Method of preparation of nanopore and uses thereof
Inventors
Ju, Jingyue • Kumar, Shiv • Tao, Chuanjuan • Chien, Minchen • Russo, James J. • Kasianowicz, John J. • Robertson, Joseph W. F.
Assignees
Columbia University in the City of New York • United States Department of Commerce
Publication Number
US-11795191-B2
Publication Date
2023-10-24
Expiration Date
2033-04-08
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Abstract
This disclosure provides systems and methods for sequencing nucleic acids using nucleotide analogues and translocation of tags from incorporated nucleotide analogues through a nanopore. In aspects, this disclosure is related to composition, method, and system for sequencing a nucleic acid using tag molecules and detection of translocation through a nanopore of tags released from incorporation of the molecule.
Core Innovation
This invention provides systems and methods for sequencing nucleic acids by detecting tags released from tagged nucleotides upon polymerization using nanopores. More particularly, nucleotides each having a unique tag capable of being cleaved during nucleotide incorporation events are polymerized with the aid of an enzyme such as a DNA polymerase linked to a nanopore, and the released tags are directed through or adjacent to the nanopore for detection, allowing determination of the nucleic acid sequence.
The problem being solved arises from the insufficient sensitivity of existing nanopore sequencing methods that detect single-stranded nucleic acids passing through nanopores. Natural nucleic acid bases (A, C, G, T, U) have similar size, shape, and charge characteristics, particularly purines (A and G) and pyrimidines (C, T, U), which results in insufficiently distinct signals for accurate base identification and sequencing. The invention addresses the need for improved nucleic acid sequencing methods that enable accurate identification and sequencing by using tagged nucleotides and detecting the tags released upon nucleotide incorporation as distinct signals through a nanopore.
Claims Coverage
The patent includes one independent claim focused on a method for nucleic acid sequencing using an array of nanopores coupled to nucleic acid polymerases. The inventive features relate to the configuration and functional aspects of the nanopores, polymerases, tagged nucleotides, and detection methods.
Method of nucleic acid sequencing using an array of addressable nanopore-polymerase sites.
The method provides an array of individually addressable sites, each with a nanopore coupled to a nucleic acid polymerase attached within 0.7 nm of the nanopore entrance. Each site polymerizes tagged nucleotides having cleavable tags detected by the nanopore after release, enabling determination of the nucleotide sequence of single-stranded DNA in an electrolyte solution with a primer hybridized to the DNA template.
Use of specific tagged nucleotide analogues with detailed structural features.
Each tagged nucleotide is a deoxyribonucleotide polyphosphate (dNPP) analogue having a defined chemical structure including a tag that can comprise oligonucleotides, additional identifiable moieties such as coumarin-based dyes, or compositions including ethylene glycol, amino acids, carbohydrates, peptides, fluorescent or chemiluminescent compounds, and other functional groups.
Attachment of polymerase and alkaline phosphatase enzymes in close proximity to the nanopore.
The polymerase is attached to the nanopore near its entrance, and alkaline phosphatase molecules are covalently attached adjacent to the polymerase at the rim of each nanopore, facilitating release and detection of tags cleaved from the tagged nucleotides.
Charge-based differentiation of tags.
The tag has a charge reverse in sign relative to the remainder of the tagged nucleotide, with the magnitude of the tag's charge equal to that of the rest of the molecule, enabling electrical discrimination of tags during translocation through the nanopore.
Use of alpha hemolysin nanopores and phi29 DNA polymerase.
The nanopore is specifically an alpha hemolysin nanopore, and the DNA polymerase used is phi29 polymerase, providing biological components engineered for sensitive nucleic acid sequencing.
Control of incorporation and detection rates for accurate sequencing.
The polymerase catalyzes incorporation at a rate slower than the rate at which tags are released and detected by the nanopore, ensuring accurate sequential detection corresponding to nucleotide sequence.
The claims cover a method and system for single molecule nucleic acid sequencing employing an array of individually addressable nanopore-polymerase complexes that detect cleavable tags from tagged nucleotide analogues. These tags include oligonucleotides with distinctive chemical moieties, are charge-engineered for discrimination, and the system incorporates enzymatic components and specific nanopore technology to achieve accurate DNA sequence determination.
Stated Advantages
The approach enables clear discrimination of the four bases by modifying nucleotides with unique cleavable tags that produce distinct electronic blockade signals in nanopores.
The method enhances detection signal strength and signal processing, improving accuracy and sensitivity for single-molecule sequencing.
Controlling the translocation rate of tags, rather than nucleic acids, allows overcoming speed limitations for nanopore sequencing and enables accurate base calling without phasing errors.
The system allows massively parallel sequencing using arrays of nanopores with individually addressable polymerase-nanopore complexes, increasing throughput and lowering cost.
Use of nanopore electrical detection provides high signal levels and avoids expensive optical detection systems, facilitating miniaturization and scaling.
Documented Applications
Sequencing of single-stranded DNA and RNA molecules by polymerase-catalyzed incorporation of tagged nucleotides that release detectable tags upon incorporation.
Massively parallel single molecule DNA sequencing by synthesis using arrays of nanopores each coupled to a polymerase enzyme.
Use of nanopores integrated with CMOS electronics for high-bandwidth, low-noise detection of released tags during sequencing.
Distinguishing nucleotides in polymerase reactions by detection of tag molecules released and translocated through nanopores for nucleotide identification.
Preparation and use of tagged nucleotide analogues with cleavable linker-tag moieties for sequencing by synthesis coupled with nanopore detection.
Fabrication of solid-state nanopore devices integrated with electronics and enzymatic components for nucleic acid sequencing applications.
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