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US 12,703,879 B2Granted
RNase H assisted in situ rolling circle amplification
Assignee10x Genomics, Inc.
InventorsYufeng Qian, Shankar Shastry
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Abstract
The present disclosure relates in some aspects to methods for analyzing a target RNA in a biological sample, such as detection of a sequence of interest in an RNA. In some aspects, provided herein are methods of using an RNase such as RNase H to provide in situ RNA detection methods having high sensitivity, efficiency, and/or specificity.
Claims19
A method for analyzing a biological sample, comprising:
- (a) contacting the biological sample with a circular or circularizable probe, wherein the circular or circularizable probe comprises a hybridization region that hybridizes to a target RNA sequence in a target RNA in the biological sample to form a duplex,
- (b) contacting the biological sample comprising the target RNA hybridized to the circular or circularizable probe with a primer oligonucleotide, a ribonuclease H (RNase H), and a polymerase, wherein the primer oligonucleotide comprises a sequence complementary to a sequence in the circular or circularizable probe, and the RNase H cleaves the target RNA in the duplex at one or more sites in the target RNA sequence;
- (c) performing rolling circle amplification (RCA) using the circular probe or a circularized probe formed by circularizing the circularizable probe as an RCA template;
- and
- (d) detecting one or more products of the RCA, thereby detecting the target RNA or a sequence thereof in the biological sample.
2 more elements
A method for analyzing a biological sample, comprising:
- (a) contacting the biological sample with a circularizable probe comprising a hybridization region that hybridizes to a target RNA sequence in a target RNA in the biological sample to form a duplex,
- (b) ligating the circularizable probe to form a circularized probe;
- (c) contacting the biological sample comprising the target RNA hybridized to the circularized probe with (i) a primer oligonucleotide that comprises a sequence complementary to a sequence in the circularized probe, (ii) a ribonuclease H (RNase H), and (iii) a Phi29 DNA polymerase,
- wherein the contacting comprises contacting the biological sample with the RNase H in a first buffer that is free or substantially free of a cofactor for the RNase H, followed by incubating the biological sample in a second buffer comprising the RNase H and the cofactor;
- (d) performing rolling circle amplification (RCA) using the circularized probe as an RCA template;
- and
- (e) detecting one or more products of the RCA, thereby detecting the target RNA or a sequence thereof in the biological sample.
4 more elements
Dependencies are inferred from the claim text.