US 12,227,796 B2
RNA templated ligation
Mats Nilsson, Drottningholm (SE); Malte Kühnemund, Stockholm (SE); and Tomasz Krzywkowski, Skarpnäck (SE)
Assigned to 10X GENOMICS, INC., Pleasanton, CA (US)
Filed by 10x Genomics, Inc., Pleasanton, CA (US)
Filed on Jul. 19, 2023, as Appl. No. 18/355,291.
Application 18/355,291 is a continuation of application No. 18/154,592, filed on Jan. 13, 2023.
Application 18/154,592 is a continuation of application No. 16/647,860, granted, now 11,597,965, issued on Mar. 7, 2023, previously published as PCT/EP2018/077161, filed on Oct. 5, 2018.
Claims priority of application No. 1716407 (GB), filed on Oct. 6, 2017; application No. 1718095 (GB), filed on Nov. 1, 2017; and application No. 1718098 (GB), filed on Nov. 1, 2017.
Prior Publication US 2023/0366010 A1, Nov. 16, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/682 (2018.01); C12N 15/11 (2006.01); C12P 19/34 (2006.01); C12Q 1/6806 (2018.01); C12Q 1/6876 (2018.01)
CPC C12Q 1/682 (2013.01) [C12N 15/11 (2013.01); C12P 19/34 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6876 (2013.01); C12N 2310/532 (2013.01)] 30 Claims
 
1. A method of detecting a target RNA molecule in situ in a cell or tissue sample, the method comprising:
(a) contacting the cell or tissue sample with a first oligonucleotide and a second oligonucleotide to hybridize the first oligonucleotide and the second oligonucleotide to the target RNA molecule,
wherein the first oligonucleotide comprises a first target binding site at a 5′ end,
wherein the second oligonucleotide comprises (i) a second target binding site at a 3′ end, and (ii) a ribonucleotide at a 3′ terminus; and
wherein a 5′ terminus of the first oligonucleotide and the 3′ terminus of the second oligonucleotide are adjacent to one another when hybridized to the target RNA molecule;
(b) ligating the first and second oligonucleotides hybridized to the target RNA molecule to generate a circularized probe,
wherein the 5′ terminus of the first oligonucleotide is ligated to the 3′ terminus of the second oligonucleotide, and
wherein the 3′ terminus of the first oligonucleotide is ligated to the 5′ terminus of the second oligonucleotide;
(c) amplifying the circularized probe by rolling circle amplification (RCA) to generate an RCA product; and
(d) detecting the RCA product at a location in the cell or tissue sample, thereby detecting the target RNA molecule at the location in the cell or tissue sample.