US 11,788,124 B2
Methods to further enhance signal amplification for the in situ detection of nucleic acids
Yuling Luo, Newark, CA (US); Xiao-Jun Ma, Newark, CA (US); Steve Chen, Newark, CA (US); Nan Su, Newark, CA (US); Emerald Doolittle, Newark, CA (US); Bingqing Zhang, Newark, CA (US); Xiaoming Wang, Newark, CA (US); Xingyong Wu, Newark, CA (US); Xiao Yan Pimentel, Newark, CA (US); and Helen Jarnagin, Newark, CA (US)
Assigned to ADVANCED CELL DIAGNOSTICS, INC., Newark, CA (US)
Appl. No. 17/46,228
Filed by ADVANCED CELL DIAGNOSTICS, INC., Newark, CA (US)
PCT Filed Apr. 8, 2019, PCT No. PCT/US2019/026279
§ 371(c)(1), (2) Date Oct. 8, 2020,
PCT Pub. No. WO2019/199643, PCT Pub. Date Oct. 17, 2019.
Claims priority of provisional application 62/667,237, filed on May 4, 2018.
Claims priority of provisional application 62/655,143, filed on Apr. 9, 2018.
Prior Publication US 2021/0032690 A1, Feb. 4, 2021
Int. Cl. C07H 21/02 (2006.01); C12Q 1/6841 (2018.01); C12Q 1/682 (2018.01); C12Q 1/6876 (2018.01)
CPC C12Q 1/6841 (2013.01) [C12Q 1/682 (2013.01); C12Q 1/6876 (2013.01); C12Q 2543/10 (2013.01)] 15 Claims
OG exemplary drawing
 
1. A composition comprising a Signal Generating Complex (SGC), wherein the composition comprises:
(A) a pair of target probes (TPs), wherein a first TP of the pair of TPs comprises a nucleic acid sequence comprising two segments, (i) a segment comprising a binding site for a first segment of a target nucleic acid, and (ii) a segment comprising a binding site for a first base pre-pre-amplifier (PPA); and wherein a second TP of the pair of TPs comprises a nucleic acid sequence comprising two segments, (i) a segment comprising a binding site for a second segment of the target nucleic acid, and (ii) a segment comprising a binding site for a second base PPA;
(B) a pair of base PPAs comprising the first base PPA and the second base PPA, wherein the first base PPA comprises a nucleic acid sequence comprising three segments, (i) a segment that binds the first TP, (ii) a segment comprising a plurality of first preamplifier binding segment repeats (PA-BSRs), and (iii) a segment comprising a binding site for a first extension pre-pre-amplifier (PPA); and wherein the second base PPA comprises a nucleic acid sequence comprising three segments, (i) a segment that binds the second TP, (ii) a segment comprising a plurality of second PA-BSRs, and (iii) a segment comprising a binding site for a second extension PPA; wherein the first PA-BSRs and the second PA-BSRs are different sequences, and wherein the segments of each of the first base PPA and the second base PPA are in the order (i), (ii), and (iii) and the order is either from 5′ to 3′ or 3′ to 5′ for the first base PPA and either from 5′ to 3′ or 3′ to 5′ for the second base PPA;
(C) a set of extension PPAs comprising the first extension PPA and the second extension PPA, wherein the first extension PPA comprises a nucleic acid sequence comprising two segments, (i) a segment that binds to the first base PPA, and (ii) a segment comprising a plurality of the first PA-BSRs; and wherein the second extension PPA comprises a nucleic acid sequence comprising two segments, (i) a segment that binds to the second base PPA, and (ii) a segment comprising a plurality of the second PA-BSRs;
(D) a plurality of pre-amplifiers (PAs), wherein the PAs comprise a nucleic acid sequence comprising three segments, (i) a segment complementary to the first PA-BSR, (ii) a segment complementary to the second PA-BSR, and (iii) a segment comprising a plurality of amplifier binding segment repeats (AMP-BSRs); wherein the segments of each of the plurality of PAs are in the order (i), (ii), and (iii) and the order is either from 5′ to 3′ or 3′ to 5′ for each of the plurality of PAs;
(E) a plurality of amplifiers (AMPs), wherein the AMPs comprise a nucleic acid sequence comprising two segments, (i) a segment complementary to the AMP-BSRs, and (ii) a segment comprising a plurality of label probe binding segment repeats (LP-BSRs); and
(F) a plurality of label probes (LPs), wherein the LPs comprise a nucleic acid sequence comprising two segments, (i) a segment complementary to the LP-BSRs, and (ii) a segment that is detectable;
wherein the SGC is formed by combining items (A) to (E) and the target nucleic acid together.