Circular probes and methods for sample analysis
Assignee10x Genomics, Inc.
InventorsZahra Kamila Belhocine, Justin Costa, Eswar Prasad Ramachandran Iyer
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Abstract
The present disclosure relates in some aspects to methods and compositions for analyzing a target nucleic acid, such as in situ detection of a region of interest in a polynucleotide in a cell or tissue sample. In some aspects, provided herein are circular probes (e.g., dumbbell probes) for analyzing a target nucleic acid, as well as methods comprising an enzymatic treatment to de-circularize unbound or non-specifically bound circular probes. Circular probes specifically bound to target nucleic acids remain intact during and after the enzymatic treatment and can be detected, e.g., via rolling circle amplification (RCA) of the circular probes and detection of the RCA products.
Claims20
A method, comprising:
- (a) contacting a biological sample comprising a plurality of ribonucleic acid (RNA) molecules with a plurality of circular probes, wherein a first circular probe of the plurality of circular probes comprises:
- (i) a first loop region comprising a sequence complementary to a first sequence of a first RNA molecule of the plurality of RNA molecules,
- (ii) a stem region comprising (1) a first strand complementary to the first RNA molecule, wherein the first strand comprises one or more ribonucleotides, and (2) a second strand comprising deoxyribonucleic acid (DNA) complementary to the first strand, and
- (iii) a second loop region;
- (b) hybridizing the first loop region to the first sequence of the first RNA molecule;
- (c) displacing the second strand from the first strand and hybridizing the first strand to the first RNA molecule, thereby obtaining a hybridized first circular probe;
- (d) after (c), contacting the biological sample with an RNAse, wherein the RNAse cleaves one or more ribonucleotides of a second circular probe of the plurality of circular probes, wherein the one or more ribonucleotides of the second circular probe are not hybridized to an RNA molecule of the plurality of RNA molecules;
- (e) performing a rolling circle amplification reaction on the hybridized first circular probe to generate a rolling circle amplification (RCA) product;
- and
- (f) detecting the RCA product, thereby detecting the first RNA molecule in the biological sample.
A method, comprising:
- (a) contacting a biological sample comprising a ribonucleic acid (RNA) molecule with a circular probe, wherein:
- the circular probe comprises a stem region, a first loop region, and a second loop region,
- the stem region comprises (i) a first strand comprising one or more ribonucleotides and (ii) a second strand complementary to the first strand, and
- a sequence in the first loop region hybridizes to a first sequence in the RNA molecule;
- (b) hybridizing a second sequence of the RNA molecule to the first strand of the stem region, wherein:
- the second strand is displaced from the first strand, the circular probe hybridizes to at least the first and second sequences in the RNA molecule, and the one or more ribonucleotides in the circular probe hybridize to complementary ribonucleotide(s) in the RNA molecule;
- (c) after (b), contacting the biological sample with an RNAse;
- (d) performing a rolling circle amplification reaction on the circular probe to generate a rolling circle amplification (RCA) product;
- and
- (e) detecting a signal or absence thereof, wherein the signal is associated with the RCA product in the biological sample.
Dependencies are inferred from the claim text.