Method for detection of rna
Assignee10x Genomics, Inc.
InventorsMalte KÜHNEMUND, Mats NILSSON, Hao Zhe LEE
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Abstract
The present invention provides a method for detection of target RNA in a sample comprising the use of a hairpin probe which unfolds upon binding to its target RNA sequence to reveal a 3′ end region which is capable firstly of binding to a circular or circularisable probe and if necessary templating ligation of a circularisable probe to circularize the probe, and secondly of priming a subsequent RCA reaction of the circularized/circular probe. The circularized or circular probe is then subjected to RCA and a RCA product is detected, in order to detect the target RNA. Also provided are probes and kits for performing such a method.
Claims24
A method for detection of target RNA in a sample comprising:
- (a) contacting said sample with at least one target-specific hairpin probe comprising;
- (i) a first domain which comprises a first region of complementarity to a target sequence in said target RNA and is capable of hybridising to said target RNA;
- (ii) a second domain which is not capable of hybridising to the target RNA, which comprises a second region of complementarity to a cognate circular or circularisable probe, and which is capable of acting as a primer for an RCA reaction;
- wherein the second domain is at least partly comprised within a hairpin structure of the probe, such that the second domain is not capable of binding to said cognate circular or circularisable probe and/or priming an RCA reaction until released from said hairpin structure, and allowing said hairpin probe to hybridise to the target RNA, wherein hybridization of said first domain to said target sequence causes the hairpin structure to open and release said second domain;
- (b) contacting said sample with a circular or circularisable probe cognate for said hairpin probe, wherein the circular or circularisable probe comprises one or more regions complementary to the second region of complementarity in the second domain, and hybridising the padlock probe to the hairpin probe at said second domain, wherein if the probe is a circularisable probe, it comprises 3′ and 5′ end regions complementary to the second region of complementarity in the second domain such that said ends are hybridised in juxtaposition for ligation, directly or indirectly, to each other;
- (c) if said probe is a circularisable probe, ligating, directly or indirectly the ends of said padlock probe to circularise the circularisable probe, thereby providing an RCA reaction template;
- (d) performing an RCA reaction using the circular probe of (b) or the RCA template of (c), wherein said RCA reaction is primed by the second domain;
- and
- (e) detecting the product of said RCA reaction.
A hairpin probe for use in detecting a target RNA molecule, said probe comprising:
- (i) a first domain which comprises a first region of complementarity to a target sequence in a target RNA and is capable of hybridising to said target RNA;
- (ii) a second domain which is not capable of hybridising to the target RNA, which comprises a second region of complementarity to a cognate circular or circularisable probe, and which is capable of acting as a primer for an RCA reaction;
- wherein the second domain is at least partly comprised within a hairpin structure of the probe, such that the second domain is not capable of binding to said cognate circular or circularisable probe and/or priming an RCA reaction until released from said hairpin structure, and wherein the hairpin structure is such that hybridisation of said first domain to said target sequence causes the hairpin structure to open and release said second domain.
Dependencies are inferred from the claim text.