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(12) United States Patent (10) Patent No.: US 9,102,835 B2 Mao Et Al

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(12) United States Patent (10) Patent No.: US 9,102,835 B2 Mao Et Al US009102835B2 (12) United States Patent (10) Patent No.: US 9,102,835 B2 Mao et al. (45) Date of Patent: *Aug. 11, 2015 (54) METHODS OF USING DYES IN (56) References Cited ASSOCATION WITH NUCLECACD STAINING ORDETECTION AND U.S. PATENT DOCUMENTS ASSOCATED TECHNOLOGY 4,883,867 A 11/1989 Lee et al. 5,118,801 A 6/1992 Lizardi et al. (71) Applicant: Biotium, Inc., Hayward, CA (US) 5,210,015 A 5/1993 Gelfand et al. 5,312,728 A 5/1994 Lizardi et al. 5,321,130 A 6/1994 Yue et al. (72) Inventors: Fei Mao, Fremont, CA (US); Wai-Yee 5,401,847 A 3, 1995 Glazer et al. Leung, San Ramon, CA (US) 5,403,928 A 4/1995 Arrhenuis 5,410,030 A 4/1995 Yue et al. (73) Assignee: BIOTIUM, INC., Hayward, CA (US) 5,436,134 A 7/1995 Haugland et al. 5,445,946 A 8, 1995 Roth et al. 5,534.416 A 7/1996 Millard et al. (*) Notice: Subject to any disclaimer, the term of this 5,538,848 A 7/1996 Livak et al. patent is extended or adjusted under 35 5,545,535 A 8, 1996 Roth et al. U.S.C. 154(b) by 0 days. 5,582,977 A 12/1996 Yue et al. 5,646,264 A 7/1997 Glazer et al. This patent is Subject to a terminal dis 5,656,449 A 8, 1997 Yue claimer. 5,658,751 A 8, 1997 Yue et al. 5,691,146 A 1 1/1997 Mayrand 5,763,162 A 6/1998 Glazer et al. (21) Appl. No.: 13/962,668 5,846,726 A 12/1998 Nadeau et al. 5,863,753 A 1/1999 Haugland et al. (22) Filed: Aug. 8, 2013 5,925,517 A 7/1999 Tyagi et al. (65) Prior Publication Data (Continued) US 2014/OO73058 A1 Mar. 13, 2014 FOREIGN PATENT DOCUMENTS EP 1344835 A2 9, 2003 EP 1348713 A2 10, 2003 Related U.S. Application Data EP 1373250 B1 8, 2006 JP 20O2327 130 11, 2002 (60) Continuation of application No. 13/541.313, filed on WO WO 2006/020947 A2 2, 2006 Jul. 3, 2012, now Pat. No. 8,753,814, which is a WO WO 2006/020947 A3 8, 2008 division of application No. 12/854,436, filed on Aug. OTHER PUBLICATIONS 11, 2010, now Pat. No. 8,232,050, which is a continuation of application No. 12/484.968, filed on U.S. Appl. No. 12/976.917, filed Dec. 22, 2010, Mao et al. Jun. 15, 2009, now Pat. No. 7,803,943, which is a U.S. Appl. No. 13/968,122, filed Aug. 15, 2013, Mao et al. continuation of application No. 1 1/377,254, filed on “Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Mar. 16, 2006, now Pat. No. 7,601498. Tools for Use at the Bench.” Electrophoresis of DNA, RNA, and Protein, Section 3A, 2002, pp. 62-80. (60) Provisional application No. 60/663,613, filed on Mar. "Nucleic Acid Stains and Products for Genomics Studies,” www. 17, 2005. biotium.com—Fluorescent Probes and Related Biochemical Reagents for Life Science, Section 9, 2005-2006, pp. 161-173. (51) Int. C. (Continued) CO7D 22/06 (2006.01) CO7D 455/04 (2006.01) Primary Examiner — Jezia Riley CO7D 47L/04 (2006.01) (74) Attorney, Agent, or Firm — Wilson Sonsini Goodrich & C09B I5/00 (2006.01) Rosati C09B II/28 (2006.01) C09B 23/04 (2006.01) (57) ABSTRACT CI2O I/68 (2006.01) Methods of using dyes and associated technology are pro GOIN L/30 (2006.01) vided. A dye, such as a monomeric dye or a dimeric dye, may GOIN 2L/64 (2006.01) be used in a nucleic acid gel staining application and/or a CO7H 9/04 (2006.01) nucleic acid detection application. Such a dye and a salt that (52) U.S. C. comprises an anion that is associated with a strong acid and a CPC ................. C09B 15/00 (2013.01); C09B II/28 cation that is associated with a strong base may be used in (2013.01); C09B 23/04 (2013.01): CI2O Such an application. A dimeric dye, Such as a dimeric dye I/6818 (2013.01); G0IN 1/30 (2013.01); G0IN capable of forming a hairpin-like structure, may be used to 21/6486 (2013.01); YIOS 435/81 (2013.01); stain and/or detect nucleic acids via a release-on-demand Y10T 436/143333 (2015.01) mechanism. A dimeric dye having low background fluores (58) Field of Classification Search cence in the absence of nucleic acids and high fluorescence in CPC ........ C09B 15700; C09B 23/04; C09B 23/06; the presence of nucleic acids, upon binding therewith, may be C09B 23/08; C09B 23/0091 used to stain and/or detect nucleic acids. USPC ................................ 546/79, 80, 88:536/26.6 See application file for complete search history. 22 Claims, 14 Drawing Sheets US 9,102,835 B2 Page 2 (56) References Cited Gaugain, et al. DNA Bifunctional Intercalators. 2. Fluorescence Properties and DNA Binding Interaction of an Ethidium Homodimer U.S. PATENT DOCUMENTS and an Acridine Ethidium Heterodimer. Biochemistry, vol. 17. No. 24, 1978, pp. 5078-5088. 5,977,344 A 11/1999 Glazer et al. 6,037,137 A 3/2000 Komoriya et al. Gerlach, et al. Annalen Dr Physik, G Folge, Band 2, 1948, pp.55-75. 6,150,097 A 11/2000 Tyagi et al. Gong, et al., New DNA Minor-Groove Binding Molecules with High 6,258,569 B1 7/2001 Livak et al. Sequence-Selectivities and Binding Affinities, Biochemical and Bio 6,277,570 B1 8, 2001 Wood et al. physical Communications, vol. 240, No. 3, 1997, pp. 557-560. 6,569,627 B2 5, 2003 Wittwer et al. Guo, et al. DNA-Dye Fluorescence Enhancement Based on Shifting 6,635,427 B2 10/2003 Wittwer et al. the Dimer-Monomer Equilibrium of Fluorescent Dye, Applied Spec 6,664.047 B1 12/2003 Haugland et al. troscopy, vol. 51, No. 7, 1997, pp. 1002-1007. 7,166,478 B2 1/2007 Stavrianopoulos et al. Hartwig, et al. Room-Temperature Palladium-Catalyzed Amination 7,387,887 B2 6, 2008. Wittwer et al. 7,601498 B2 10/2009 Mao et al. of Aryl Bromides and Chlorides and Extended Scope of Aromatic 7,776,567 B2 8, 2010 Mao et al. C N Bond Formation with a Commercial Ligand. J. Org. Chem. 7,803,943 B2 9, 2010 Mao et al. vol. 64, 1999, pp. 5575-5580. 8,232,050 B2 7, 2012 Mao et al. Haugland. Handbook of Fluorescent Probes and Research Prod 2003,000831.6 A1 1/2003 Smith et al. ucts—Ninth Edition: Nucleic Acid Detection and Genomics Tech 2003/0092062 A1 5/2003 Reddy et al. nology. Molecular Probes, Chapter 8, 2002, pp. 265-352. 2004/O132046 A1 7/2004 Westman et al. 2005/0239096 A1 10/2005 Beaudet et al. Higuchi, et al. Simultaneous Amplification and Detection of Specific 2006/0211028 A1 9, 2006 Mao et al. DNA Sequences. Bio/Technology, vol. 10, Apr. 1992, pp. 413-417. 2006/0211029 A1 9, 2006 Mao et al. Holland, et al. Detection of Specific Polymerase Chain Reaction 2010, OOO9454 A1 1/2010 Mao et al. Product by Utilizing the 5'-->3' Exonuclease Activity of Thermus 2013,016.7309 A1 7, 2013 Mao et al. Aquaticus DNA Polymerase. Proc. Natl. Acad. Sci. USA. vol. 88, Aug. 1991, pp. 7276-7280. OTHER PUBLICATIONS Ishiguro, et al. Fluorescence Detection of Specific Sequence of Nucleic Acids by Oxazole Yellow-Linked Oligonucleotides. Homo Adkins, et al. Visualization of DNA in Agarose Gels as Migrating geneous Quantitative Monitoring of in vitro Transcription. Nucleic Colored Bands: Applications for Preparative Gels and Educational Acids Research, vol. 24, No. 24, 1996, pp. 4992-4997. Demonstrations. Analytical Biochemistry, vol. 240, Article No. Jackobsen, et al. Site selective bis-intercalation of a homodimeric 0325, 1996, pp. 17-23. thiazole orange dye in DNA oligonucleotides. Nucleic Acids Res. Albert, A. The Acridines. Their Preparation, Physical, Chemical, and 1995; 23(5):753-60. Biological Properties, and Uses. Angew. Chem. internat. Edi, vol. 6, Joseph, et al. Tuning of Intercalation and Electron-Transfer Processes No. 10, 1967, 1 page. Between DNA and Acridinium Derivatives through Steric Effects. Atwell, etal. Potential antitumor agents. 45. Synthesis, DNA-binding Bioconjugate Chem... vol. 15, 2004, pp. 1230-1235. interaction, and biological activity of triacridine derivatives. J Med Kapuscinski, et al. Fluorescent Complexes of DNA with DAPI 4.6- Chem. Jan. 1986:29(1):69-74. diamidine-2-phenyl indole.2HCI or DCI 4",6-dicarboxyamide-2- Barak, et al. Fluorescent Low Density Lipoprotein for Observation of phenyl indole. Nucleic Acid Research, vol. 5, No. 10, Oct. 1978, pp. Dynamics of Individual Receptor Complexes on Cultured Human 3775-3799. Fibroblasts, The Journal of Cell Biology, vol.90, 1981, pp. 595-604. Karsai, et al. Evaluation of a Homemade SYBR Green I Reaction Bengtsson, et al. A New Minor Groove Binding Asymmetric Mixture for Real-Time PCR Quantification of Gene Expression. Cyanine Reporter Dye for Real-Time PCR. Nucleic Acid Research, Biotechniques, Apr. 2002; 32(4): 790-2, 794-6. vol. 31, No. 8, 2003, pp. 1-5. Khairutdinov, et al. Photophysics of Cyanine Dyes: Subnanosecond Benson, et al. Heterodimeric DNA-Binding Dyes Designed for Relaxation Dynamics in Monomers, Dimers, and H- and J-Aggre Energy Transfer: Synthesis and Spectroscopic Properties. Nucleic gates in Solution. J. Phys. Chem. B. vol. 101, No. 14, 1997, pp. Acids Research, vol. 21, No. 24, 1993, pp.
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