US 12,467,081 B2
Encoded endonuclease assays
Jeffrey Brodin, San Diego, CA (US); Lorenzo Berti, San Diego, CA (US); Donald Brian Eidson, San Diego, CA (US); Christian Schlegel, San Diego, CA (US); Angela Blum, San Diego, CA (US); Rachel Schowalter, San Diego, CA (US); Ludovic Vincent, San Diego, CA (US); Pieter Van Rooyen, San Diego, CA (US); Gavin Stone, San Diego, CA (US); and Hatim T. Allawi, Middleton, WI (US)
Assigned to Pleno, Inc.; and Exact Sciences Corporation
Filed by Pleno, Inc., San Diego, CA (US); and Exact Sciences Corporation, Madison, WI (US)
Filed on Jan. 5, 2023, as Appl. No. 18/150,669.
Application 18/150,669 is a continuation of application No. PCT/US2022/037791, filed on Jul. 21, 2022.
Application PCT/US2022/037791 is a continuation of application No. PCT/US2021/060647, filed on Nov. 23, 2021.
Claims priority of provisional application 63/346,186, filed on May 26, 2022.
Claims priority of provisional application 63/317,838, filed on Mar. 8, 2022.
Claims priority of provisional application 63/234,635, filed on Aug. 18, 2021.
Claims priority of provisional application 63/222,963, filed on Jul. 17, 2021.
Claims priority of provisional application 63/183,876, filed on May 4, 2021.
Claims priority of provisional application 63/157,924, filed on Mar. 8, 2021.
Claims priority of provisional application 63/126,414, filed on Dec. 16, 2020.
Claims priority of provisional application 63/116,997, filed on Nov. 23, 2020.
Prior Publication US 2023/0295739 A1, Sep. 21, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6827 (2018.01); C12N 9/22 (2006.01); C12Q 1/6874 (2018.01); C12Q 1/6886 (2018.01); G16B 30/20 (2019.01)
CPC C12Q 1/6827 (2013.01) [C12N 9/22 (2013.01); C12Q 1/6874 (2013.01); C12Q 1/6886 (2013.01); G16B 30/20 (2019.02); C12Q 2600/154 (2013.01); C12Q 2600/156 (2013.01)] 27 Claims
 
1. A method of conducting an assay for nucleic acid targets, the method comprising:
(a) combining dual probes with a sample composition potentially comprising nucleic acid targets to form cleavable ternary nucleic acid complexes, wherein:
(i) each of the dual probes comprise two probes;
(ii) a first probe of each of the dual probes comprises a first targeting sequence that is complementary to a first portion of a nucleic acid target;
(iii) a second probe of each of the dual probes comprises:
(1) a second targeting sequence; and
(2) a non-targeting recognition element sequence,
wherein the second targeting sequence is complementary to a second portion of the nucleic acid target and wherein the second targeting sequence of the second probe overlaps by at least one nucleotide with the first targeting sequence of the first probe;
(iv) binding of the dual probe to the nucleic acid target with no mismatches between the nucleic acid target and the second targeting sequence of the second probe at a nucleic acid target site of interest results in a cleavable ternary nucleic acid complex, wherein the cleavable ternary nucleic acid complex comprises the non-targeting recognition element sequence of the second probe;
(v) binding of the dual probe to a non-target nucleic acid with mismatches between the non-target nucleic acid and the second targeting sequence of the second probe results in an uncleavable ternary nucleic acid complex;
(b) releasing from the cleavable ternary nucleic acid complex the non-targeting recognition element sequence, thereby forming a recognition element fragment;
(c) combining the released recognition element fragment with an additional nucleic acid sequence to create a circular nucleic acid, wherein the circular nucleic acid comprises a target-associated code and additional sequence elements, wherein the combining the released recognition element fragment with the additional nucleic acid sequence comprises ligating the released recognition element fragment to a coded oligonucleotide probe, wherein the coded oligonucleotide probe comprises a code comprising at least one segment encoding one or more symbols that correspond to a sequence of one or more nucleotides, and wherein the ligating comprises ligating ends of the released recognition element fragment to the coded oligonucleotide probe to produce a circular modified recognition element comprising the recognition element and the coded oligonucleotide probe; and
(d) performing a detection event to identify one or more detected codes of the target-associated code;
wherein the detected code indicates the presence of the nucleic acid target in the sample composition.