Nucleic acid sequences using tags
This disclosure provides chips, systems and methods for sequencing a nucleic acid sample. Tagged nucleotides are provided into a reaction chamber comprising a nanopore in a membrane. An individual tagged nucleotide of the tagged nucleotides can contain a tag coupled to a nucleotide, which tag is detectable with the aid of the nanopore. Next, an individual tagged nucleotide of the tagged nucleotides can be incorporated into a growing strand complementary to a single stranded nucleic acid molecule derived from the nucleic acid sample. With the aid of the nanopore, a tag associated with the individual tagged nucleotide can be detected upon incorporation of the individual tagged nucleotide. The tag can be detected with the aid of the nanopore when the tag is released from the nucleotide.
1. A method for generating a nanopore-polymerase complex, the method comprising:
(a) obtaining a nanopore composition enriched for nanopores having a first subunit and a second subunit in a ratio of 1:n−1, wherein n is the total number of subunits comprising the nanopore;
(b) attaching a polymerase to the first subunit to obtain the nanopore-polymerase complex,
wherein the polymerase is attached to the first subunit by a protein-protein tag.
2. The method of claim 1 , wherein the first subunit comprises a purification tag.
3. The method of claim 1 , wherein the protein-protein tag is generated by a SpyTag/SpyCatcher system.
4. The method of claim 1 , wherein the nanopore composition of (a) is an ion exchange fraction of a mixture of nanopores, wherein the mixture contains different ratios of the first subunit and second subunit.
5. The method of claim 1 , further comprising:
(c) inserting the nanopore-polymerase complex into a membrane.
6. The method of claim 1 , wherein the nanopore is 80% homologous to alpha-hemolysin.
7. A reaction chamber for performing nanopore-based nucleic acid sequencing, the reaction chamber comprising a nanopore in a membrane disposed adjacent or in proximity to an electrode, wherein the nanopore comprises a first subunit and a plurality of second subunits, wherein the ratio of first subunit to second subunits is 1:n−1, wherein ‘n’ is the total number of subunits comprising the nanopore, and wherein a single polymerase is attached to the first subunit,
wherein the polymerase is attached to the first subunit by a protein-protein tag.
8. The reaction chamber of claim 7 , wherein the first subunit comprises a purification tag.
9. The reaction chamber of claim 7 , wherein the protein-protein tag is generated by a SpyTag/SpyCatcher system.
10. The reaction chamber of claim 7 , wherein the nanopore is 80% homologous to alpha-hemolysin.