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US 2026/0098291 A1Published application
Multiplexed single molecule rna visualization with a two-probe proximity ligation system
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsNikolay Samusik, Felice Bava, Yury Goltsev, Garry P. Nolan
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Abstract
SNAIL provides cost-efficient detection of specific nucleic acids in single cells, and may be combined with flow cytometry to simultaneously analyze large numbers of cells for a plurality of nucleic acids, e.g. at least one, to up to 5, up to 10, up to 15, up to 20 or more transcripts can be simultaneously analyzed, at a rate of up to about 50, 100, 250, 500 or more cells/second. The methods require only two primers for amplification, and may further include a detection primer.
Claims20
A method comprising:
- (a) contacting a sample comprising a nucleic acid with a pair of probes under conditions to allow for specific hybridization of the pair of probes to the nucleic acid, wherein the pair of probes comprises:
- (i) a first probe, wherein the first probe comprises a first complementarity region, a second complementarity region, and a third complementarity region;
- and
- (ii) a second probe, wherein the second probe comprises a fourth complementarity region, a fifth complementarity region, and a sixth complementarity region;
- and
- wherein the first complementarity region is complementary to a first portion of the nucleic acid, the second complementarity region is complementary to the fourth complementarity region, the third complementarity region is complementary to the sixth complementarity region, the fifth complementarity region is complementary to a second portion of the nucleic acid, wherein the first complementarity region and fifth complementarity region are hybridized to the nucleic acid;
- (b) performing an extension reaction with the first probe serving as a primer and the second probe servicing as a template to generate an extended product;
- and
- (c) imaging the extended product.
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Dependencies are inferred from the claim text.