US 12,234,503 B2
Methods for multiplex imaging using labeled nucleic acid imaging agents
Stephanie Rae Hennek, Medford, MA (US); Mael Manesse, Medford, MA (US); Abdul Mohammed, Cambridge, MA (US); Xi Chen, West Newton, MA (US); and Michael Jules Natan, Weston, MA (US)
Assigned to Ultivue, Inc., Cambridge, MA (US)
Filed by Ultivue, Inc., Cambridge, MA (US)
Filed on Feb. 19, 2020, as Appl. No. 16/794,686.
Application 16/794,686 is a continuation of application No. 15/836,322, filed on Dec. 8, 2017, abandoned.
Claims priority of provisional application 62/445,896, filed on Jan. 13, 2017.
Claims priority of provisional application 62/432,511, filed on Dec. 9, 2016.
Prior Publication US 2020/0256867 A1, Aug. 13, 2020
Int. Cl. C12Q 1/6816 (2018.01); C12Q 1/68 (2018.01); C12Q 1/6804 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6818 (2018.01); C12Q 1/682 (2018.01); C12Q 1/6823 (2018.01); C12Q 1/6832 (2018.01); C12Q 1/6874 (2018.01); G01N 33/543 (2006.01); G01N 33/58 (2006.01)
CPC C12Q 1/6816 (2013.01) [C12Q 1/68 (2013.01); C12Q 1/6804 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6818 (2013.01); C12Q 1/682 (2013.01); C12Q 1/6823 (2013.01); C12Q 1/6832 (2013.01); C12Q 1/6874 (2013.01); G01N 33/543 (2013.01); G01N 33/54306 (2013.01); G01N 33/54333 (2013.01); G01N 33/58 (2013.01); G01N 33/587 (2013.01)] 19 Claims
 
1. A method to test a tissue sample for the location of at least four distinctive targets comprising:
(1) contacting a tissue sample being tested for the presence of at least four distinctive targets with at least four distinctive corresponding target-specific binding partners, wherein each target-specific binding partner of different specificity is linked to a different docking strand, and wherein each docking strand is an oligonucleotide;
(2) contacting the tissue sample with circular DNA amplifier strands having complementarity to the different docking strands;
(3) contacting the tissue sample with DNA polymerase, nucleotides, and buffer solution for rolling circle amplification, and amplifying simultaneously the different docking strands with rolling circle amplification to produce concatemers comprising repeats of the different docking strands;
(4) contacting the tissue sample with imager strands having complementarity to the different docking strands, wherein the imager strands comprise fluorescent labels, wherein step (3) occurs in the same step as step (4); and
(5) imaging the tissue sample to detect bound imager strands, thereby detecting the location of the at least four distinctive targets in the tissue sample.