Methods and compositions for in situ sequencing
Assignee10x Genomics, Inc.
InventorTrevor STOCKDALE
Life of this patent
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Abstract
The present disclosure relates in some aspects to methods, systems, and kits for using diphosphonic acid to promote N—O bond cleavage in a 3′ O—NH2 nucleotide in a cell or tissue sample. In some aspects, diphosphonic acid is used to promote N—O bond cleavage in a 3′ O—NH2 blocked reversible terminator during multiple cycles of a nucleic acid sequencing reaction in a cell or tissue sample. In some embodiments, the chemical reaction of the 3′ O—NH2 blocked reversible terminator with diphosphonic acid releases free alcohols and phosphoric acid derivatives, which are not damaging to the cell or tissue sample.
Claims25
A method of generating a nucleic acid strand comprising a free 3′-OH in a cell or tissue sample comprising reacting a blocked nucleic acid strand comprising a 3′-O—NH2 at a 3′ terminus with a diphosphonic acid of Formula (P1) or (P2):
- or a salt of Formula (P1) or (P2).
A method of de-blocking a 3′ terminal nucleotide of a priming strand of a sequencing reaction, comprising:
- (a) providing a cell or tissue sample comprising:
- a priming strand bound to a template nucleic acid molecule, wherein the priming strand comprises a blocked nucleotide comprising a 3′-O—NH2 at a 3′ terminus;
- and
- b) reacting the —O—NH2 of the blocked nucleotide with a diphosphonic acid of Formula (P1) or (P2):
- or a salt of Formula (P1) or (P2),
- thereby generating a free 3′-OH at the 3′ terminus of the priming strand in the cell or tissue sample.
A method of sequencing at least a portion of a template nucleic acid in a cell or tissue sample, the method comprising:
- (a) performing a cycle of a nucleic acid sequencing reaction, comprising:
- using a polymerase, incorporating a blocked nucleotide comprising a 3′-O—NH2 into a priming strand bound to the template nucleic acid, and
- identifying a nucleotide in the template nucleic acid;
- and
- (b) reacting the blocked nucleotide incorporated into the priming strand with a diphosphonic acid of Formula (P1) or (P2):
- or a salt of Formula (P1) or (P2),
- thereby generating a free 3′-OH in the priming strand in the cell or tissue sample.
Dependencies are inferred from the claim text.