁺ m/z - 216.04 (MH⁺), 238.03 (M + Na⁺). ¹R NMR (300 MHz; d₆-DMSO) δ/ppm 2.59 (2H, t, J = 7.0), 2.98 (2H, t, J = 7.0), 7.23 (IH, ddd, J = 7.1, 4.8, 1.2), 7.75 (IH, d, J = 8.0), 7.8 (IH, td, J = 7.9, 1.4), 8.4 (IH, d, J = 4.9), 12.43 (IH, broad s). ¹³C NMR (300MHz, d₆-DMSO) δ/ppm 33.6, 33.7, 119.2, 121.1, 137.7, 149.5, 159.0, 172.7.

B) Preparation of N-Succinimidyl 3-(2-pyridyldithio)-propanoate (SPDP)

3-(2-Pyridyldithio)-propanoic acid (3.45 g, 0.016 mol) in anhydrous DCM was cooled to 0⁰C in an ice bath. 7V-hydroxysuccinimde (2.03 g, 0.0176 mol) was added followed by dicyclohexylcarbodiimide (3.64 g, 0.0176 mol), both in anhydrous DCM. The solution was brought back up to room temperature and left stirring for 3 hours. The formation of a product was followed by silica gel TLC. After the reaction had reached completion the solution was filtered under vacuum from the urea precipitate and the solvent removed from the filtrate under reduced pressure to yield a yellow oil. This crude material was purified via silica gel column chromatography using dichloromethane/methanol (50/2) as the eluent. The fractions containing the product were combined and the solvent removed to yield a colourless oil that crystallized overnight at -20⁰C (3.93 g, 0.0126 mol, 79% yield). Calculated mass: 312.36. ESI⁺ m/z - 313.35 (MH⁺), 335.36 (M + Na⁺). ¹R NMR (300 MHz; d₆-DMSO) δ/ppm 2.81 (4H, s), 3.12 (4H, m, AB_sys), 7.25 (IH, ddd, J = 6.4, 4.8, 1.6), 7.76 (IH, d, J = 7.1), 7.82 (IH, td, J = 7.5, 1.6), 8.48 (IH, d, J = 4.9). ¹³C NMR (300 MHz; d₆-DMSO) δ/ppm 25.4, 30.2, 32.8, 119.5, 121.4, 137.8, 149.7, 158.5, 167.4, 170.0.

C) Preparation CysHiSδ Peptide

The CHHHHHH peptide was synthesised using standard automated fmoc SPPS chemistry on a Syro automated peptide synthesiser using HBTU coupling chemistry

(no HOBt was included) on a pre-loaded Wang resin. The program allowed for a 4 fold excess of amino acids and 10% above this value was dissolved into solution. Once the synthesis was complete the peptides were deprotected and cleaved from the resin using a TFA/TES/EDT/H2O solution (resin in solution for 3 hours). The peptide solution was then collected and precipitated in diethyl ether, allowed to precipitate at -20⁰C, centrifuged to a pellet and redissolved. This process was repeated three times. The pellet was then dissolved in the minimum volume of water, frozen in liquid N₂ and freeze dried overnight. The peptide was further purified via RP-HPLC - 5 mg was dissolved in 300 μL of 0.1% TFA water. 50 - 100 μL aliquots were purified per run using a 5 ml/min semi prep column; gradient 2 - 10% B over 15min (A - 0.1% TFA water, B 0.1% TFA acetonitrile). The fractions containing the correct peak were pooled and freeze dried overnight to leave a white powder - single peak on analytical HPLC. Calculated mass ES⁺ ESI: (m/z) - 473.22 (MH²⁺), 944.99 (MH⁺), 966.91 (M + Na⁺).

D) Preparation of a 27mer Oligonucleotide - Peptide conjugate (CHe)

4.4 mg of N-Succinimidyl 3-(2-pyridyldithio)-propanoate (SPDP) was dissolved in 112 μL of DMSO (0.125M). To 25 nmol of internally amine modified DNA (27 mer - seq.

5 ' - ACA TTC CTA ACA TCA CTA ACT AAT CTT - 3 ' T denotes Thymine modified with primary amine, cone. 1 mM in 1OmM Tris, ImM EDTA, pH 8.0) was added 65 μL OfNaHCO₃ (0.1 M, 50 mM NaCl, pH 9.0). 10 μL (50eq.) of the SPDP solution was added to the buffered DNA producing a white precipitate which immediately dissipates upon agitation. This reaction was left at room temp for 2 hours (10% DMSO; 0.25mM).

After this period the solution was eluted through a size exclusion sephadex column (attached to the AKTA purification system) to remove excess SPDP. Using a 5 mL column with an isocratic gradient of 20 mM Tris 50 mM NaCl, pH 8.0, the DNA was eluted at 2 ml and collected. 1.9 mg of the CH₆ peptide was dissolved in 100 μL of Tris buffer - 0.02M (20 mM Tris, 50 mM NaCl, pH 8.0). To the DNA sample (eluted in 2 ml of eluent) 20 eq. (12.5 μL) of the peptide solution was added to each 12.5 nmole aliquot (final cone. 12.5 μM). The sample was agitated at room temp for 2 hrs before the volume was reduced on the vac. centrifuge to approx. 500 μL. The POC was purified via anion-exchange chromatography (1 ml/min HiTrapp Q FF col.) using a linear gradient (0 - 40% B) over 90 CV (buffer A - 20 mM Tris, pH 8.0; buffer B - 20 mM Tris, 2M NaCl, pH 8.0). The fractions containing the product (elution time 10 ml) were pool into 2 ml and desalted using 4 mL (5KDa MW cut-off) desalting columns. E) Analysis of the Peptide - Oligonucleotide conjugate

For MALDI MS preparation 1/3 vol. of 1OM ammonium acetate was added to the oligonucleotide followed by 2.5 vol. of ethanol. This solution was left overnight to precipitate out after which time the suspension was centrifuged at max. rpm for 15 min. The pellet was redissolved in 60 μL of nano-pure water, and the precipitation repeated.

2', 4', 6'- Trihydroxyacetophenone (THAP) matrix (25 mg) was weighed out and dissolved in 250 μL of methanol. Imidazole (0.2 g) was dissolved in 10 ml of water and vortexed until dissolved. The oligonucleotide pellet was dissolved in 50 μL of nano- pure water and 2 μL of the matrix solution was mixed well with 2 μL of the oligonucleotide sample and this solution was then spotted onto the MALDI plate. 1 μL of co-matrix (in this case imidazole) was added to the droplet and the sample was allowed to crystallize before being analysed via MALDI MS. A sample of unmodified 27mer oligonucleotide was also analysed as a comparative control; 27mer unmodified oligonucleotide L (m/z) 8361.99 (M^1"), 4183.43 9 (M^2") observed (actual mass 8380.6 g.mol^"1 - 0.2% error). POC sample; L (m/z) 9453.89 (M^1"), 4729.1 (M^2") observed (actual mass 9411 g.mol^"1 0.4% error).

The POC was also analysed via Ni²⁺ affinity columns on the AKTA system (step gradient from 0% B 10 column volumes (CV) to 100% B lOCV using the following buffers: A - 0.1 M Na₂HPO₄, 50 mM NaCl, pH 8.0; B - 0.1 M Na₂HPO₄, 50 mM NaCl, IM Imidazole, pH 8.0). The Histidine peptide-DNA conjugate eluted with the imidazole-containing buffer.

10 μL of the POC sample was diluted to 1 ml and the concentration confirmed by UV/Vis spectroscopy (A₂6o - 0.571, extinction co-efficient - 268 200 L/(mol.cm), cone. - 2.1 μM; final yield 42%).

Example 2

Preparation of 54mer DNA oligonucleotide carrying two peptide tags T₄ Ligase

CHHHHHH CGGGGGG

Scheme for the generation of DNA oligonucleotides carrying two peptide tags

The procedure detail above was used to modify two 27mer oligonucleotides with internal amine modifications with CG₆ and CH₆ peptides respectively. The second POC also contained a 5 ' phosphate group to allow ligation:

POCiCG₆ 5' - ACA TTC CTA ACA T(CysGfy₆)CA CTA ACTAAT CTT- 3' POC₂CH₆ 5' - /5Phos/TCA ACT CGA TAC GT(CysHis₆)A CCTATCAAT CTA - 3' CompOligo 5' - A TCG AGT TGA AAG ATT AGT T - 3'

To 50 μL 0.1M Tris, 0.1M NaCl, ImM EDTA, pH 8.0 was added 25 μL of POCiCG₆ (12.625 nmol), 24 μL of POC₂ CH₆ (12.625 nmol) and 12.625 μL of compOligo (12.625 nmol). The solutions were heated in a PCR block to 95 ⁰C for 2min and cooled in the block to room temp, before briefly being stored in the fridge.

1.6 mg of SPDP was dissolved in 51.28 μL of DMSO and 461.52 μL of nano-pure water was added (0.01M; 10% DMSO). A cloudy suspension resulted which dissipated upon agitation. The sample was agitated at room temp, for 15 mins to hydro lyse the activated ester. It was observed at this time that some compound had come out of solution so a further 175 μL of DMSO was added (7.45 mM; 33% DMSO). To 15 μL of the T4 Ligase enzyme (ImM DTT - 15 nmol) was added 4 μL of the hydrolysed SPDP (30 nmol) and left at room temp, for 10 mins (7% DMSO).

The following buffer was made up to 10 mL; 50 mM Tris. HCl, 10 mM MgCl₂, mM ATP, pH 7.5 and cooled to 16 ⁰C. To 370.4 μL of this solution the hybridized DNA solution was added followed by the T4 solution, and left overnight (0.025 mM). The sample was diluted up to 1 mL in the following buffer A and run through an anion - exchange col. using a gradient of 0 - 40% B over 90 CV in buffers; A - 2OmM Tris, 8M Urea, pH 8.0; B - 2OmM Tris, 2M NaCl, 8M Urea, pH 8.0. The short linking oligo eluted at 16 ml while the longer DNA strand with two chemical tags eluted at 21 ml. The solution was then desalted using 4 mL, 5KDa MW cutt-off desalting column and prepared for MALDI by precipitating in 2.5 vol absolute ethanol in the presence of 1/3 vol. 1OM ammonium acetate. A broad M^1" peak at 18232.5 m/z was observed (18236 m/z expected) along with a sharp M^3" peak at 6181.7 m/z (6075.66 m/z expected). A 15% TBE polyacrylamide gel was also run confirming that the gel shift of the double- modified DNA strand.

Example 3

The chemical synthesis of a chemically modified deoxyribonucleotide which can be incorporated into a DNA strand using template-directed enzymatically catalyzed DNA synthesis (5-acteylene-deoxyuridine 5' triphosphate and its derivative carrying a histidine-tag)

Synthesis of 5-ethynyl-2^f-deoxyuridine, Click reaction with azido-acetic acid, and formation of 5-ethynyl-5^f-triphosphate-2^f-deoxyuridine

A) Synthesis of 5-ethynyl-2^f-deoxyuridine

5-iodo-2'-deoxyuridine (370 mg, 1.04 mmol), palladium tetrakis triphenylphosphine (113mg, 0.098 mmol), copper iodide (47mg, 0.25mmol) were placed in a dried flask flushed with argon. Anhydrous DMF (12.0 mL) was added via syringe, followed by triethylamine (0.4 mL, 2.9 mmol) and trimethylsilylacetylene (0.7ImL, 5.0 mmol). The mixture was stirred at room temperature for 3h30, then the solvents were removed under vaccuo and the residue was submitted to flash column chromatography (10% MeOH in DCM), affording 381mg of yellow foam. The yellow foam was diluted with THF (4 mL), to which was added a solution of TBAF in THF (1.2 mL, IM, 1.2 mmol). The reaction mixture was strirred at room temperature for 3h. The resulting precipitate was removed by filtration and rinsed with THF, then 10% MeOH in DCM (2 mL). The filtrates were concentrated and purified by flash column chromatography (10% MeOH in DCM) to afford 191 mg (73%) of white powder.

B) Click reaction of 5-ethynyl-2^f-deoxyuridine with azido-acetic acid

5-ethynyl-2'-deoxyuridine (12 mg, 0.05 mmol) and azido-acetic acid (6mg, 0.06 mmol) were dissolved in a NaH₂PO₄ buffer (pH 6.2, 0.12M, 1 mL) and heated at 37°C to ensure dissolution of the reagents. Then a freshly made sodium ascorbate aqueous solution (25 μL, IM) and a freshly made CuSO4 aqueous solution (25 μL, 0.1M) were added and the reaction mixture was maintained at 37°C for 130min, at which point, analytical HPLC of a sample of the solution indicated complete consumption of the starting material nucleoside. The mixture was filtered through microfilter (minisart 0.20μM) and the filtrates were purified by HPLC.

C) Synthesis of 5-ethynyl-5^ftriphosphate-2^f-deoxyuridine

5-ethynyl-2'deoxyuridine (55 mg, 0.22 mmol) and proton sponge (71 mg, 0.33 mmol) were placed in a dried 2-neck flask, which was then flushed with argon. Trimethyl phosphate (2.0 mL) was added via syringe and the mixture was stirred at room temperature for 10 min or until the species fully dissolved. The reaction mixture was then brought to the desired temperature using an ice-salt bath (-13⁰C experiment), and POC13 (30 μL, 33 mmol) was added via syringe. After stirring 20 min, a solution of tetrabutylammonium pyrophosphate in anhydrous DMF (0.5M, 2 mL, 1 mmol) and tributylamine (0.16 mL). The mixture was stirred for 5 min, after which it was hydro lysed with a solution of TEAB (pH 7.0, 0.1M, 10 mL).The product was purified on a ion exchange column (Sephadex A-25 DEAE), followed by HPLC.

Example 4

PCR amplification of a 500 bp DNA fragment using 5-acteylene-deoxyuridine 5' triphosphate and the chemical modification of the DNA product with a hexahistidine tag.

Incorporation of 5-ethynyl-5^ftriphosphate-2^f-deoxyuridine into DNA via template- directed DNA polymerisation

A) PCR amplification

PCR amplification of 500 bp fragments were carried out using 50 μL reaction mixtures each containing 10x thermopol buffer (NEB) (5 μL), lOOμM Primer forward 5150 (2 μL) (5'-CCAACA GGT GCAAAT GTT TAC GGT C), lOOμM primer reverse 5578 (2 μL) (5'-ATG CTA GTT ATT GCT CAG CGG TGG), 25 nM SbsB T433C template (J. Duranton, C. Boudier, D. Belorgey, P. Mellet, J. G Bieth, JBC 2000, 275, 3787) (0.2 μL), DMSO (8 μL), Deep vent exo- polymerase (NEB) (1 μL), sterile water (26.8 μL), and dNTP mix (5 μL). Three versions of dNTP mix were used :

PCR amplification was performed using a thermocycler with the following conditions: step 1 (2 min at 94°C); 30 cycles of step 2 (1 min at 95°C, 1 min at 54°C, 1 min at 72°C), step 3 (10 min at 72°C). The three DNA samples were analysed by polyacrylamide gel electrophoresis (10%) and ethidium bromide staining. Samples 1 and 3 displayed a DNA band at 500 bp, sample 2 did not show any DNA band.

B) Click-reaction of ethynyl-base containing DNA with azido-modified peptides

The Click reaction was carried following a published procedure (Org. Lett. 2006, 8, 3639-3642) by mixing PCR mix 1 and 3 (45 μL) with 1 mM CuSO₄ solution (5 μL), 10 mM ascorbate solution (5 μL), and 20 mM peptide azido-GGGHHHH (4.5 μL). The mixtures were incubated at 37°C for 90 min, and analysed by gel electrophoresis (10% polyacrylamide gel). Sample from the ethynyl-base containing sample showed an upshifted band compared to the band from the sample without ethynyl groups.

Example 5

The single channel current analysis of POCs, data about the extent of current blockade and the blockade duration for all POCs

Analysis of chemically modified DNA strands carrying one or two peptide tags Single-channel current recordings were performed by using a planar lipid bilayer apparatus as described. (Braha, O., Walker, B., Cheley, S., Kasianowicz, J. J., Song, L., Gouaux, J. E., and Bayley, H. (1997) Chem Biol 4, 497-505) Briefly, a bilayer of 1,2- diphytanoyl-sft-glycerophosphocholine (Avanti Polar Lipids) was formed on an aperture (80 μm in diameter) in a Teflon septum (Goodfellow Corporation, Malvern, PA) separating the cis and trans chambers of the apparatus. Each compartment contained 1.0 ml of 2 M KCl, 20 mM TrisΗCl pH 7.5. Gel-purified heptameric αHL protein (final concentration 0.01-0.1 ng/ml) was added to the cis compartment, and the electrolyte in the cis chamber was stirred until a single channel inserted into the bilayer. Transmembrane currents were recorded at a holding potential of +100 mV (with the cis side grounded) by using a patch-clamp amplifier (Axopatch 200B, Axon Instruments, Union City, CA). For analysis, currents were low-pass filtered at 10 kHz and sampled at 50 kHz by computer with a Digidata 1200 A/D converter (Axon Instruments), as described (Movileanu, L., Howorka, S., Braha, O., and Bayley, H. (2000) Nat Biotechnol 18, 1091-1095). Single-channel conductances were determined by fitting the peaks in amplitude histograms to Gaussian functions.

A typical current single-channel current trace with events caused by hexa-arginine modified oligonucleotide of 27 bases in length is shown in Fig. 7A. Analysis of more than 400 events yielded a characteristic current blockade of more than 205 pA which constitutes more than 99% of the open channel current. The average duration of the events was obtained form the fitting of the dwell-time distribution with a single exponential and yielded a value of 12 ms. By comparison, events of the DNA strand carrying a hexa-histidine tag (Fig. 7B) were characterised by a current blockade of 94% and an average duration of 1.84 ms. By contrast, the DNA oligonucleotide without any peptide tag had an average duration of 0.14 ms and a current blockade of 80% (Fig. 7C). The recordings demonstrate that a chemical tag such as a short oligopeptide slows down the passage of a DNA strand through the pore. In addition, tags of the same length but different chemical composition such as hexaarginine and hexahistidine led to different current signatures.

Additional current recordings also showed that the blockade duration can be varied by changing the length of the peptide. Three peptides with 2, 4 or 6 histidines were tested and the blockade characteristics for 500 events each are summarized in the table. Within this histidine peptide series, the blockade durations correlated directly with the length of the peptides.

Peptide . ...

„ tau-off/ms

Sequence no peptide 0.14

CHHHHHH 1.84

CHHHH 1.57

CHH 0.66

Finally, DNA strands carrying two peptide tags, one with a hexa-histidine the other with a tri-tyrosine tag from example 2 was analysed with single channel current recordings. A typical event is shown in Fig. 7D.

Example 6

Identification of drug-resistance conferring point mutations in the protease gene of the HIV-2 virus

The approach of primer extension is used to detect single nucleotide polymorphisms in biomedically relevant sequences. In particular, a single point mutation in the HIV-2 gene encoding for the protease is detected. The first point mutation at codon position 90 from TTG -> ATG encodes leads to an amino acid change from leucine (L) to methionine (M). The second point mutation at codon position 23 from CTA -> ATA encodes for an amino acid change from leucine (L) to iso leucine (I).

The section of the HIV-2 sequence around position 90 of the wild type is: 5'-AT TGG AAG AAA TCT GTT GAC TCA GAT TGG TTG CAC TTT AA-3' and for the drug resistance conferring mutation is

5'-AT TGG AAG AAA TCT GAT GAC TCA GAT TGG TTG CAC TTT AA-3'.

Performing a primer extension using a primer with the sequence

5' TT AAA GTG CAA CCA ATC TGA GTC A-3' and nucleotides dATP, dCTP, ethynyl-dUTP, the following primer extension product are formed: For the wild-type sequence, the product

5' TT AAA GTG CAA CCA ATC TGA GTC A-ACA is formed. For the mutated sequence,

5' TT AAA GTG CAA CCA ATC TGA GTC A-UCA is formed.

The extended bases are indicated in bold. The U base carries the ethynyl group.

For the second point mutation, the sequence around position 23 is 5 ' AAA GGA AGC TCT ATT AGA TAC AGG AGC AGA TGA TAC AGT while the sequence for the single-point mutant is 5' AAA GGA AGC TAT ATT AGA TAC AGG AGC AGA TGA TAC AGT.

Performing a primer extension using a primer with the sequence 5 ' ACT GTA TCA TCT GCT CCT GTA TCT AAT A-3 ' and nucleotides dATP, dGTP, and ethynyl-dUTP, the following primer extension product are formed:

For wild-type, the product is

5' ACT GTA TCA TCT GCT CCT GTA TCT AAT A-GAG and for the mutant the product is 5 ' ACT GTA TCA TCT GCT CCT GTA TCT AAT A-UAG.

The ethynl containing uridine base in the extended products is modified with His- tagged peptide as described in example 3 and 4, and the tagged extended primers products are purified as described in example 1. In addition, the histidine-tag causes a specific current signature in the recordings. The primer sequence itself contains additional peptide sequences and gives rise to characteristic current signatures as described in example 5, and allows us to distinguish between the primers for position 90 and 23.

Example 7

Single channel current analysis of chemically modified DNA carrying a single peptide tag

A DNA strand oligonucleotide Ol of 27 bases in length was modified with the hexahistidine tag H₆Ci at an internal base (Figure 8B). The resulting peptide-DNA conjugate H₆Ci-Ol was analyzed in nanopore recordings. In the absence of DNA, the wild-type CcHL pore yielded a conductance of 1900 ± 120 pS (n = 4; n, number of independent recordings) when a positive potential was applied at the trans side (Figure 8C and 8A). The addition of unmodified oligonucleotide Ol to the cis side of the pore (Figure 8A) led to short high-amplitude blockades (Figure 8D). The high-amplitude events were characterized by an average amplitude of 91.7 ± 1.1 % relative to the open pore current, and a duration, τ₀fτ, of 0.18 ± 0.06 ms (n = 3). The short events represent the fast translocation of individual strands from the cis to the trans side of the pore (Figure 8D) (Kasianowicz et al, Proc. Nat. Acad. Sci. U S A 1996, 93, 13770; Butler et al, Biophys. J. 2006, 90, 190). The recordings also displayed blockades with 50% amplitude, which were not pursued further as they likely represent the reversible threading of a strand into and the escape from the cis opening rather than the complete translocation to the trans side.

When modified DNA strand H₆Ci-Ol was analyzed, events of two different types were observed. Type I events (Figure 8E) had a high-amplitude blockade, Ah of 96.8 ± 0.5 % with an average duration, τ_off-h, of 1.83 ± 0.26 ms. Due to the this very defined blockade, type I events certainly represent complete pore translocation. By comparison, type II events (Figure 8E) started with a mid-amplitude level, A_m, of 56.6 ± 2.6 % with a duration, τ_off_-m, of 1.34 ± 0.36 ms. This medium level was followed (Figure 8E) by a high-amplitude blockade, A_h, of 97.4 ± 0.9 % and a duration, τ_off-h, of 1.96 ± 0.48 ms. The mid-amplitude blockade of type II events possibly stems from mis-folded strands which reside in the internal cavity but eventually thread into the inner constriction. Due to the uncertainty in the assignment of the mid-level blockade, we focused our further investigation on the more clearly defined type I events which only exhibited high- amplitude blockades. A comparison of type I events from H₆Ci-Ol with unmodified DNA shows that the histidine tag slowed down translocation by a factor of 10 and increased the current amplitude by 5 %.

Several lines of evidence support the notion that the current blockades of the histidine- modified strand are caused by the steric hindrance encountered when a wide peptide- DNA segment passes the narrow inner constriction (Figure 8E). First, histidine tags with six, four and two residues led to correspondingly shorter high-amplitude blockades in type I events (Table 1, H_xC-Ol, x = 6, 4, 2) (type II events, Supporting Information). This implies that the peptide is elongated and aligned parallel to the DNA strand while being translocated. Second, tags composed of less bulky glycine did not exhibit the same length-dependence indicating that the small amino acid does not reach the critical size threshold required for continually slowing down DNA (type I, Table 1, G_xCi-Ol, x = 6, 4, 2)(type II, Supporting Information). Third, Ol carrying a H₆Ci tag at a terminal rather than an internal position did not greatly retard DNA passage as shown by a short event time of 0.23 ± 0.10 ms (Figure lF)(Table 1, H₆C-Ol -term.). The absence of a major retardation is attributed to the fact that the peptide can sequentially pass the pore after the DNA strand without the formation of a bulky peptide-DNA segment. The peptides tags are certainly the molecular reason of the retardation and may exert their effect by either hindered diffusion or an increase in friction mediated by steric, electrostatic, polar, and/or hydrophobic interactions. (Mathe et al, Proc. Nat. Acad. ScL USA 2005, 102, 12377; Kathawalla et al, Macromolecules 1989, 22, 1215).

Table 1. Characteristics of type I translocation events of DNA carrying a single chemical tag^[a]

Modified DNA A_h [%]^[b] χ_off._h [ms]^[c]

Ol^ld] 91.7 ± 1.1 0.18 ± 0.06

H₆Ci-Ol 96.8 ± 0.5 1.91 ± 0.23

H₄Ci-Ol 96.0 ± 0.6 1.57 ± 0.29 H₂Ci-Ol 93.0 ± 0.7 0.82 ± 0.16

G₆Ci-Ol 91.4 ± 0.6 0.56 ± 0.15

G₄Ci-Ol 92.5 ± 0.5 0.55 ± 0.12

G₂Ci-Ol 92.7 ± 0.4 0.53 ± 0.13

H₆C-Ol -term. ^[d] 93.9 ± 0.7 0.23 ± 0.10

H₆Ci-01 / pH 6.4 96.9 ± 0.7 2.18 ± 0.42

Y₃Ci-Ol /step 92.4 ± 0.6 / 97.8 ± 0.5 0.46 ± 0.15/ 0.35 ± 0.13 Y₃Ci-Ol /slope 94.8 ± 0.7 1.00 ± 0.22

Y₃Ci-O2 /step 91.8 ± 0.6 / 98.9 ± 0.7 0.43 ± 0.12 / 0.39 ± 0.08 Y₃Ci-O2 /slope 96.7 ± 0.8 0.97 ± 0.13

[a] The recordings were conducted at 2 M KCl, 20 mM Tris, pH 8.0, filtered at 10 kHz and sampled at 50 kHz unless stated otherwise. The number of events analyzed for each type of DNA was between 1500 and 2000. n = 3. [b] The relative amplitude was calculated using A = (Ioc - he) I foe, where /oc and he are the conductance levels from the open and blocked channel, respectively, /oc and he were derived using all-point histograms, [c] The average duration represents the mono-exponential fit of the dwell- time histogram, [d] Filtered at 30 kHz and sampled at 100 kHz.

Example 8 Single channel current analysis of chemically modified DNA carrying a two peptide tags

Additional peptides were examined to demonstrate that strand retardation is a general feature of bulky amino acids and not only restricted to histidines. An additional aim was to identify tags which give rise to current signatures distinguishable from the histidine blockades. Two different peptides were investigated. The first peptide, R₇Ci, was composed of seven arginines. In the nanopore analysis, type I events Of R₇Ci-Ol (Figure 9A) exhibited a duration of 25 ± 5 ms and an amplitude of 98.9 ± 0.6 %. (Table 1, R₇Ci-Ol). This blockade is higher and longer than that Of H₆Ci-Ol (Table 1). The more pronounced blockade Of R₇Ci-Ol is possibly due to the longer amino acid side chain of arginine compared to histidine (Figure 9E), or to the folding back of the positively charged arginine onto the negatively charged DNA backbone to generate a compact and bulkier DNA-peptide segment. The second peptide investigated was Y₃C₁. Tyrosine has an uncharged aromatic side chain (Figure 9E). Translocation OfY₃Ci-Ol led to type I events with two current levels (Figure 9B, inset, dotted lines). The first level at 92.4 ± 0.6 % (Figure 9B, inset, top dotted line) is similar to the blockade amplitude of unmodified DNA. It is therefore very likely that this level stems from a DNA strand which is threaded into the inner constriction but kept from passing the β- barrel because the bulky peptide has not yet entered the narrow pore region. The second level at 97.8 ± 0.5 % (Fig. 9B, inset, bottom dotted line) is ascribed to a state where the peptide-DNA segment has entered the inner constriction and translocates the β-barrel. The step signature was observed for 60 % of type I Y₃Ci-Ol events. In the remaining events, the transition between the two current levels resembled a slope (Figure 9C, inset). This could reflect a peptide-DNA segment which is being gradually rather than abruptly pulled into the inner constriction. Importantly, the step-like blocking effect of Y₃Ci was independent of the DNA sequence around the modified base because the same event characteristics were also seen for Y₃Ci-O2 with a different sequence (Table 1; Y₃Ci-Ol /step vs Y₃Ci-O2 /step).

DNA strands with two separate chemical tags were tested to prove that tags act independently and give rise to corresponding distinct current modulations. The first strand was a 37-mer Y₃/Y₃-O3 in which two Y₃Ci peptides are tethered to two modified bases separated by 13 nucleotides. Similar to single modified Y₃Ci-Ol strand, double modified DNA gave rise to unresolved slope events (Supporting Information) as well as fully resolved step-like events (Figure 9D). In the latter events, the blockade amplitude fluctuates twice between two levels sequentially from event segments 1 to 4 (Figure 9D, event segments numbered red). The average current levels for segments 1 and 3, and 2 and 4 are 92 and 99%, respectively (Table 2). The step-like signature is in line with expectations for two Y₃Ci peptides because one peptide is known to cause a blockade step from 92 % to 98 % (Table 1, Y₃Ci-I /step). The signature of Y₃/Y₃-O3 in Figure 9D strongly suggests that the current alterations reflect the sequential pulling of a DNA strand through the pore as illustrated schematically in Figure 9F (numbers correspond to segments in Figure 2D). Table 2. Characteristics of type I translocation events of Y3/Y3-O3 and Y3/Y3-O4 carrying tags separated by 13 and 27 nt, respectively^

Segments 1 2 3 4

03 A_h [%]^[b] 92.2 ± 1.1 99.7 ± 0.7 92.3 ± 0.9 99.7 ± 0.6

O3 τ_off-h [ms]^[c] 3.43 ±0.67 0.80 ±0.22 0.26 ±0.08 0.61 ±0.15

04 Λ [%]^[b 92.8 ± 1.1 98.2 ± 0.8 91.1 ± 1.5 98 ± 0.7

04 τ₀f_f-h [ms]^[c] 1.34 ±0.75 0.64 ±0.26 0.67 ±0.21 0.74 ±0.37

[a] The recordings were conducted at 2 M KCl, 20 mM Tris, pH 8.0, filtered at 10 kHz and sampled at 50 kHz. [b] and [c] are defined as in Table 1.

The interpretation of the stepped events as the sequential pulling is supported by the finding that 54-mer DNA strand Y₃/Y₃-O4 with a separation of 27 nt showed a similar current modulations. The two current levels were identical within experimental error to the values observed for Y₃/Y₃-O3 (Table 2). The duration of event segment 3 was, however, longer for Y₃/Y₃-O4 than for Y₃/Y₃-O3 (Table 2). This positive correlation between duration and tag separation indicates that a longer DNA strand take more time to pass the pore. 37-mer Y₃/Y₃-O5 with a separation of seven nt also displayed the step- behaviour. The percentage of stepped events as well as the quality of the current steps was lower than for Y₃/Y₃-O3 and Y₃/Y₃-O4. This reduced resolution agrees with molecular models showing that the tag with an extended length of 2.8 nm bridges the gap between two tagged bases separated by 7 nt or 2.2 nm.

These examples demonstrate a new nanopore-based strategy to enable the detection of separate bases in DNA strands. Using a model system of DNA olignucleotides modified with peptides, it is demonstrated that chemical tags attached to bases are capable of causing characteristic current signatures for strands translocating through nanopores. These experiments show that blockade duration, amplitude and signature can be tuned by changing the length, charge and size of the tags. The current modulations are independent of the surrounding DNA sequence and different tags on a strand retain their characteristic signatures. This is the first time that (i) pore recordings have detected one or two separate bases in translocating individual DNA strands, and that (ii) chemically modified DNA has been used to infer base-specific information.

Claims

Claims

1 A method of detecting the presence or absence of one or more bases or the number of repeats of one or more bases in an analyte nucleic acid molecule, comprising providing a membrane having first and second sides and containing a nanoscale pore having a lumen spanning the membrane; providing a fluid comprising the analyte nucleic acid molecule and an ionic salt on the first side of the membrane and a solution comprising an ionic salt on the second side of the membrane; and applying a potential difference across the membrane and measuring the resulting current over time, wherein the analyte nucleic acid molecule is chemically modified to modulate the duration and/or amplitude of the measured current and thereby allow identification of the analyte nucleic acid molecule and detection of the presence or absence of the one or more bases or the number of repeats of the one or more bases.

2 A method according to claim 1 wherein the analyte nucleic acid molecule comprises an extended primer oligonucleotide consisting of an original primer portion and an extended oligonucleotide portion, which is chemically modified in the original primer portion and optionally in the extended oligonucleotide portion, the presence or extent of chemical modification in the extended oligonucleotide portion being related to the presence or absence of the one or more bases or the number of repeats of the one or more bases.

3 A method of detecting the presence or absence of one or more bases or the number of repeats of one or more bases in an analyte nucleic acid molecule, comprising: providing a sample of nucleic acid molecules and a chemically modified primer complementary to a portion of the analyte nucleic acid molecule; allowing the primer to bind to analyte nucleic acid molecules in the sample and to extend wherein one or more chemically modified nucleotides are incorporated into the extended primer oligonucleotide when one or more complementary bases are present in the bound analyte nucleic acid molecule; providing a membrane having first and second sides and containing a nanoscale pore having a lumen spanning the membrane; providing a fluid comprising the chemically modified extended primer oligonucleotide and an ionic salt on the first side of the membrane and a solution comprising an ionic salt on the second side of the membrane; and applying a potential difference across the membrane and measuring the resulting current over time, wherein the extent of chemical modification of the extended primer oligonucleotide modulates the duration and/or amplitude of the measured current and thereby allows identification of the analyte nucleic acid molecule and detection of the presence or absence of the one or more bases or the number of repeats of the one or more bases in the analyte nucleic acid molecule.

4 A method according to claim 3 wherein two or more primers are used, wherein each primer is complementary to a portion of a nucleic acid molecule in the sample and wherein the chemical modification of each primer modulates the duration and/or amplitude of the measured current in a different way and thereby allows identification of the analyte nucleic acid molecule and detection of the presence or absence of the one or more bases or the number of repeats of the one or more bases in the analyte nucleic acid molecule.

5 A method according to any preceding claim wherein the one or more bases which are detected in the analyte nucleic acid molecule is an SNP or a mutation in the analyte nucleic acid molecule or wherein the one or more bases which are detected are part of a highly repetitive sequence in the analyte nucleic acid molecule.

6 A method according to any preceding claim wherein the analyte nucleic acid molecule is single stranded DNA or single stranded RNA.

7 A method according to any preceding claim wherein the lumen of the nanopore a diameter of less than 100 nm, preferably less than 10 nm.

8 A method according to any preceding claim wherein the nanopore is an organic pore, preferably a protein pore. 9 A method according to any preceding claim wherein the nanopore is an inorganic pore, preferably composed of silica, alumina, titanium, gold, platinum, zirconia, silicon nitride or a combination thereof.

10 A method according to any preceding claim wherein the membrane is a lipid bilayer.

11 A method according to any preceding claim wherein the chemical modification is smaller than 1.5 nm diameter, more preferably smaller than 1 nm diameter.

12 A method according to any preceding claim wherein the chemical modification is a peptide tag, preferably consisting of from 2 to 6 amino acids.

13 A method according to claim 12 wherein the peptide tag is selected from hexahistidine, hexaarginine, hexaaspartic acid, histidine(4), histidine(2) and tyrosine(3).

14 A method according to any of claims 2 to 13 wherein the primer is modified with one or more chemical tags which are different to the one or more chemical tags used to detect the presence or absence of the one or more bases or the number of repeats of the one or more bases.

15 A method according to any of claims 2 to 14 wherein the primer is modified with a chemical tag which allows the extended primer oligonucleotide to be isolated from other nucleic acid molecules.

16 A method according to claim 15 wherein the chemical tag is selected from peptides, phenyl rings and substituted derivatives thereof, condensed aromatic systems and substituted derivatives thereof, saturated organic cyclic compounds, spiro-molecule and other bicyclic compounds, tricyclic compounds, monomeric and oligomeric carbohydrates and substituted derivatives thereof, organometallic compounds and substituted derivatives thereof, inorganic compounds, porphyrins and substituted organic derivatives thereof. 17 A method according to any preceding claim wherein each type of base which is detected is labelled with a different chemical tag which gives rise to a specific current signature.

18 A method according to claim 17 wherein part or all of the nucleic acid molecule is sequenced.

19 A method according to any preceding claim wherein the nanoscale pore is an engineered pore, preferably an engineered protein pore, which provides enhanced discrimination between the duration and/or amplitude of the measured current signal produced by each chemical tag.

20 A method of mRNA profiling comprising: providing a sample comprising a first mRNA species and a second mRNA species, a chemically modified first primer complementary to a portion of said first mRNA species and a chemically modified second primer complementary to a portion of said second mRNA species; allowing the primers to bind to the mRNA species in the sample and to extend wherein one or more chemically modified nucleotides is incorporated into the extended primer oligonucleotides when one or more complementary bases are present in the bound mRNA species; providing a membrane having first and second sides and containing a nanoscale pore having a lumen spanning the membrane; providing a fluid comprising the chemically modified extended primer oligonucleotides and an ionic salt on the first side of the membrane and a solution comprising an ionic salt on the second side of the membrane; and applying a potential difference across the membrane and measuring the resulting current over time, wherein the chemical modification of the extended primer oligonucleotides modulates the duration and/or amplitude of the measured current and thereby allows identification and quantification of the first and second mRNA species. 21 A method according to claim 20 wherein the chemical modification of the first primer is different to the chemical modification of the second primer.

22 A method according to claim 20 wherein different chemically modified nucleotides are incorporated into the first and second mRNA species.

23 A method according to claims 20 to 22 wherein the first and second mRNA species are selected from different mRNA species expressed in the same cell type, or the same mRNA species expressed in different cell types or different organisms.

24 A method of sequencing a nucleic acid molecule comprising: providing a sample comprising the nucleic acid molecule and a chemically modified primer complementary to a portion of said nucleic acid molecule; allowing the primer to bind to the nucleic acid molecule in the sample and to extend wherein one or more chemically modified nucleotides is incorporated into the extended primer oligonucleotide when one or more complementary bases are present in the bound nucleic acid molecule, wherein each type of chemically modified nucleotide is labelled with a different chemical tag which gives rise to a specific current signature; providing a membrane having first and second sides and containing a nanoscale pore having a lumen spanning the membrane; providing a fluid comprising the chemically modified extended primer oligonucleotides and an ionic salt on the first side of the membrane and a solution comprising an ionic salt on the second side of the membrane; and applying a potential difference across the membrane and measuring the resulting current over time, wherein the chemical modification of the extended primer oligonucleotides modulates the duration and/or amplitude of the measured current and thereby allows identification of one or more nucleotides in the nucleic acid molecule.