IP Library › Granted Patent US 12,209,273
Granted Patent B2
US 12,209,273 · App. 17/346,077 · Granted Jan 28, 2025

Nucleic acid assays using click chemistry bioconjugation

Inventors: Felice Alessio Bava (Pleasanton, CA); David M. Patterson (Oakland, CA); Meiliana Tjandra (Dublin, CA); Yi Luo (Pleasanton, CA)
Assignee: 10X GENOMICS, INC.
C12Q1/6816
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Quick Facts
Patent No.
US 12,209,273
App. No.
17/346,077
Granted
Jan 28, 2025
Kind
B2
Abstract

Provided herein are methods of sequencing comprising click chemistry bioconjugation. In some embodiments, target polynucleotide sequences on the same or different molecules are contacted with and hybridize to probes comprising click functional groups. Probes (e.g., reading probes) hybridizing to adaptor sequences adjacent to different sequences of interest (e.g., barcodes to be sequenced) can be hybridized simultaneously in large pools. In some embodiments, the provided methods achieve multiplexing without requiring separate hybridization of probes (e.g., reading probes) for each sequencing-by-ligation cycle, thereby reducing total hybridization time which is typically a most time-consuming step in in situ technologies. In some aspects, the hybridized probes (e.g., reading probes) are clicked onto detectable probes to analyze a sequence of a target polynucleotide in a sequencing-by-ligation fashion.

Claims (67)

1. A method for analyzing a sample, comprising:

(a) contacting the sample with a first probe and a second probe, wherein:

the sample comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises a first adaptor sequence and a first target polynucleotide sequence adjacent to the first adaptor sequence, and the second nucleic acid sequence comprises a second adaptor sequence and a second target polynucleotide sequence adjacent to the second adaptor sequence, and

the first probe comprises click functional group C1 and hybridizes to the first adaptor sequence, and the second probe comprises click functional group C2 and hybridizes to the second adaptor sequence;

(b) contacting the sample with a first detectable probe, wherein the first detectable probe comprises click functional group C1′ and hybridizes to the first target polynucleotide sequence, thereby juxtaposing C1 and C1′;

(c) reacting C1 with C1′ in a click reaction, thereby ligating the first probe to the first detectable probe to form a first ligated product hybridized to the first nucleic acid sequence, wherein the click reaction between C1 and C1′ is orthogonal to a click reaction involving C2; and

(d) detecting a first signal associated with the first ligated product, wherein the first signal is indicative of a sequence of interest in the first target polynucleotide sequence.

2. The method of claim 1 , further comprising:

(b′) contacting the sample with a second detectable probe, wherein the second detectable probe comprises click functional group C2′ and hybridizes to the second target polynucleotide sequence, thereby juxtaposing C2 and C2′.

3. The method of claim 2 , further comprising:

(c′) reacting C2 with C2′ in a click reaction, thereby ligating the second probe to the second detectable probe to form a second ligated product hybridized to the second nucleic acid sequence, wherein the click reaction between C1 and C1′ is orthogonal to the click reaction between C2 and C2′.

4. The method of claim 3 , further comprising:

(d′) detecting a second signal associated with the second ligated product, wherein the second signal is indicative of a sequence of interest in the second target polynucleotide sequence.

5. The method of claim 4 , wherein the first detectable probe comprises an interrogatory region complementary to the sequence of interest in the first target polynucleotide sequence, and/or the second detectable probe comprises an interrogatory region complementary to the sequence of interest in the second target polynucleotide sequence.

6. The method of claim 5 , wherein the first detectable probe is among a plurality of first detectable probes contacted with the sample in step (b), wherein:

the plurality of first detectable probes are of formula N x B y N z , wherein N is a degenerate base and B is an interrogatory base, x, y, and z are integers independent of each other, wherein x is 0 or greater, y is 1 or greater, and z is 0 or greater, and

each of the plurality of first detectable probes: (i) comprises click functional group C1′ and a different interrogatory region, and (ii) is labeled with a detectable label corresponding to one or more of the different interrogatory regions.

7. The method of claim 6 , further comprising, prior to the reacting step (c), a step of removing first detectable probes comprising interrogatory regions that are not complementary to the sequence of interest in the first target polynucleotide sequence, while the first detectable probe remains hybridized to the first target polynucleotide sequence.

8. The method of claim 5 , wherein the second detectable probe is among a plurality of second detectable probes contacted with the sample in step (b′), wherein:

the plurality of second detectable probes are of formula N a B b N c , wherein N is a degenerate base and B is an interrogatory base, a, b, and c are integers independent of each other, wherein a is 0 or greater, b is 1 or greater, and c is 0 or greater, and

each of the plurality of second detectable probes: (i) comprises click functional group C2′ and a different interrogatory region, and (ii) is labeled with a detectable label corresponding to one or more of the different interrogatory regions.

9. The method of claim 8 , further comprising, prior to the reacting step (c′), a step of removing second detectable probes comprising interrogatory regions that are not complementary to the sequence of interest in the second target polynucleotide sequence, while the second detectable probe remains hybridized to the second target polynucleotide sequence.

10. The method of claim 5 , wherein the sequence of interest in the first target polynucleotide sequence is a single nucleotide or dinucleotide sequence.

11. The method of claim 5 , wherein the sequence of interest in the second target polynucleotide sequence is a single nucleotide or dinucleotide sequence.

12. The method of claim 4 , further comprising, after the detecting in step (d) or (d′):

(i) a step of cleaving the first or second detectable probe, respectively; and/or

(ii) a step of unhybridizing the first or second ligated product or a portion thereof from the first or second nucleic acid sequence, respectively.

13. The method of claim 3 , wherein the click reaction between C1 and C1′ and the click reaction between C2 and C2′ are independently selected from the group consisting of a nucleophilic addition reaction, a cyclopropane-tetrazine reaction, a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, an alkyne hydrothiolation reaction, an alkene hydrothiolation reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron-demand Diels-Alder (IED-DA) reaction, a cyanobenzothiazole condensation reaction, an aldehyde/ketone condensation reaction, and Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, and/or

wherein the click reaction between C1 and C1′ and/or the click reaction between C2 and C2′ are independently selected from the group consisting of a template-dependent reaction and a template-independent reaction.

14. The method of claim 3 , wherein:

(i) the click functional group C1 is on the 5′ of the first probe and the click functional group C1′ is on the 3′ of the first detectable probe, or the click functional group C1 is on the 3′ of the first probe and the click functional group C1′ is on the 5′ of the first detectable probe; and/or

(ii) the click functional group C2 is on the 5′ of the second probe and the click functional group C2′ is on the 3′ of the second detectable probe, or the click functional group C2 is on the 3′ of the second probe and the click functional group C2′ is on the 5′ of the second detectable probe.

15. The method of claim 3 , wherein C1/C1′ and/or C2/C2′ are independently selected from the group consisting of:

(i) 3′-azido/5′-alkynyl;

(ii) 3′-alkynyl/5′-azido;

(iii) 3′-azido/5′-cyclooctynyl;

(iv) 3′-cyclooctynyl/5′-azido;

(v) 3′-tetrazine/5′-dienophile;

(vi) 3′-dienophile/5′-tetrazine;

(vii) 3′-thiol/5′-alkynyl;

(viii) 3′-alkynyl/5′-thiol;

(ix) 3′-cyano/5′-1,2-amino thiol;

(x) 3′-1,2-amino thiol/5′-cyano;

(xi) 3′-nitrone/5′-cyclooctynyl; and

(xii) 3′-cyclooctynyl/5′-nitrone.

16. The method of claim 3 , wherein the first probe and the second probe are hybridized to the first and second nucleic acid sequences, respectively, prior to contacting the sample with the first and second detectable probes.

17. The method of claim 2 , wherein the first detectable probe and/or the second detectable probe comprise a fluorescent label or an overhang that hybridizes to a fluorescently labeled probe.

18. The method of claim 1 , wherein the first probe and/or the second probe remain hybridized to the first and second adaptor sequences, respectively, during one or more additional cycles of contacting the sample with additional detectable probes, additional click reactions, and additional signal detections.

19. The method of claim 1 , wherein the first target polynucleotide sequence and the second target polynucleotide sequence are barcode sequences or sub-barcode sequences corresponding to an analyte.

20. The method of claim 1 , wherein the first nucleic acid sequence and the second nucleic acid sequence are sequences of one or more rolling circle amplification (RCA) products generated in situ in the sample.

21. A method for analyzing a sample, comprising:

(a) contacting the sample with a first probe and a second probe, wherein:

the sample comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises a first adaptor sequence and a first target polynucleotide sequence adjacent to the first adaptor sequence, and the second nucleic acid sequence comprises a second adaptor sequence and a second target polynucleotide sequence adjacent to the second adaptor sequence, and

the first probe comprises click functional group C1 and hybridizes to the first adaptor sequence, and the second probe comprises click functional group C2 and hybridizes to the second adaptor sequence;

(b) contacting the sample with a plurality of first detectable probes of formula N x B y N z ,

wherein N is a degenerate base and B is an interrogatory base, x, y, and z are integers independent of each other, x is 0 or greater, y is 1 or 2, and z is 0 or greater,

wherein each of the plurality of first detectable probes comprises: (i) click functional group C1′, (ii) interrogatory region B y , and (iii) a detectable label corresponding to one or more different interrogatory regions in the plurality of first detectable probes, and

wherein the first detectable probe comprising the interrogatory region complementary to a corresponding sequence of interest in the first target polynucleotide sequence hybridizes to the first nucleic acid sequence, thereby juxtaposing C1 of the first probe and C1′ of the first detectable probe;

(c) reacting C1 with C1′ in a click reaction, thereby ligating the first probe to the first detectable probe to form a first ligated product hybridized to the first nucleic acid sequence;

(d) detecting a signal associated with the first ligated product, wherein the signal is indicative of the sequence of interest in the first target polynucleotide sequence;

(e) contacting the sample with a plurality of second detectable probes of formula N a B b N c ,

wherein N is a degenerate base and B is an interrogatory base, a, b, and c are integers independent of each other, a is 0 or greater, b is 1 or 2, and c is 0 or greater,

wherein each of the plurality of second detectable probes comprises: (i) click functional group C2′, (ii) interrogatory region B b , and (iii) a detectable label corresponding to one or more different interrogatory regions in the plurality of second detectable probes, and

wherein the second detectable probe comprising the interrogatory region complementary to a corresponding sequence of interest in the second target polynucleotide sequence hybridizes to the second nucleic acid sequence, thereby juxtaposing C2 of the second probe and C2′ of the second detectable probe;

(f) reacting C2 with C2′ in a click reaction, thereby ligating the second probe to the second detectable probe to form a second ligated product hybridized to the second nucleic acid sequence, wherein the click reaction between C1 and C1′ is orthogonal to the click reaction between C2 and C2′; and

(g) detecting a signal associated with the second ligated product, wherein the signal is indicative of the sequence of interest in the second target polynucleotide sequence.

22. The method of claim 21 , wherein the second probe remains hybridized to the second adaptor sequence in steps (b) through (g).

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 17, 2021
From: BAVA, FELICE ALESSIO; PATTERSON, DAVID M.; TJANDRA, MEILIANA; LUO, YI
To: 10X GENOMICS, INC.
Reel/Frame 057206/0270 →
Continuity (2)
Provisional Application 63038616 · Jun 12, 2020
Related Publication 20210388423A1 · Dec 16, 2021
References Cited (358)
US 4318846A · Khanna et al. · 1982 [cited by applicant]
US 4757141A · Fung et al. · 1988 [cited by applicant]
US 5066580A · Lee · 1991 [cited by applicant]
US 5091519A · Cruickshank · 1992 [cited by applicant]
US 5151507A · Hobbs, Jr. et al. · 1992 [cited by applicant]
US 5188934A · Menchen et al. · 1993 [cited by applicant]
US 5366860A · Bergot et al. · 1994 [cited by applicant]
US 5599675A · Brenner et al. · 1997 [cited by applicant]
US 5635352A · Urdea et al. · 1997 [cited by applicant]
US 5688648A · Mathies et al. · 1997 [cited by applicant]
US 5695940A · Drmanac et al. · 1997 [cited by applicant]
US 5750341A · Macevicz · 1998 [cited by applicant]
US 5800996A · Lee et al. · 1998 [cited by applicant]
US 5847162A · Lee et al. · 1998 [cited by applicant]
US 5990479A · Weiss et al. · 1999 [cited by applicant]
US 6054274A · Sampson et al. · 2000 [cited by applicant]
US 6172218B1 · Brenner · 2001 [cited by applicant]
US 6207392B1 · Weiss et al. · 2001 [cited by applicant]
US 6251303B1 · Bawendi et al. · 2001 [cited by applicant]
US 6265552B1 · Schatz · 2001 [cited by applicant]
US 6291187B1 · Kingsmore et al. · 2001 [cited by applicant]
US 6306597B1 · Macevicz · 2001 [cited by applicant]
US 6319426B1 · Bawendi et al. · 2001 [cited by applicant]
US 6322901B1 · Bawendi et al. · 2001 [cited by applicant]
US 6323009B1 · Lasken et al. · 2001 [cited by applicant]
US 6344329B1 · Lizardi · 2002 [cited by applicant]
US 6368801B1 · Faruqi · 2002 [cited by applicant]
US 6391937B1 · Beuhler et al. · 2002 [cited by applicant]
US 6423551B1 · Weiss et al. · 2002 [cited by applicant]
US 6426513B1 · Bawendi et al. · 2002 [cited by applicant]
US 6444143B2 · Bawendi et al. · 2002 [cited by applicant]
US 6534266B1 · Singer · 2003 [cited by applicant]
US 6576291B2 · Bawendi et al. · 2003 [cited by applicant]
US 6969488B2 · Bridgham et al. · 2005 [cited by applicant]
US 7255994B2 · Lao · 2007 [cited by applicant]
US 7345159B2 · Ju et al. · 2008 [cited by applicant]
US 7473767B2 · Dimitrov · 2009 [cited by applicant]
US 7534991B2 · Miller et al. · 2009 [cited by applicant]
US 7544794B1 · Benner · 2009 [cited by applicant]
US 7555155B2 · Levenson et al. · 2009 [cited by applicant]
US 7566537B2 · Balasubramanian et al. · 2009 [cited by applicant]
US 7655898B2 · Miller · 2010 [cited by applicant]
US 7893227B2 · Wu et al. · 2011 [cited by applicant]
US 7906285B2 · Drmanac · 2011 [cited by applicant]
US 7910304B2 · Drmanac · 2011 [cited by applicant]
US 7941279B2 · Hwang et al. · 2011 [cited by applicant]
US 7989166B2 · Koch et al. · 2011 [cited by applicant]
US 8124751B2 · Pierce et al. · 2012 [cited by applicant]
US 8199999B2 · Hoyt et al. · 2012 [cited by applicant]
US 8268554B2 · Schallmeiner · 2012 [cited by applicant]
US 8330087B2 · Domenicali · 2012 [cited by applicant]
US 8415102B2 · Geiss et al. · 2013 [cited by applicant]
US 8431691B2 · Mckernan et al. · 2013 [cited by applicant]
US 8460865B2 · Chee et al. · 2013 [cited by applicant]
US 8462981B2 · Determan et al. · 2013 [cited by applicant]
US 8481258B2 · Church et al. · 2013 [cited by applicant]
US 8519115B2 · Webster et al. · 2013 [cited by applicant]
US 8551710B2 · Bernitz et al. · 2013 [cited by applicant]
US 8632975B2 · Vander Horn et al. · 2014 [cited by applicant]
US 8658361B2 · Wu et al. · 2014 [cited by applicant]
US 8771950B2 · Church et al. · 2014 [cited by applicant]
US 8986926B2 · Ferree et al. · 2015 [cited by applicant]
US 9201063B2 · Sood et al. · 2015 [cited by applicant]
US 9273349B2 · Nguyen et al. · 2016 [cited by applicant]
US 9371563B2 · Geiss et al. · 2016 [cited by applicant]
US 9371598B2 · Chee · 2016 [cited by applicant]
US 9376717B2 · Gao et al. · 2016 [cited by applicant]
US 9404155B2 · Bortner · 2016 [cited by applicant]
US 9512422B2 · Barnard et al. · 2016 [cited by applicant]
US 9541504B2 · Hoyt · 2017 [cited by applicant]
US 9551032B2 · Landegren et al. · 2017 [cited by applicant]
US 9624538B2 · Church et al. · 2017 [cited by applicant]
US 9650406B2 · Zhou et al. · 2017 [cited by applicant]
US 9714446B2 · Webster et al. · 2017 [cited by applicant]
US 9714937B2 · Dunaway · 2017 [cited by applicant]
US 9727810B2 · Fodor et al. · 2017 [cited by applicant]
US 9778155B2 · Gradinaru et al. · 2017 [cited by applicant]
US 9783841B2 · Nolan et al. · 2017 [cited by applicant]
US 9889422B2 · Smith et al. · 2018 [cited by applicant]
US 9909167B2 · Samusik et al. · 2018 [cited by applicant]
US 10032064B2 · Hoyt · 2018 [cited by applicant]
US 10059990B2 · Boyden et al. · 2018 [cited by applicant]
US 10126242B2 · Miller et al. · 2018 [cited by applicant]
US 10138509B2 · Church et al. · 2018 [cited by applicant]
US 10179932B2 · Church et al. · 2019 [cited by applicant]
US 10227639B2 · Levner et al. · 2019 [cited by applicant]
US 10246700B2 · Dunaway et al. · 2019 [cited by applicant]
US 10266888B2 · Daugharthy et al. · 2019 [cited by applicant]
US 10267808B2 · Cai · 2019 [cited by applicant]
US 10309879B2 · Chen et al. · 2019 [cited by applicant]
US 10317321B2 · Tillberg et al. · 2019 [cited by applicant]
US 10364457B2 · Wassie et al. · 2019 [cited by applicant]
US 10370698B2 · Nolan et al. · 2019 [cited by applicant]
US 10415080B2 · Dunaway et al. · 2019 [cited by applicant]
US 10457980B2 · Cai et al. · 2019 [cited by applicant]
US 10465235B2 · Gullberg et al. · 2019 [cited by applicant]
US 10494662B2 · Church et al. · 2019 [cited by applicant]
US 10495554B2 · Deisseroth et al. · 2019 [cited by applicant]
US 10501777B2 · Beechem et al. · 2019 [cited by applicant]
US 10501791B2 · Church et al. · 2019 [cited by applicant]
US 10510435B2 · Cai et al. · 2019 [cited by applicant]
US 10526649B2 · Chen et al. · 2020 [cited by applicant]
US 10545075B2 · Deisseroth et al. · 2020 [cited by applicant]
US 10550429B2 · Harada et al. · 2020 [cited by applicant]
US 10580128B2 · Miller · 2020 [cited by applicant]
US 10640816B2 · Beechem et al. · 2020 [cited by applicant]
US 10640826B2 · Church et al. · 2020 [cited by applicant]
US 10669569B2 · Gullberg et al. · 2020 [cited by applicant]
US 10746981B2 · Tomer et al. · 2020 [cited by applicant]
US 10774372B2 · Chee et al. · 2020 [cited by applicant]
US 10774374B2 · Frisén et al. · 2020 [cited by applicant]
US 10794802B2 · Gradinaru et al. · 2020 [cited by applicant]
US 10802262B2 · Tomer et al. · 2020 [cited by applicant]
US 10815519B2 · Husain et al. · 2020 [cited by applicant]
US 10829814B2 · Fan et al. · 2020 [cited by applicant]
US 10844426B2 · Daugharthy et al. · 2020 [cited by applicant]
US 10858698B2 · Church et al. · 2020 [cited by applicant]
US 10872679B2 · Cai et al. · 2020 [cited by applicant]
US 10964001B2 · Miller · 2021 [cited by applicant]
US 11174281B1 · Graham et al. · 2021 [cited by applicant]
US 11287422B2 · Previte et al. · 2022 [cited by applicant]
US 11434525B2 · Glezer · 2022 [cited by applicant]
US 11459603B2 · Tyagi et al. · 2022 [cited by applicant]
US 11499185B2 · Vijayan et al. · 2022 [cited by applicant]
US 11643679B2 · Glezer et al. · 2023 [cited by applicant]
US 11999999B2 · Ju et al. · 2024 [cited by applicant]
US 20020045045A1 · Adams et al. · 2002 [cited by applicant]
US 20030013091A1 · Dimitrov · 2003 [cited by applicant]
US 20030017264A1 · Treadway et al. · 2003 [cited by applicant]
US 20070166708A1 · Dimitrov et al. · 2007 [cited by applicant]
US 20100015607A1 · Geiss et al. · 2010 [cited by applicant]
US 20100047924A1 · Webster et al. · 2010 [cited by applicant]
US 20100055733A1 · Lutolf et al. · 2010 [cited by applicant]
US 20100112710A1 · Geiss et al. · 2010 [cited by applicant]
US 20100261026A1 · Ferree et al. · 2010 [cited by applicant]
US 20100262374A1 · Hwang et al. · 2010 [cited by applicant]
US 20110223585A1 · Gullberg et al. · 2011 [cited by applicant]
US 20130288249A1 · Gullbert · 2013 [cited by applicant]
US 20130323729A1 · Landegren et al. · 2013 [cited by applicant]
US 20140371088A1 · Webster · 2014 [cited by applicant]
US 20160024555A1 · Church et al. · 2016 [cited by applicant]
US 20160108458A1 · Frei et al. · 2016 [cited by applicant]
US 20160305856A1 · Boyden et al. · 2016 [cited by applicant]
US 20160376642A1 · Landegren et al. · 2016 [cited by applicant]
US 20170009278A1 · Söderberg et al. · 2017 [cited by applicant]
US 20170029872A1 · Bhattacharyya et al. · 2017 [cited by applicant]
US 20170081489A1 · Rodriques et al. · 2017 [cited by applicant]
US 20170101672A1 · Luo et al. · 2017 [cited by applicant]
US 20170220733A1 · Zhuang et al. · 2017 [cited by applicant]
US 20170253918A1 · Kohman · 2017 [cited by applicant]
US 20180052081A1 · Kohman · 2018 [cited by applicant]
US 20180080876A1 · Rockel et al. · 2018 [cited by applicant]
US 20180208967A1 · Larman et al. · 2018 [cited by applicant]
US 20180237864A1 · Imler et al. · 2018 [cited by applicant]
US 20180320226A1 · Church et al. · 2018 [cited by applicant]
US 20190017106A1 · Frisen et al. · 2019 [cited by applicant]
US 20190032121A1 · Daugharthy et al. · 2019 [cited by applicant]
US 20190032128A1 · Chen et al. · 2019 [cited by applicant]
US 20190055594A1 · Samusik et al. · 2019 [cited by applicant]
US 20190112599A1 · Church et al. · 2019 [cited by applicant]
US 20190119735A1 · Deisseroth et al. · 2019 [cited by applicant]
US 20190155835A1 · Daugharthy et al. · 2019 [cited by applicant]
US 20190161796A1 · Hauling et al. · 2019 [cited by applicant]
US 20190177718A1 · Church et al. · 2019 [cited by applicant]
US 20190177800A1 · Boutet et al. · 2019 [cited by applicant]
US 20190194709A1 · Church et al. · 2019 [cited by applicant]
US 20190218608A1 · Daugharthy et al. · 2019 [cited by applicant]
US 20190249248A1 · Beechem et al. · 2019 [cited by applicant]
US 20190264270A1 · Zhuang et al. · 2019 [cited by applicant]
US 20190271028A1 · Khafizov et al. · 2019 [cited by applicant]
US 20190276881A1 · Zhuang et al. · 2019 [cited by applicant]
US 20190339203A1 · Miller et al. · 2019 [cited by applicant]
US 20190367969A1 · Belhocine et al. · 2019 [cited by applicant]
US 20200010891A1 · Beechem et al. · 2020 [cited by applicant]
US 20200071751A1 · Daugharthy et al. · 2020 [cited by applicant]
US 20200123597A1 · Daniel · 2020 [cited by applicant]
US 20200140920A1 · Pierce et al. · 2020 [cited by applicant]
US 20200224243A1 · Desai et al. · 2020 [cited by applicant]
US 20200224244A1 · Nilsson et al. · 2020 [cited by applicant]
US 20200239946A1 · Dewal · 2020 [cited by applicant]
US 20200332368A1 · Ferree et al. · 2020 [cited by applicant]
US 20200354774A1 · Church et al. · 2020 [cited by applicant]
US 20200354782A1 · Dewal · 2020 [cited by applicant]
US 20200362398A1 · Kishi et al. · 2020 [cited by applicant]
US 20200393343A1 · Kennedy-Darling et al. · 2020 [cited by applicant]
US 20210017587A1 · Cai et al. · 2021 [cited by applicant]
US 20210115504A1 · Cai et al. · 2021 [cited by applicant]
US 20210238662A1 · Bava · 2021 [cited by applicant]
US 20210238674A1 · Bava · 2021 [cited by applicant]
US 20210254140A1 · Stahl et al. · 2021 [cited by applicant]
US 20210262018A1 · Bava et al. · 2021 [cited by applicant]
US 20210277460A1 · Bava · 2021 [cited by applicant]
US 20210340621A1 · Daugharthy et al. · 2021 [cited by applicant]
US 20210388423A1 · Bava et al. · 2021 [cited by applicant]
US 20210388424A1 · Bava · 2021 [cited by applicant]
US 20220049302A1 · Daugharthy et al. · 2022 [cited by applicant]
US 20220049303A1 · Busby et al. · 2022 [cited by applicant]
US 20220083832A1 · Shah · 2022 [cited by applicant]
US 20220084628A1 · Shah · 2022 [cited by applicant]
US 20220084629A1 · Shah · 2022 [cited by applicant]
US 20220136049A1 · Bava et al. · 2022 [cited by applicant]
US 20220186300A1 · Bava · 2022 [cited by applicant]
US 20220195498A1 · Kuhnemund et al. · 2022 [cited by applicant]
US 20220213529A1 · Kuhnemund et al. · 2022 [cited by applicant]
US 20220228200A1 · Bava · 2022 [cited by applicant]
US 20220235403A1 · Costa · 2022 [cited by applicant]
US 20220282306A1 · Bava et al. · 2022 [cited by applicant]
US 20220282316A1 · Bava et al. · 2022 [cited by applicant]
US 20220282319A1 · Verheyen et al. · 2022 [cited by applicant]
US 20220372570A1 · Costa · 2022 [cited by applicant]
US 20220380838A1 · Kuhnemund et al. · 2022 [cited by applicant]
US 20220403458A1 · Bava et al. · 2022 [cited by applicant]
US 20230012607A1 · Kuhnemund et al. · 2023 [cited by applicant]
US 20230013775A1 · Chen · 2023 [cited by applicant]
US 20230015226A1 · Chen · 2023 [cited by applicant]
US 20230026886A1 · Chen · 2023 [cited by applicant]
US 20230031305A1 · Hernandez Neuta et al. · 2023 [cited by applicant]
US 20230031996A1 · Hernandez Neuta et al. · 2023 [cited by applicant]
US 20230035685A1 · Hernandez Neuta et al. · 2023 [cited by applicant]
US 20230037182A1 · Bava et al. · 2023 [cited by applicant]
US 20230039148A1 · Verheyen et al. · 2023 [cited by applicant]
US 20230041485A1 · Hernandez Neuta et al. · 2023 [cited by applicant]
US 20230044650A1 · Dockter · 2023 [cited by applicant]
US 20230057571A1 · Costa et al. · 2023 [cited by applicant]
US 20230061438A1 · Astier et al. · 2023 [cited by applicant]
US 20230061542A1 · Kuhnemund et al. · 2023 [cited by applicant]
US 20230084407A1 · Neuta et al. · 2023 [cited by applicant]
US 20230159997A1 · Belhocine et al. · 2023 [cited by applicant]
US 20230160794A1 · Dockter et al. · 2023 [cited by applicant]
US 20230183787A1 · Bava et al. · 2023 [cited by applicant]
US 20230242974A1 · Costa et al. · 2023 [cited by applicant]
US 20230279465A1 · He et al. · 2023 [cited by applicant]
US 20230279475A1 · Kuhnemund et al. · 2023 [cited by applicant]
US 20230279480A1 · Kuhnemund et al. · 2023 [cited by applicant]
US 20230287478A1 · Bava · 2023 [cited by applicant]
US 20230314327A1 · Hoffman · 2023 [cited by applicant]
US 20230314328A1 · Costa · 2023 [cited by applicant]
US 20230323427A1 · Levin · 2023 [cited by applicant]
US 20230323430A1 · Shastry · 2023 [cited by applicant]
US 20230323437A1 · Chen · 2023 [cited by applicant]
US 20230374573A1 · Qian et al. · 2023 [cited by applicant]
US 20230374580A1 · Costa · 2023 [cited by applicant]
US 20230416821A1 · Bava et al. · 2023 [cited by applicant]
US 20240002902A1 · Jakobsen et al. · 2024 [cited by applicant]
US 20240026426A1 · Costa · 2024 [cited by applicant]
US 20240026427A1 · Kuhnemund et al. · 2024 [cited by applicant]
US 20240026439A1 · Sasaki et al. · 2024 [cited by applicant]
US 20240026448A1 · Costa · 2024 [cited by applicant]
US 20240035070A1 · Christopherson · 2024 [cited by applicant]
US 20240035071A1 · Delaney et al. · 2024 [cited by applicant]
US 20240035072A1 · Christopherson · 2024 [cited by applicant]
US 20240043910A1 · Shastry · 2024 [cited by applicant]
US 20240043914A1 · Chen · 2024 [cited by applicant]
US 20240060119A1 · Bava et al. · 2024 [cited by applicant]
US 20240084373A1 · Shastry · 2024 [cited by applicant]
US 20240084378A1 · Marks et al. · 2024 [cited by applicant]
US 20240101978A1 · Boghospor · 2024 [cited by applicant]
US 20240132938A1 · Kuhnemund et al. · 2024 [cited by applicant]
US 20240141418A1 · Mielinis · 2024 [cited by applicant]
US 20240150816A1 · Feng et al. · 2024 [cited by applicant]
US 20240158852A1 · Belhocine et al. · 2024 [cited by applicant]
US 20240167081A1 · Bava et al. · 2024 [cited by applicant]
US 20240175082A1 · Costa · 2024 [cited by applicant]
US 20240175083A1 · Bava et al. · 2024 [cited by applicant]
US 20240191297A1 · Christopherson et al. · 2024 [cited by applicant]
US 20240209330A1 · Shastry et al. · 2024 [cited by applicant]
US 20240218424A1 · Costa et al. · 2024 [cited by applicant]
US 20240218437A1 · Belhocine et al. · 2024 [cited by applicant]
WO 2017143155A2 · 2017 [cited by applicant]
WO 2019199579A1 · 2019 [cited by applicant]
WO 2020056381A1 · 2020 [cited by applicant]
WO 2020076976A1 · 2020 [cited by applicant]
WO 2020076979A1 · 2020 [cited by applicant]
WO 2020096687A1 · 2020 [cited by applicant]
WO 2020099640A1 · 2020 [cited by applicant]
WO 2020117914A1 · 2020 [cited by applicant]
WO 2020123742A1 · 2020 [cited by applicant]
WO WO2020123316 · 2020 [cited by applicant]
WO 2020142490A1 · 2020 [cited by applicant]
WO 2020240025A1 · 2020 [cited by applicant]
WO 2020254519A1 · 2020 [cited by applicant]
WO WO2021123282 · 2021 [cited by applicant]
WO WO2021123286 · 2021 [cited by applicant]
WO WO2021138676 · 2021 [cited by applicant]
WO WO2021155063 · 2021 [cited by applicant]
WO WO2021168326 · 2021 [cited by applicant]
WO WO2023108139 · 2023 [cited by applicant]
WO WO2023141476 · 2023 [cited by applicant]
WO WO2023172915 · 2023 [cited by applicant]
WO WO2023192302 · 2023 [cited by applicant]
WO WO2024148300 · 2024 [cited by applicant]
Bibikova et al., “Quantitative gene expression profiling in formalin-fixed, paraffin-embedded tissues using universal bead arrays,” Am J Pathol. Nov. 2004;165(5):1799-807. [cited by applicant]
Capodieci et al., “Gene expression profiling in single cells within tissue,” Nat Methods. (2005) 2(9): 663-5. [cited by applicant]
Conze et al., “Single molecule analysis of combinatorial splicing,” Nucleic Acids Res. (2010) 38(16): e163. [cited by applicant]
Femino et al., “Visualization of single RNA transcripts in situ,” Science. (1998) 280(5363): 585-90. [cited by applicant]
Gavrilovic et al., “Automated classification of multicolored rolling circle products in dual-channel wide-field fluorescence microscopy,” Cytometry A. (2011) 79(7): 518-27. [cited by applicant]
Geiss et al., “Direct multiplexed measurement of gene expression with color-coded probe pairs,” Nat Biotechnol. (2008) 26(3): 317-25. [cited by applicant]
Gunderson et al. “Decoding randomly ordered DNA arrays.” Genome research 14.5 (2004): 870-877. [cited by applicant]
Han et al., “Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules,” Nat Biotechnol. (2001) 19(7): 631-5. [cited by applicant]
Itzkovitz et al., “Single-molecule transcript counting of stem-cell markers in the mouse intestine,” Nat Cell Biol. (2011) 14(1): 106-14. [cited by applicant]
Itzkovitz et al., “Validating Transcripts with Probes and Imaging Technology,” Nat Methods. (2011) 8(4 Suppl): S12-S19. [cited by applicant]
Lagunavicius et al., “Novel application of Phi29 DNA polymerase: RNA detection and analysis in vitro and in situ by target RNA-primed RCA,” RNA. (2009) 15(5):765-71. [cited by applicant]
Larsson et al. “In situ detection and genotyping of individual mRNA molecules,” Nat Methods. (2010) 7(5):395-397. [cited by applicant]
Levsky et al., “Fluorescence in situ hybridization: past, present and future,” J Cell Sci. (2003) 116(Pt 14): 2833-8. [cited by applicant]
Levsky et al., “Single-cell gene expression profiling,” Science. (2002) 297(5582): 836-40. [cited by applicant]
Maierhorfer et al., “Multicolor deconvolution microscopy of thick biological specimens,” Am J Pathol. (2003) 162(2): 373-9. [cited by applicant]
Meade et al. “Multiplexed DNA detection using spectrally encoded porous SiO2 photonic crystal particles,” Anal Chem. (2009) 81(7): 2618-25. [cited by applicant]
Raj et al., “Imaging individual mRNA molecules using multiple singly labeled probes,” Nat Methods. (2008) 5(10): 877-879. [cited by applicant]
Sun et al., “Composite organic-inorganic nanoparticles as Raman labels for tissue analysis,” Nano Lett. (2007) 7(2): 351-6. [cited by applicant]
Wählby et al., “Sequential immunofluorescence staining and image analysis for detection of large numbers of antigens in individual cell nuclei,” Cytometry. (2002) 47(1): 32-41. [cited by applicant]
Weibrecht et al., “Simultaneous visualization of both signaling cascade activity and end-point gene expression in single cells,” PLoS One. (2011) 6(5): e20148. [cited by applicant]
Wilson et al., “Encoded microcarriers for high-throughput multiplexed detection,” Angew Chem Int Ed Engl. (2006) 18;45(37): 6104-17. [cited by applicant]
Zhao et al., “Advances of multiplex and high throughput biomolecular detection technologies based on encoding microparticles,” Sci China Chem. (2011) 54(8):1185. [cited by applicant]
Allawi et al., “Thermodynamics and NMR of internal G.T mismatches in DNA,” Biochemistry, (1997) 36:10581-94. [cited by applicant]
Archer et al., “Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage,” BMC Genomics. (2014) 15(1):401. [cited by applicant]
Baner et al., “Signal amplification of padlock probes by rolling circle replication,” Nucleic Acids Res. (1998) 26 (22):5073-5078. [cited by applicant]
Berger et al., “Universal bases for hybridization, replication and chain termination,” Nucleic Acid Res. (2000) 28(15): 2911-2914. [cited by applicant]
Bio et al., “Click and photo-unclick chemistry of aminoacrylate for visible light-triggered drug release,” Chem. Commun. (2012) 48(52); 6517-6519. [cited by applicant]
Bolognesi et al., “Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections,” J. Histochem. Cytochem. (2017); 65(8): 431-444. [cited by applicant]
Chen et al., “Nanoscale imaging of RNA with expansion microscopy,” Nat Methods. (2016) 13:679-684. [cited by applicant]
Chen, F. et al. “Expansion Microscopy,” Science (2015) 347(6221):543-548. [cited by applicant]
Dean et al., “Rapid Amplification Of Plasmid And Phage DNA Using Phi29 DNA Polymerase And Multiply-Primed Rolling Circle Amplification,” Genome Research (2001) 11:1095-1099. [cited by applicant]
Fang et al., “Fluoride-Cleavable Biotinylation Phosphoramidite for 5′-end-Labelling and Affinity Purification of Synthetic Oligonucleotides,” Nucleic Acids Res. (2003) 31(2): 708-715. [cited by applicant]
Faruqi et al., “High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification,” BMC Genomics. (2001) 2:4. [cited by applicant]
Gartner et al., “The Generality of DNA-Templated Synthesis as a Basis for Evolving Non-Natural Small Molecules,” J. Am. Chem. Soc. (2001), 123(28); 6961-6963. [cited by applicant]
Glass et al., “SIMPLE: a sequential immunoperoxidase labeling and erasing method,” J Histochem Cytochem. (2009) 57(10); 899-905. [cited by applicant]
Goh, J.J.L. et al. (Jul. 2020, e-pub. Jun. 15, 2020). “Highly Specific Multiplexed RNA Imaging In Tissues With Split-FISH,” Nat Methods 17(7):689-693. [cited by applicant]
Goransson, J. et al. (Jan. 2009, e-pub. Nov. 25, 2008). “A Single Molecule Array For Digital Targeted Molecular Analyses,” Nucleic Acids Res 37(1):e7, 9 pages. [cited by applicant]
Guo et al., “Four-color DNA sequencing with 3′-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides,” Proc Natl Acad Sci U S A. (2008) 105(27): 9145-50. [cited by applicant]
Gyllborg et al., “Hybridization-based in situ sequencing (HybISS) for spatially resolved transcriptomics in human and mouse brain tissue,” Nucleic Acids Res. (2020) 48(19): e112. [cited by applicant]
Henegariu et al., “Custom fluorescent-nucleotide synthesis as an alternative method for nucleic acid labeling,” Nature Biotechnol. (2000) 18:345. [cited by applicant]
Jamur et al., “Permeabilization of cell membranes,” Method Mol. Biol. (2010) 588: 63-66 (abstract only). [cited by applicant]
Ju et al., “Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators,” Proc Natl Acad Sci U S A. (2006) 103(52): 19635-40. [cited by applicant]
Kanehisa, “Use of statistical criteria for screening potential homologies in nucleic acid sequences,” Nucleic Acids Res. (1984) 12:203-213. [cited by applicant]
Lakowicz et al., “Silver particles enhance emission of fluorescent DNA oligomers,” Bio Techniques (2003) 34(1); 62-66. [cited by applicant]
Lee, J.H. et al. (Mar. 21, 2014, e-pub. Sep. 21, 2014). “Highly Multiplexed Subcellular RNA Sequencing In Situ”, Science 343(6177):1360-1363. [cited by applicant]
Lin et al., “Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method,” Nat Commun. (2015) 6:8390. [cited by applicant]
Liu, S. et al. (2021, e-pub. Mar. 8, 2021). “Barcoded Oligonucleotides Ligated On RNA Amplified For Multiplexed And Parallel In Situ Analyses,” Nucleic Acids Res. 49(10):e58, 15 pages. [cited by applicant]
Lizardi et al., “Mutation detection and single-molecule counting using isothermal rolling-circle amplification,” Nat Genet. (1998) 19(3): 225-232. [cited by applicant]
McGinn et al., “New technologies for DNA analysis—a review of the READNA Project,” N Biotechnol. (2016) 33(3): 311-30. doi: 10.1016/j.nbt.2015.10.003. [cited by applicant]
Mitra et al., “Fluorescent in situ sequencing on polymerase colonies,” Anal. Biochem. (2003) 320; 55-65. [cited by applicant]
Moffitt et al., “RNA Imaging with Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH),” Methods in Enzymology, (2016) 572; 1-49. [cited by applicant]
Nallur et al., “Signal amplification by rolling circle amplification on DNA microarrays,” Nucleic Acids Res. (2001) 29 (23): e118. [cited by applicant]
Patterson et al., “Finding the right (bioorthogonal) chemistry,” ACS Chem. Biol. (2014) 9(3): 592-605. [cited by applicant]
Payne, A.C. et al. (Feb. 26, 2021, e-pub Dec. 31, 2020). “In Situ Genome Sequencing Resolves DNA Sequence And Structure In Intact Biological Samples,” Science 371(6532):1-19, 20 pages. [cited by applicant]
Pirici et al., “Antibody elution method for multiple immunohistochemistry on primary antibodies raised in the same species and of the same subtype,” J Histochem Cytochem. (2009) 57(6); 567-75. [cited by applicant]
Rouhanifard, S.H. et al. (Nov. 12, 2018, e-pub. May 13, 2019). “Clampfish Detects Individual Nucleic Acid Molecules Using Click Chemistry-Based Amplification,” Nat Biotechnol, 17 pages. [cited by applicant]
Schweitzer et al. “Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection,” Proc. Natl Acad. Sci. USA (2000) 97:10113-119. [cited by applicant]
Schweitzer et al., “Multiplexed protein profiling on microarrays by rolling-circle amplification,” Nature Biotech, (2002) 20:359-365. [cited by applicant]
Seckute et al., “Rapid oligonucleotide-templated fluorogenic tetrazine ligations,” Nucleic Acids Res. (2013) 41(15); e148. [cited by applicant]
Seo et al., “Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides,” PNAS (2005) 102(17); 5926-5931. [cited by applicant]
Shendure et al., “Accurate multiplex polony sequencing of an evolved bacterial genome,” Science (2005) 309 (5741); 1728-1732. [cited by applicant]
Takei, Y. et al. (Feb. 2021, e-pub Jan. 27, 2021). “Integrated Spatial Genomics Reveals Global Architecture Of Single Nuclei,” Nature 590(7845):344-350, 53 pages. [cited by applicant]
Vandernoot et al., “cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications,” Biotechniques, (2012) 53(6) 373-80. [cited by applicant]
Wang et al., “Three-dimensional intact-tissue sequencing of single-cell transcriptional states,” Science. (2018) 361 (6400): eaat5691. [cited by applicant]
Wetmur, “DNA Probes: Applications of the Principles of Nucleic Acid Hybridization,” Critical Reviews in Biochemistry and Molecular Biology, (1991) 26(91); 227-259. [cited by applicant]
Wu, C. et al. (Nov. 28, 2018). “RollFISh Achieves Robust Quantification Of Single-Molecule RNA Biomarkers In Paraffin-Embedded Tumor Tissue Samples,” Commun Biol. 1:(209):1-8. [cited by applicant]
Xiong et al., “Stepwise “Click” Chemistry for the Template Independent Construction of a Broad Variety of Cross-Linked Oligonucleotides: Influence of Linker Length, Position, and Linking Number on DNA Duplex Stability,”… [cited by applicant]
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