Nanopore sequencing using charge blockade labels
First Claim
1. A method for determining sequence information about a template nucleic acid molecule comprisingproviding a substrate having at least one nanopore, the at least one nanopore having a top opening and a bottom opening, and having a single polymerase enzyme attached proximal to an opening, the polymerase enzyme complexed with a primed template nucleic acid;
- contacting the substrate with a sequencing reaction mixture comprising reagents required for polymerase mediated nucleic acid synthesis including two or more different types of nucleotide analogs, each comprising a current blockade label attached to the phosphate portion of the nucleotide analogs such that the current blockade label is cleaved upon incorporation of the nucleotide into a growing strand,providing a voltage drop across the nanopore such that the current blockage label produces a change in current through the nanopore;
measuring the current through the nanopore over time to detect the incorporation of nucleotides into the growing strand; and
identifying the type of nucleotide incorporated into the growing strand using current blockage characteristics, thus determining sequence information about the template nucleic acid molecule.
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Abstract
The invention relates to devices and methods for nanopore sequencing. The invention includes compositions and methods of nucleic acid sequencing using a single polymerase enzyme complex comprising a polymerase enzyme and a template nucleic acid attached proximal to a nanopore, and nucleotide analogs in solution comprising charge blockade label that are attached to the polyphosphate portion of the nucleotide analog such that the charge blockade labels are cleaved when the nucleotide analog is incorporated into a growing nucleic acid and the charge blockade label is detected by the nanopore to determine the presence and identity of the incorporated nucleotide and thereby determine the sequence of a template nucleic acid.
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Citations
27 Claims
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1. A method for determining sequence information about a template nucleic acid molecule comprising
providing a substrate having at least one nanopore, the at least one nanopore having a top opening and a bottom opening, and having a single polymerase enzyme attached proximal to an opening, the polymerase enzyme complexed with a primed template nucleic acid; -
contacting the substrate with a sequencing reaction mixture comprising reagents required for polymerase mediated nucleic acid synthesis including two or more different types of nucleotide analogs, each comprising a current blockade label attached to the phosphate portion of the nucleotide analogs such that the current blockade label is cleaved upon incorporation of the nucleotide into a growing strand, providing a voltage drop across the nanopore such that the current blockage label produces a change in current through the nanopore; measuring the current through the nanopore over time to detect the incorporation of nucleotides into the growing strand; and identifying the type of nucleotide incorporated into the growing strand using current blockage characteristics, thus determining sequence information about the template nucleic acid molecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A system for nucleic acid sequencing comprising:
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a substrate having at least one nanopore, the at least one nanopore having a top opening and a bottom opening, and having a single polymerase enzyme attached proximal to an opening, the polymerase enzyme complexed with a primed template nucleic acid; a sequencing reaction mixture in contact with the substrate comprising reagents required for polymerase mediated nucleic acid synthesis including two or more types of nucleotide analogs, each comprising a current blockade label; drive electrodes in contact with the reaction mixture on either side of the substrate for producing a voltage drop across the at least one nanopore; one or more measurement electrodes connected to electronic measurement equipment for measuring the current through the nanopore over time; and a computer for identifying the type of nucleotide incorporated into the growing strand. - View Dependent Claims (27)
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Specification