Anchored primary nucleic acid probes and methods thereof; ribonuclease-insensitive methods for determining cellular nucleic acid in a biological sample
AssigneeVizgen, Inc.
InventorsJiang He, Lizhi He
Life of this patent
Estimating the term…
Abstract
The present disclosure is generally directed to anchoring primary nucleic acid probes in a polymer matrix, which are configured to hybridize to cellular nucleic acid, embedding in a polymer matrix, clearing cellular components including the cellular nucleic acid and imaging. The disclosure describes methods for imaging cellular RNA in an RNase insensitive method.
Claims25
A method of anchoring a primary nucleic acid probe within a matrix and clearing cellular components comprising:
- a. contacting a biological sample with a primary nucleic acid probe and an anchoring agent, wherein the primary probe comprises an acrydite moiety and hybridizes with a target nucleic acid and the anchoring agent forms a covalent bond with the primary nucleic acid probe;
- b. embedding the biological sample in a polymer matrix wherein the primary nucleic acid probe and the anchoring agent each form a covalent bond with the polymer matrix;
- and
- c. clearing cellular components from the polymer matrix wherein the primary nucleic acid probe hybridized to the target nucleic acid remains anchored in the polymer matrix to form a matrix anchored primary nucleic acid probe sample.
(canceled)
15-48. (canceled)
A method for imaging a nucleic acid target within a matrix and clearing cellular components comprising:
- a. contacting a biological sample with a primary nucleic acid probe and an anchoring agent, wherein the primary probe comprises an acrydite moiety and hybridizes with a target nucleic acid and the anchoring agent forms a covalent bond with the primary nucleic acid probe;
- b. embedding the biological sample in a polymer matrix wherein the primary nucleic acid probe and the anchoring agent each form a covalent bond with the polymer matrix;
- c. clearing non-target cellular components from the polymer matrix wherein the primary nucleic acid probe hybridized to the target nucleic acid remains anchored in the polymer matrix to form a matrix anchored primary nucleic acid probe sample;
- and,
- d. contacting the anchored primary nucleic acid probe with a plurality of secondary nucleic acid probes comprising a fluorescent label and a recognition sequence that hybridizes to a sequence of the primary nucleic acid probe and imaging the target nucleic acids.
50-156. (canceled)
A plurality of primary nucleic acid probe pools each of the primary nucleic acid probes comprising:
- a target sequence, one or more read sequences and an acrydite moiety, wherein each pool of primary nucleic acid probes is configured to hybridize to a distinct RNA species in a sample and each pool of primary probes encode a N-bit code that was assigned to each distinct RNA species, wherein each assigned N-bit code is a valid codeword.
158-167. (canceled)
173-184. (canceled)
Dependencies are inferred from the claim text.