Nanopore sequencing complexes
AssigneeGenia Technologies, Inc.
InventorsTimothy Kellogg Craig, Christos Tzitzilonis, Alexander H. Yang, Liv E. Jensen, Marshall Porter, Charlotte Yang, Corissa Harris, Matt Dipetro
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Abstract
A method is provided for preparing nanopore sequencing complexes in membranes for sequencing of polymers, e.g., polynucleotides and polypeptides. The nanopore sequencing complex is formed by the sequential linking of an enzyme to a nanopore that is inserted in a membrane, and of a polymer to the enzyme. Alternatively, the nanopore sequencing complex is formed by linking a preformed enzyme-polymer complex to a nanopore that is inserted in a membrane. The enzyme polymer complex is interchangeable.
Claims24
1-15. (canceled)
A method for preparing a nanopore sequencing complex, the method comprising:
- (a) inserting a nanopore into a membrane;
- (b) contacting the nanopore with a sequencing enzyme;
- (c) attaching the enzyme to the nanopore to form an enzyme-nanopore complex;
- and
- (d) binding a polymer to the enzyme-nanopore complex to provide a nanopore sequencing complex.
A method for sequencing a nucleic acid sample, comprising:
- (a) preparing a nanopore sequencing complex in a lipid bilayer according to claim 1;
- (b) providing tagged nucleotides or nucleotide analogs to the nanopore sequencing complex, wherein the tag of the tagged nucleotide is detectable with the aid of the nanopore;
- (c) carrying out a polymerization reaction with the aid of the polymerase coupled to the nanopore in the nanopore-sequencing complex, thereby incorporating an individual tagged nucleotide of the tagged nucleotides into a growing strand complementary to a sample ssDNA template;
- and
- (d) detecting, with the aid of the nanopore, a tag associated with the individual tagged nucleotide during incorporation of the individual tagged nucleotide, wherein the tag is detected with the aid of the nanopore while the nucleotide is associated with the polymerase, thereby providing a sequence of the nucleic acid sample.
A biochip for sequencing a nucleic acid sample, the biochip comprising:
- an array of a plurality of nanopore sequencing complexes disposed in a membrane and prepared according to claim 1, a nanopore of the plurality being disposed adjacent or in proximity to an electrode, wherein the nanopore is individually addressable and has a single polymerase-polynucleotide complex attached to the nanopore;
- and wherein an individual nanopore detects the tag associated with the tagged nucleotide during incorporation of the nucleotide into a growing nucleic acid chain by the polymerase.
A method for preparing a nanopore sequencing complex, the method comprising:
- (a) inserting a nanopore into a membrane;
- (b) combining a sequencing enzyme with a polymer to provide an enzyme-polymer complex;
- and
- (c) attaching the enzyme-polymer complex to the pore, thereby providing a nanopore sequencing complex.
Dependencies are inferred from the claim text.