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(12) United States Patent (10) Patent No.: US 9,624,534 B2 Kuersten (45) Date of Patent: *Apr

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(12) United States Patent (10) Patent No.: US 9,624,534 B2 Kuersten (45) Date of Patent: *Apr USOO9624534B2 (12) United States Patent (10) Patent No.: US 9,624,534 B2 Kuersten (45) Date of Patent: *Apr. 18, 2017 (54) AMPLIFICATION AND DETECTION OF 8, 192,941 B2 6/2012 Kuersten RBONUCLEC ACDS 2003/0119004 A1 6/2003 Wenz et al. 2004/0219565 A1 11/2004 Kauppinen et al. 2005/0272O75 A1 12/2005 Jacobsen et al. (71) Applicant: APPLIED BIOSYSTEM, LLC, 2006,0003337 A1 1/2006 Brandis et al. Carlsbad, CA (US) 2006/0051789 A1 3/2006 Kazakov et al. 2006, OO63181 A1 3/2006 Green et al. (72) Inventor: R. Scott Kuersten, Fitchburg, WI (US) 2006/0211000 A1 9/2006 Sorge et al. 2006, O246464 A1 11/2006 Xia 2007,0009944 A1 1/2007 Bowater et al. (73) Assignee: APPLIED BIOSYSTEMS, LLC, 2007, OO15187 A1 1/2007 Lao et al. Carlsbad, CA (US) 2007/0020644 A1 1/2007 Kolykhalov et al. 2008.0070238 A1 3/2008 Klussmann et al. (*) Notice: Subject to any disclaimer, the term of this 2010.0029511 A1 2/2010 Raymond et al. patent is extended or adjusted under 35 2010, O297728 A1 1 1/2010 Ziman et al. U.S.C. 154(b) by 0 days. FOREIGN PATENT DOCUMENTS This patent is Subject to a terminal dis claimer. EP O32O3O8 6, 1989 WO WO-90/01069 2, 1990 WO WO-03/093.504 11/2003 (21) Appl. No.: 15/209,090 WO WO-2006/033020 3, 2006 WO WO-2007/044.066 4/2007 (22) Filed: Jul. 13, 2016 WO WO-2007/050990 5/2007 WO WO-2009/091719 T 2009 (65) Prior Publication Data WO WO-2010/048605 4/2010 WO WO-2010/094040 8, 2010 US 2016/0312277 A1 Oct. 27, 2016 OTHER PUBLICATIONS Related U.S. Application Data Ambros, et al., “MicroRNAs and Other Tiny Enfogenous RNAs in (60) Division of application No. 14/571,142, filed on Dec. C. elegans'. Current Biology, vol. 13, Issue 10, May 13, 2003, 15, 2014, now Pat. 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Bentwich, et al., “Identification of hundreds of conserved and 14, 2008, provisional application No. 61/034,833, nonconserved human micro RNAs'. Nature Genetics, vol. 37, No. filed on Mar. 7, 2008, provisional application No. 7, Jun. 19, 2005, 766-770. 61/039,460, filed on Mar. 26, 2008, provisional (Continued) application No. 61/047,549, filed on Apr. 24, 2008. Primary Examiner — Kenneth Horlick (51) Int. Cl. CI2P 19/34 (2006.01) (57) ABSTRACT CI2O I/68 (2006.01) Compositions, methods, and kits for detecting one or more CI2N IS/10 (2006.01) species of RNA molecules are disclosed. In one embodi (52) U.S. Cl. ment, a first adaptor and a second adaptor are ligated to the CPC ....... CI2O I/6855 (2013.01); C12N 15/1096 RNA molecule using a polypeptide comprising double (2013.01) Strand specific RNA ligase activity, without an intervening (58) Field of Classification Search purification step. The ligated product is reverse transcribed, USPC ...................................... 435/91.1, 6.12, 91.2 then at least some of the ribonucleosides in the reverse See application file for complete search history. transcription product are removed. Primers are added and amplified products are generated. In certain embodiments, (56) References Cited the sequence of at least part of at least one species of U.S. PATENT DOCUMENTS amplified product is determined and at least part of the corresponding RNA molecule is determined. In some 5,185,243 A 2f1993 Ullman et al. embodiments, at least Some of the amplified product species 6,013,445. A 1/2000 Albrecht et al. are detected, directly or indirectly, allowing the presence 6,506,561 B1 1/2003 Cheval et al. 6,511,810 B2 1/2003 Bi et al. and/or quantity of the RNA molecule of interest to be 6,797.470 B2 9/2004 Barany et al. determined. 6,806,049 B1 10/2004 Maekawa et al. 7,153,658 B2 12/2006 Andersen et al. 20 Claims, 16 Drawing Sheets US 9,624,534 B2 Page 2 (56) References Cited Ho, et al., “Structure and Mechanism of RNA Ligase”. Structure, Vo. 12, 2004, 327-339. Hutvagner, "Small RNA asymmetry in RNAi: Function in RISC OTHER PUBLICATIONS assembly and gene regulation’, FEBS Letters, vol. 579, 2005, Berezikov, et al., “Approaches to microRNA discovery”. Nature 5850-5857. Genetics Supplement, vol. 38, Supplemental, Jun. 2006, 2-7. Illumina, Inc., “Preparing Samples for Analysis of Small RNA'. Berezikov, et al., “Diversity of microRNAs in human and chim Part # 1125 1913 Rev. 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