Method of preparation of nanopore and uses thereof

A method for determining the nucleotide sequence of a single-stranded DNA or RNA, the method comprising: (a) contacting the single-stranded DNA or RNA with a conductance measurement system comprising: (i) a first and second compartment with a first and second electrolyte solutions separated by a physical barrier, which barrier has at least one pore with a diameter in the nanometer scale; (ii) a means for applying an electric field across the barrier; (iii) a means for measuring a change in electric field; (iv) at least one pore-bound polymerase adjacent to the pore entrance, and optionally (v) more than one pore-bound phosphatase enzyme and a compound having the structure **(See formula)** wherein the tag comprises an oligonucleotide, where R1 is OH, where R2 is H or OH, where X is O, NH, S, or CH2, where Z is O, S, or BH3, where base is adenine, guanine, cytosine, thymine, or a derivative of one of these bases, and where n is 1, 2, 3, or 4, and optionally wherein the tag has a charge that is of inverse sign relative to the charge on the rest of the compound, wherein the single-stranded DNA or RNA is in an electrolyte solution in contact with the pore-bound polymerase, and wherein the DNA o Single-stranded RNA has a primer hybridized to a portion of it, under conditions that allow the polymerase to catalyze incorporation of the compound into the primer if it is complementary to the nucleotide residue of the single-stranded DNA or RNA that is immediately 5' to a nucleotide residue of the single-stranded DNA or RNA hybridized to the 3' terminal nucleotide residue of the primer, so that a DNA or RNA extension product is formed, where if the compound is not incorporated, iteratively repeat the contact with different compounds until one compound is incorporated, provided that (1) the type of base in the compound is different from the type of base in each of the previous compounds, and (2) the type of tag on the compound is different from the type of tag on each of the previous compounds, wherein incorporation of the compound results in the release of a polyphosphate having a tag attached to it, optionally wherein the pore-bound phosphatase enzyme cleaves the polyphosphate tag to release the tag; (b) determining which compound has been incorporated into the primer to form the DNA or RNA extension product in step (a) by applying an electric field across the barrier and measuring an electronic shift across the pore resulting from the translocation of the label generated in step (a) through the pore, and detecting the label with the help of at least one electrode, wherein detecting said label with the help of the electrode comprises: (i) passing an ionic current through said first electrolyte solution, said pore, and said second electrolyte solution at an electrical potential between said first and said second electrolyte solutions; (ii) measuring the ion current passing through said pore and recording the duration of changes in the ion current as a conductance time series, wherein said conductance time series spans periods of time when said pore is unobstructed by said tag and also periods of time when said tag produces pulses of reduced conductance; and (iii) delineating segments of the conductance time series in regions statistically consistent with the conductance level of unclogged pore, pulses of reduced conductance, and statistically stationary segments in individual pulses of reduced conductance; wherein the electronic change is different for each type of tag, thus identifying the nucleotide residue in the single-stranded DNA or RNA complementary to the incorporated compound; and (c) iteratively performing steps (a) and (b) for each nucleotide residue of the single-stranded DNA or RNA that is sequenced, thereby determining the nucleotide sequence of the single-stranded DNA or RNA.