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-10246479-B2
Publication Date
2019-04-02
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
The invention provides systems and methods for sequencing nucleic acids by using nucleotide analogues with attached tags and detecting the translocation of these tags through a nanopore. Specifically, the methods involve polymerization of tagged nucleotides by a nucleic acid polymerase, whereby during polymerization a tag is released from the nucleotide and subsequently passes through or near a nanopore. The nanopore, disposed in a membrane adjacent to an electrode, detects the distinct electrical signals associated with each tag, enabling identification of each nucleotide incorporated during sequencing.
The problem solved addresses limitations of existing nucleic acid sequencing methods, particularly nanopore sequencing of single stranded nucleic acids, where the similarity in size, shape, and charge among purines (adenine and guanine) and among pyrimidines (cytosine, thymine, and uracil) leads to insufficiently distinct signals, resulting in inadequate sensitivity and accuracy. The invention recognizes the need for improved methods that can accurately identify individual nucleotides by utilizing unique tag molecules released upon nucleotide incorporation and detected via nanopores, thus improving the sequencing accuracy and sensitivity.
The invention also provides for high-density arrays of individually addressable nanopores, each coupled to a polymerase, to enable massively parallel nucleic acid sequencing. It includes methods of releasing tags from tagged nucleotide analogues during polymerization events and detecting these tags via nanopores with support from integrated circuits and complementary metal-oxide semiconductor (CMOS) technology for sensitive and rapid electrical detection. The methods achieve single-molecule sequencing without requiring DNA amplification and can detect nucleotide incorporation events with high statistical accuracy (e.g., greater than 4 sigma). The tagged nucleotides may be designed with various chemical linkers and diverse tags such as polyethylene glycol (PEG) polymers of distinct lengths, enabling discrimination between bases based on unique electrical blockade signatures as the tags pass through the nanopore.
Claims Coverage
The claims include one independent claim directed to a tagged nucleotide comprising a tag cleavable in a nucleotide polymerization event and detectable by a nanopore, along with various dependent claims and related claims addressing compositions and uses of such tagged nucleotides for sequencing.
Tagged nucleotide with cleavable and detectable tag
A tagged nucleotide comprising a tag capable of being cleaved during nucleotide polymerization and detected with the aid of a nanopore, wherein the nucleotide has a defined chemical structure with the tag attached.
Distinct tagging for different nucleotide types
A composition comprising four tagged nucleotides where each nucleotide type (adenine, guanine, cytosine, thymine or uracil) has a different tag enabling differentiation during sequencing.
Non-fluorescent detectability of tags
The tag does not comprise a fluorophore and is detectable by its charge, shape, or size, or combinations thereof, including the use of additional identifiable moieties like coumarin-based dyes.
Tag chemical variety and charge properties
Tags may comprise entities such as ethylene glycol, amino acids, carbohydrates, peptides, dyes, chemiluminescent compounds, nucleotides, alkyl or similar groups, with tags having a charge opposite to the rest of the nucleotide, optionally balanced by lysine or arginine residues, and a positive charge that changes upon tag removal.
Oligonucleotide tags and structural variations
The tag may comprise oligonucleotides approximately 30 bases in length, which can include abasic sites or modified bases, with the 5′-OH of the oligonucleotide attached to the terminal phosphate of the tagged nucleotide analogue.
The claims collectively cover tagged nucleotides with unique detectable tags cleavable during polymerization and methods for their use in nanopore sequencing, supporting high-accuracy base identification via tag-based electrical detection.
Stated Advantages
The method can identify individual nucleotide incorporation events with high accuracy, enabling single-molecule sequencing without requiring DNA amplification.
The use of unique chemical tags overcomes the insufficient signal distinction between purines and pyrimidines in nanopore sequencing.
High-density arrays of individually addressable nanopores enable massively parallel sequencing, increasing throughput and reducing cost.
Tags designed to optimize charge, size, and shape provide distinct electrical blockade signatures for accurate base discrimination.
Polymerase attachment to nanopores and integration with CMOS electronics allow sensitive, high-bandwidth electrical detection suitable for real-time sequencing.
Documented Applications
Sequencing nucleic acid molecules including DNA and RNA using tagged nucleotide analogues and nanopore detection.
Massively parallel nucleic acid sequencing using arrays of nanopores with polymerase attached to each nanopore.
Single-molecule sequencing by synthesis methods that detect released tags during polymerase extension events.
Use of tagged nucleotides with distinct tags to identify incorporated bases based on electrical signals in nanopores.
Real-time monitoring of nucleotide incorporation with high accuracy for applications in genetic disease diagnosis and pathogen research.
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