IP Library › Granted Patent US 12,195,798
Granted Patent B2
US 12,195,798 · App. 17/035,436 · Granted Jan 14, 2025

Methods and compositions for single cell genomics

Inventors: Jian-Bing Fan (San Diego, CA); Kun Zhang (San Diego, CA)
Assignees: Illumina, Inc.; The Regents of the University of California
C12Q1/6874C12Q1/6841
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Quick Facts
Patent No.
US 12,195,798
App. No.
17/035,436
Granted
Jan 14, 2025
Kind
B2
Abstract

Presented are methods and compositions for obtaining sequence information from one or more individual cells. The methods are useful for obtaining sequence information for a single nucleotide sequence, and for multiplex generation of sequence information from one or more individual cells.

Claims (19)

1. A method of obtaining sequence information of at least one nucleic acid molecule from an individual cell comprising:

(a) amplifying a plurality of nucleic acid molecules in situ within a permeabilized and fixed individual cell, thereby generating a plurality of amplified nucleic acid molecules;

(b) providing a plurality of different padlock probes and annealing the plurality of amplified nucleic acid molecules in situ to the plurality of different padlock probes, each of the plurality of different padlock probes is designed to anneal to an amplified nucleic acid molecule of the plurality of amplified nucleic acid molecules, wherein each of the different padlock probes comprises two sequence-specific capturing arms located on its 5′ and 3′ ends, wherein the two sequence-specific capturing arms are ligatable when each of the two sequence-specific capturing arms is annealed to the amplified nucleic acid of the plurality of amplified nucleic acid molecules,

wherein each of the plurality of different padlock probes further comprises a plurality of decoder sequences that does not anneal to the amplified nucleic acid molecules,

wherein a code is generated from decoding the decoder sequences of each of the plurality of different padlock probes by sequential hybridizations of each of the plurality of different decoder sequences with a plurality of fluorescently labeled decoder probes, wherein each of the plurality of fluorescently labeled decoder probes has a different fluorescent label, and each of the plurality of different padlock probes is identified by matching the code with a reference code that is assigned for one of the plurality of different padlock probes, and wherein each of the plurality of different padlock probes hybridizes to a different target nucleic acid sequence and has a different reference code;

(c) hybridizing a labeled decoder probe of the plurality of fluorescently labeled decoder probes to one decoder sequence of said decoder sequences of the plurality of different padlock probes in situ, wherein a label on the labeled decoder probe of the plurality of fluorescently labeled decoder probes provides a detectable fluorescent signal, and removing the labeled decoder probe of plurality of fluorescently labeled decoder probes that is not hybridized to the one decoder sequence of said decoder sequences of the plurality of different padlock probes;

(d) detecting a plurality of fluorescent signals in situ;

(e) removing the hybridized labeled decoder probe of the plurality of fluorescently labeled decoder probes in situ;

(f) repeating steps (c) to (e) for a desired number of times using a different labeled decoder probe of the plurality of fluorescently labeled decoder probes that can hybridize to another decoder sequence of said decoder sequences of the plurality of different padlock probes; and

(g) determining the identity of the plurality of different padlock probes based upon matching each code generated from decoding the decoder sequences of each of the plurality of different padlock probes with a reference code that is assigned for one of the plurality of different padlock probes, thereby generating the sequence information of the at least one nucleic acid from the individual cell.

2. The method of claim 1 , wherein said amplifying step is carried out by rolling circle amplification.

3. The method of claim 1 , wherein said plurality of different padlock probes is a rolling circle amplification probe.

4. The method of claim 1 , wherein said plurality of amplified nucleic acid molecules is immobilized within said permeabilized and fixed individual cell.

5. The method of claim 1 , wherein the plurality of nucleic acid molecules is RNA templates.

6. The method of claim 1 , wherein the plurality of nucleic acid molecules is DNA.

7. The method of claim 1 , wherein the plurality of nucleic acid molecules comprises a disease-causing mutation.

8. The method of claim 7 , wherein said disease-causing mutation is a base substitution.

9. The method of claim 7 , wherein said disease-causing mutation is an insertion mutation.

10. The method of claim 7 , wherein said disease-causing mutation is a deletion mutation.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 23, 2020
From: FAN, JIAN-BING
To: ILLUMINA, INC.
Reel/Frame 054149/0900 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 23, 2020
From: ZHANG, KUN
To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Reel/Frame 054150/0025 →
Continuity (3)
Continuation 14742027 · Jun 17, 2015
Provisional Application 62014538 · Jun 19, 2014
Related Publication 20210087625A1 · Mar 25, 2021
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