GENOMIC SEQUENCING USING MODIFIED PROTEIN PORES AND IONIC LIQUIDS
First Claim
1. A method of detecting the presence of one or more analytes in a sample, comprising the steps of:
- dissolving the one or more analytes in the sample in water or a buffer solution comprising an ionic salt to form a solution;
placing the solution in a cis compartment of a single-channel sensor;
contacting the solution with a pore assembly comprising a genetically modified bacterial transmembrane protein toxin;
applying an electrical potential to the single-channel sensor;
determining an ionic current across the electrical potential;
measuring one or more transient blockades in the ionic current; and
comparing the transient blockades in the ionic current to one or more known transient current blockades to determine the identity of the one or more analytes.
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Accused Products
Abstract
The present invention is a nanopore stochastic sensing system comprising modified protein pores for detection and sequencing of oligonucleotides. The system comprises a genetically modified protein pore with a variety of non-covalent bonding recognition sites to significantly slow down the translocation of ssDNA in the pores. The present invention also describes identification and application of DNA fingerprints, which are a sequence of small current modulation events for the determination of the sequence of ssDNA molecules. In separate embodiments the present invention describes a system and a method for the detection of monovalent cations, liquid explosives, water-insoluble compounds, biomolecules and oligonucleotides. The system comprising a wild-type or genetically modified protein pore with or without a molecular adaptor. Analyte samples and mixtures are added along with specially synthesized ionic liquids.
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Citations
44 Claims
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1. A method of detecting the presence of one or more analytes in a sample, comprising the steps of:
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dissolving the one or more analytes in the sample in water or a buffer solution comprising an ionic salt to form a solution; placing the solution in a cis compartment of a single-channel sensor; contacting the solution with a pore assembly comprising a genetically modified bacterial transmembrane protein toxin; applying an electrical potential to the single-channel sensor; determining an ionic current across the electrical potential; measuring one or more transient blockades in the ionic current; and comparing the transient blockades in the ionic current to one or more known transient current blockades to determine the identity of the one or more analytes. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method of detecting the presence of one or more analytes in a liquid sample, comprising the steps of:
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contacting one or more analytes in the liquid sample with boromycin to form an analyte-boromycin mixture; incubating the analyte-boromycin mixture for at least 30 minutes at room temperature; placing the analyte-boromycin mixture in a trans compartment of a single-channel sensor; contacting the analyte-boromycin mixture with a pore assembly comprising a synthetic membrane or wild type or modified bacterial transmembrane protein covalently or non-covalently coupled with an agent that modifies ionic current; applying a potential to the chamber; determining a current across the applied potential; measuring one or more transient blockades in the ionic current; and comparing the transient blockades in the ionic current to one or more known transient current blockades to determine the identity of the one or more analytes. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. An organic ion conducting solution composition comprising:
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a solvent comprising an organic ion conducting molecule, the molecule comprising; one or more heterocyclic rings comprising one or more heteroatoms; one or more side-chains attached to the one or more heteroatoms; and one or more negatively charged groups associated with the one or more of heteroatoms to form an ion conducting solution. - View Dependent Claims (23, 24, 25, 26)
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27. An organic ion conducting solution composition comprising:
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a solvent comprising an organic ion conducting molecule, the molecule comprising; one or more acyclic heteroatoms; one or more side-chains attached to the one or more heteroatoms; and one or more negatively charged groups are associated with the one or more of heteroatoms to form an organic ion conducting solution. - View Dependent Claims (28, 29, 30)
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31. A method of synthesizing an organic ion conducting solution, comprising the steps of:
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heating a mixture comprising the acyclic or heterocyclic compound and the side-chain derivative with stirring at 60°
C. or greater for at least 6 hours to form the organic ionic compound;dissolving the organic ionic compound in water; removing the excess acyclic or heterocyclic compound and the side-chain derivative by organic solvent extraction; repeating the solvent extraction process; and evaporating the water using a rotary evaporator to isolated the organic ionic liquid.
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32. A method of synthesizing butylymethylimidazolium chloride, comprising the steps of:
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heating a mixture comprising 1-methylimidazole and 1-chlorobutane with stirring at 60°
C. or greater for at least 6 hours to form the butylmethylimidazolium chloride;dissolving the butylymethylimidazolium chloride in water; removing the excess 1-methylimidazole and 1-chlorobutabe by solvent extraction with ethyl acetate; repeating the solvent extraction with ethyl-acetate; and evaporating the water using a rotary evaporator to isolated the butylymethylimidazolium chloride.
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33. A method of slowing down translocation of one or more analytes in a nanopore sensor with a genetically modified bacterial transmembrane protein pore assembly by a technique comprising one or more of the following approaches:
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forming a weak non-covalent bond between the analytes and the protein pore assembly; employing an organic salt solution; changing the concentration of an ionic salt in the buffer; changing temperature of the nanopore sensor assembly; changing the pH of a buffer system; and varying a dielectric field. - View Dependent Claims (34, 35, 36, 37, 38)
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39. A method for generating an oligonucleotide fingerprint comprising the steps of:
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dissolving one or more oligonucleotides in water or a buffer solution containing an ionic salt to form a solution; placing the oligonucleotide solution in a cis compartment of a single-channel sensor; contacting the solution with a pore assembly comprising a genetically modified bacterial transmembrane protein toxin; applying an electric potential to the sensor; determining an ionic current across the electric potential; measuring one or more transient blockades in the ionic current; and identifying one or more current modulations or sub-states in the ionic current.
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40. A method of sequencing from an oligonucleotide fingerprint comprising the steps of:
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determining one or major current value states (I1 and I0) and an amplitude Δ
I (=I1−
I0) from an all-points histogram;determining one or more probability values of the major current value states (i.e., PI 0 and PI1 ); andcomparing the major current value states and the probability values with different oligonucleotides to identify molecules with identical base compositions with different sequences. - View Dependent Claims (41, 42)
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43. A single-channel, dual-chamber molecular analysis device comprising:
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a cis chamber; a trans chamber; a boundary layer comprising a lipid bi-layer or any natural or synthetic membrane on a Teflon septum separating the cis and trans chambers; a genetically modified bacterial transmembrane protein pore attached to the boundary layer; a conducting electrolyte in the chamber; and a terminus for establishing electrical connectivity between the cis and trans chambers.
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44. A method for fabricating a single-channel, dual-chamber molecular analysis device, comprising the steps of:
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depositing a bilayer comprising two individual monolayers of a lipid molecule in an aperture of a Teflon septum; forming the bilayer at an air-water interface by hydrophobic apposition and the joining of the hydrocarbon chains of at least one individual monolayer; monitoring the bilayer formation using a function generator; adding a pore selected from a wild type bacterial transmembrane protein or a modified bacterial transmembrane protein to the bilayer or utilizing a porous synthetic membrane; and adding the conducting electrolyte to the chambers.
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Specification