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US 2024/0002932 A1Published application
Click chemistry-based dna photo-ligation for manufacturing of high-resolution dna arrays
Assignee10x Genomics, Inc.
InventorsJonathan CHENG, David Michael Patterson
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Abstract
The present disclosure relates in some aspects to methods and compositions for light-controlled in situ surface patterning of a substrate using a Click chemistry reaction. In some embodiments, a method disclosed herein comprises a copper-catalyzed alkyne-azide (CuAAC) click chemistry reaction for photocontrollable ligation of nucleic acid molecules. A large diversity of barcodes can be created in molecules on the substrate via sequential rounds of light exposure, hybridization, and ligation.
Claims28
A method for providing an array, comprising:
- (a) contacting a lawn of polynucleotides immobilized on a substrate with a first barcode sequence, wherein the polynucleotides comprise a 3′-alkyne or 3′-azido group, and wherein the first barcode sequence comprises an oligonucleotide with a 5′-azido or 5′-alkyne group that is a reaction partner for a Click reaction with the 3′-alkyne or 3′-azido group of the immobilized polynucleotides;
- (b) masking a first region of the lawn of polynucleotides on the substrate such that only a second region of the lawn of polynucleotides on the substrate are capable of being exposed to light;
- (c) irradiating the second region with a first ultraviolet light in the presence of a Cu (II) compound and a photoinitiator, thereby generating Cu(I), wherein the Cu(I) catalyzes the Click reaction between the 3′-alkyne or 3′-azido groups of the immobilized polynucleotides in the second region with the 5′-azido or 5′-alkyne groups of the first barcode sequence to generate a Click product comprising a 1,2,3-triazole group, wherein molecules of the first barcode sequence in proximity to the immobilized polynucleotides in the first region are not catalyzed by the Cu(I) to undergo the Click reaction,
- thereby providing on the substrate an array comprising the first and second regions of the lawn of polynucleotides, wherein the immobilized polynucleotides in the second region is barcoded with the first barcode sequence and the immobilized polynucleotides in the first region is not barcoded with the first barcode sequence.
1 more element
16-20. (canceled)
23-26. (canceled)
29-32. (canceled)
(canceled)
A method for providing an array, comprising:
- (a) irradiating a substrate comprising an unmasked first region and a masked second region, whereby a photoresist in the first region is degraded to render immobilized polynucleotide molecules in the first region available for hybridization and/or ligation, whereas immobilized polynucleotide molecules in the second region are protected by a photoresist from hybridization and/or ligation, wherein polynucleotide molecules in the first and second regions comprise a 3′-alkyne or 3′-azido group;
- (b) contacting the immobilized polynucleotide molecules in the first region with a first barcode sequence, wherein the first barcode sequence comprises an oligonucleotide with a 5′-azido or 5′-alkyne group that is a reaction partner for a Click reaction with the 3′-alkyne or 3′-azido group of the immobilized polynucleotides in the first region, and wherein polynucleotide molecules in the second region do not receive the barcode sequence;
- (c) irradiating the first region of the substrate with a first ultraviolet light in the presence of a Cu (II) compound and a photoinitiator, thereby generating Cu(I), wherein the Cu(I) catalyzes the Click reaction between the 3′-alkyne or 3′-azido groups of the immobilized polynucleotides in the first region of the substrate with the 5′-azido or 5′-alkyne groups of the first barcode sequence to generate a Click product comprising a 1,2,3-triazole group,
- thereby providing on the substrate an array comprising different polynucleotide molecules in the first and second regions.
1 more element
38-77. (canceled)
Dependencies are inferred from the claim text.