DNA sequencing with non-fluorescent nucleotide reversible terminators and cleavable label modified nucleotide terminators
Inventors
Ju, Jingyue • Kim, Dae Hyun • Guo, Jia • Meng, Qinglin • Li, Zengmin • Cao, Huanyan
Assignees
Columbia University in the City of New York
Publication Number
US-12331352-B2
Publication Date
2025-06-17
Expiration Date
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Abstract
This invention provides a process for sequencing nucleic acids using 3′ modified deoxynucleotide analogues or 3′ modified deoxyinosine triphosphate analogues, and 3′ modified dideoxynucleotide analogues having a detectable marker attached thereto.
Core Innovation
The patent addresses limitations of existing sequencing technologies by describing the need for new sequencing methods to overcome plateaued improvements in Sanger sequencing, limitations of electrophoresis-based separation, read-length limits, and the demand for next-generation sequencing that reduces cost and increases parallelization. The background states that electrophoresis-based separation causes deterioration of resolution due to band compressions and secondary-structure-induced mobility effects, which contribute to maximum read-length limits and that new methods must be developed to meet high-throughput sequencing demands.
The invention provides processes and compositions for sequencing nucleic acids using 3′ modified deoxynucleotide analogues or 3′ modified deoxyinosine triphosphate analogues, and 3′ modified dideoxynucleotide analogues having a detectable marker attached thereto. The methods include contacting nucleic acids with ddNTP analogues having a fluorophore attached via a cleavable linker and dNTP analogues having a cleavable chemical group at the 3′ O atom, identifying the fluorophore of incorporated ddNTP analogues to determine base identity, cleaving the fluorophore and cleavable chemical groups, and iteratively repeating these steps to determine consecutive nucleotide residues; the disclosure also provides kits, compounds and nucleic acids with a 3′-attached cleavable group for use in sequencing.
Claims Coverage
The patent as provided includes one independent claim. The main inventive features claimed relate to a deoxyribonucleic acid bearing at its 3′ end a defined phosphodiester-linked compound and related embodiments concerning the cleavable group, detectable markers, attachment to solid surfaces, and associated linkers and cleavage modalities.
Deoxyribonucleic acid having a 3′-end phosphodiester-linked compound
A deoxyribonucleic acid having attached at a 3′ end thereof, by a phosphodiester bond, a compound having the structure [as recited in the claim], wherein R′ is a cleavable chemical group.
Cleavable chemical group embodiments at the 3′ end
Embodiments wherein R′ is a nitrobenzyl group, an allyl group or a methylazido group, and wherein the cleavable chemical group is photocleavable or chemical cleavable.
Attachment of the deoxyribonucleic acid to solid surfaces
Embodiments wherein the deoxyribonucleic acid is attached to a solid surface, including attachment via 1,3-dipolar azide-alkyne cycloaddition chemistry, via a polyethylene glycol molecule, via an azido linkage, an alkynyl linkage, or biotin-streptavidin interaction, and wherein the solid surface can be a chip, bead, well, capillary tube, slide, wafer, filter, fiber, porous media, or column made of materials such as gold, quartz, silica, plastic, glass, diamond, silver, metal, or polypropylene.
Detectable marker cleavably linked to base and marker types
Embodiments wherein the base has a detectable marker cleavably linked thereto via a cleavable linker, wherein the detectable marker is a dye, a fluorophore, a chromophore, a combinatorial fluorescence energy transfer tag, a mass tag, or an electrophore, and wherein the fluorophore can be a cyanine dye, a green fluorescent dye, a red fluorescent dye or a rhodamine dye.
Linker cleavage by tris(2-carboxyethyl)phosphine and specific linker
An embodiment reciting that the linker is cleaved by contacting the linker with tris(2-carboxyethyl)phosphine, and embodiments reciting that the linker is photocleavable or chemical cleavable and that the linker can be 2-nitrobenzyl.
Copy number ranges of nucleic acid bound to surface
Embodiments reciting that about 1000 or fewer copies of the deoxyribonucleic acid are bound to the solid surface and embodiments reciting that 2×107, 1×107, 1×106 or 1×104 or fewer copies of the deoxyribonucleic acid are bound to the solid surface.
The independent claim centers on a deoxyribonucleic acid chemically modified at its 3′ end with a phosphodiester-linked, cleavable chemical group; dependent claim features specify the nature of R′, attachment to solid substrates and attachment chemistries, cleavable linkers and cleavage means, detectable markers and their types, and copy-number embodiments for surface-bound nucleic acids.
Stated Advantages
Combines advantageous aspects of Sanger and sequencing-by-synthesis (SBS) approaches to sequence DNA unambiguously.
Eliminates the need for electrophoretic DNA fragment separation by producing Sanger sequencing fragments on a sequencing by synthesis platform.
Enables massive parallel readout capability via high-density microarray and simplifies sample preparation.
Template 'walking' and primer resetting restore templates terminated with ddNTPs and can extend read length, potentially two- to three-fold or more.
Photocleavable reversible moieties regenerate natural nucleotides at the terminal 3′ end with no traces of modification, minimizing adverse effects on polymerase incorporation.
Photocleavage introduces no additional chemical reagents, eliminating possible chemical residue contamination during subsequent polymerase reactions.
Terminator-based interrogation allows accurate determination of homopolymeric regions of DNA.
High detection sensitivity with strong fluorescence signal-to-background ratios reported (approximately 20:1) enabling low amounts of fluorophore for detection.
Documented Applications
Methods for determining the identity of each of a series of consecutive nucleotide residues in a nucleic acid using combinations of ddNTP analogues with fluorophores and dNTP analogues with cleavable 3′ groups.
Sequencing nucleic acids immobilized on solid substrates such as chips, beads, wells, slides or columns (SBS on a chip).
Kits for sequencing a nucleic acid comprising ddNTP analogues and dNTP analogues described herein and instructions for use in sequencing.
Use of 3′ modified deoxyinosine triphosphate (dITP) analogues as a universal nucleotide in the hybrid Sanger/SBS method.
Sequencing RNA mutatis mutandis using appropriate ddNTPs and dNTPs or analogues thereof.
Hybrid sequencing approaches including (a) a Sanger/SBS hybrid using cleavable fluorescent dideoxynucleotides with 3′-O-modified reversible terminators and (b) SBS with cleavable fluorescent nucleotide reversible terminators (C-F-NRTs) in combination with cleavable nucleotide reversible terminators (C-NRTs).
Template 'walking' and primer resetting strategies for extending read length, including multiple specific 'walking' methods and strategies involving universal bases or multiple primer insertion.
Application to sequencing of various library types including genomic DNA, cDNA, polony PCR and emulsion PCR amplified templates as stated.
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