US 11,718,874 B2
Hybridization chain reaction methods for in situ molecular detection
Evan R. Daugharthy, Cambridge, MA (US); and George M. Church, Brookline, MA (US)
Assigned to President and Fellows of Harvard College, Cambridge, MA (US)
Filed by President and Fellows of Harvard College, Cambridge, MA (US)
Filed on Oct. 25, 2018, as Appl. No. 16/170,751.
Application 16/170,751 is a continuation of application No. PCT/US2017/029333, filed on Apr. 25, 2017.
Claims priority of provisional application 62/326,959, filed on Apr. 25, 2016.
Prior Publication US 2019/0218608 A1, Jul. 18, 2019
Int. Cl. C12Q 1/6876 (2018.01); C12Q 1/682 (2018.01); C12N 15/115 (2010.01); C12Q 1/6806 (2018.01); C12Q 1/6816 (2018.01)
CPC C12Q 1/6876 (2013.01) [C12N 15/115 (2013.01); C12Q 1/682 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6816 (2013.01); C12N 2310/16 (2013.01); C12N 2320/10 (2013.01)] 16 Claims
 
1. A method for identifying a plurality of target analytes in a sample, comprising:
(a) for each target analyte of said plurality of target analytes, contacting said sample with a probe coupled to a linker to bind said probe to said target analyte, wherein said linker is unique to said target analyte;
(b) subsequently adding a first hybridization chain reaction (HCR) initiator comprising an initiator sequence to said sample and contacting said sample with said first HCR initiator under conditions sufficient to permit said first HCR initiator to bind to said linker, wherein said first HCR initiator is separate from said probe, and wherein upon contacting said sample with said first HCR initiator, said linker couples said probe with said first HCR initiator;
(c) contacting said sample with HCR amplifiers to trigger a hybridization chain reaction, generating a first amplification product coupled to said probe;
(d) detecting said first amplification product;
(e) disrupting or reversing binding between said first HCR initiator and said linker;
(f) contacting said sample with a second HCR initiator comprising a second initiator sequence under conditions sufficient to permit said second HCR initiator to bind to said linker, wherein said second HCR initiator is separate from said probe, and wherein upon contacting said sample with said second HCR initiator, said linker couples said probe with said second HCR initiator;
(g) contacting said sample with additional HCR amplifiers to trigger an additional hybridization chain reaction, thereby generating a second amplification product coupled to said probe;
(h) detecting said second amplification product, and
(i) using at least signals from detecting said first amplification product and signals from detecting said second amplification product to generate a combined time-ordered composite signal unique for the each target analyte.