Method of oligonucleotide synthesis
Inventors
Chen, Michael Chun Hao • Mcinroy, Gordon Ross
Assignees
Publication Number
US-12442028-B2
Publication Date
2025-10-14
Expiration Date
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Abstract
The invention relates to methods and kits for the synthesis and assembly of oligonucleotides into contiguous strands. The oligonucleotides can be synthesised and assembled in the same device, allowing production of strands longer than can be prepared using base by base synthesis alone.
Core Innovation
Described herein are methods and kits for the synthesis and assembly of oligonucleotides into contiguous strands. The oligonucleotides can be synthesised and assembled in the same device, allowing production of strands longer than can be prepared using base by base synthesis alone. The device operates by synthesising a large number of oligonucleotides of different sequence, wherein the sequences can be designed such that the ends of the sequences can assemble to form one or more overlapping regions which can then be used to link together multiple of the synthesised strands into a contiguous sequence.
The background identifies that current DNA synthesis technology is practically impossible to efficiently synthesise a DNA strand greater than 200 nucleotides in length and that there is therefore a need for a new method to efficiently prepare long strands of oligonucleotides. The invention addresses this need by providing a method in which a device with a plurality of immobilised initiation oligonucleotide sequences is used to synthesise immobilised oligonucleotide sequences of at least n bases using cycles of extension of reversibly blocked nucleotide monomers, selectively cleaving subsets of the immobilised oligonucleotide sequences into solution, hybridizing cleaved oligonucleotides to form splints that hybridize to immobilised oligonucleotides, and joining cleaved oligonucleotides to immobilised oligonucleotides to prepare contiguous oligonucleotide sequences of increased length (for example at least 2n, 3n, 4n, 5n, 10n and greater).
Claims Coverage
Two independent claims were identified. The following inventive features are extracted from those independent claims and their recited components.
Device with immobilised initiation oligonucleotide sequences containing cleavage sites
A device comprising a plurality of immobilised initiation oligonucleotide sequences, one or more of which contains a cleavage site, used as the starting point for synthesis and selective release.
Synthesis by cycles of extension of reversibly blocked nucleotide monomers
Using the initiation oligonucleotide sequences to synthesise a plurality of immobilised oligonucleotide sequences of at least n bases in length using cycles of extension of reversibly blocked nucleotide monomers.
Selective cleavage of multiple immobilised oligonucleotides into solution
Selectively cleaving a defined number of the immobilised oligonucleotide sequences of at least n bases in length into solution whilst leaving one or more of the immobilised oligonucleotide sequences attached to the device.
Hybridization to form splints between cleaved and immobilised oligonucleotides
Hybridizing the cleaved oligonucleotides to each other to form splints and hybridizing one end of a splint to an immobilised oligonucleotide sequence so as to create splinted structures on the immobilised sequences.
Joining cleaved oligonucleotides to immobilised oligonucleotides to prepare contiguous sequences
Joining at least one of the cleaved oligonucleotides to the immobilised oligonucleotide sequences to prepare contiguous oligonucleotide sequences of increased length (e.g., preparing two separate contiguous sequences of at least 2n bases and further joining to prepare a contiguous sequence of at least 4n bases).
Iterative further cleavage, hybridization and joining to extend length
Performing a further cleavage step of selectively cleaving immobilised oligonucleotide sequences of at least 2n bases into solution, further hybridizing cleaved oligonucleotides to form splints on immobilised sequences of at least 2n bases, and further joining cleaved oligonucleotides of at least 2n bases to immobilised oligonucleotides to prepare a further contiguous oligonucleotide sequence of at least 4n bases.
Kit comprising immobilised initiators, reversibly blocked nucleotides, enzymes and ligase
A kit comprising a plurality of 5'-immobilised initiation oligonucleotides (one or more containing uracil or 8-oxoguanine), 3'-reversibly blocked nucleoside triphosphates and a template independent polymerase, uracil DNA glycosylase or formamidopyrimidine DNA glycosylase, optionally a mismatch recognising protein, and a nucleic acid ligase.
Specified 3'-reversible blocking groups on nucleoside triphosphates
Reversibly blocked nucleoside triphosphates wherein the 3'-reversible block is selected from groups including 3'-O—CH2N3, 3'-O—CH2CHCH2, 3'-O—CH2CH2CN, 3'-O—N═C(CH3)2, 3'-O—NH2, 3'-phosphate, 3'-ortho-nitrobenzyl, or 3'-para-nitrobenzyl as recited in the kit and method claims.
The independent claims recite a method that combines (1) on-device synthesis of immobilised oligonucleotides using cycles of extension of reversibly blocked nucleotide monomers, (2) selective cleavage of subsets of those oligonucleotides into solution, (3) splinted hybridization between cleaved and immobilised strands, and (4) joining steps to produce progressively longer contiguous oligonucleotide sequences, and a kit that provides immobilised initiators, specified 3'-reversibly blocked nucleotides, a template independent polymerase, cleavage enzymes, optional mismatch recognising proteins, and a ligase.
Stated Advantages
Allows production of strands longer than can be prepared using base by base synthesis alone by synthesising and assembling oligonucleotides in the same device.
Enables preparation of contiguous oligonucleotide sequences of increased length through selective cleavage, splinted hybridisation and joining, permitting lengths such as at least 2n, 3n, 4n, 5n, 10n and greater (examples given include strands longer than 200 bases and strands longer than 1250 bases).
Provides a parallelised assembly approach on a single solid support or device, allowing multiple sequences to be linked together to form longer contiguous strands and rapid assembly of longer fragments.
Documented Applications
Synthesis and assembly of oligonucleotides into contiguous strands longer than can be prepared using base-by-base synthesis alone.
In situ DNA synthesis for gene assembly or hybridization microarrays (identified in the background as an application area for controlled single-stranded DNA synthesis).
Use of synthesized contiguous oligonucleotide products for protein expression, where the nucleic acid products can be moved to a module capable of performing protein expression via cell-free or cell-based protein synthesis.
Release of assembled material by copying/amplification to produce non-immobilised copies (amplification of contiguous oligonucleotides using primers in solution to obtain non-immobilised products).
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