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(12) United States Patent (10) Patent No.: US 9,546,389 B2 Li (45) Date of Patent: Jan

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(12) United States Patent (10) Patent No.: US 9,546,389 B2 Li (45) Date of Patent: Jan USOO9546,389 B2 (12) United States Patent (10) Patent No.: US 9,546,389 B2 Li (45) Date of Patent: Jan. 17, 2017 (54) METHODS AND SYSTEMS FOR NUCLEIC (56) References Cited ACIDAMPLIFICATION U.S. PATENT DOCUMENTS (71) Applicant: Coyote Bioscience Co., Ltd., Beijing 5,759,821. A * 6/1998 Teasdale .............. C12O 1/6806 (CN) 435,912 2004/0209331 A1* 10, 2004 Ririe ....................... BO1L 3,505 435,912 (72) Inventor: Xiang Li, Beijing (CN) 2008/0085541 A1 4/2008 Spangler 2012/0308990 A1* 12/2012 TeMaat ..................... BO1L, 7,52 (73) Assignee: COYOTE BIOSCIENCE CO.,LTD., 435/3 Beijing (CN) 2013/0022963 A1 1/2013 Exner et al. FOREIGN PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 CN 101597652. A 12/2009 U.S.C. 154(b) by 0 days. CN 103074349 A 5, 2013 JP EP 1069 190 A2 * 1 2001 ............. C12O 1/686 WO WO-2012109604 A1 8, 2012 (21) Appl. No.: 14/963,986 OTHER PUBLICATIONS (22) Filed: Dec. 9, 2015 Callahan et al. Use of a portable real-time reverse transcriptase (65) Prior Publication Data polymerase chain reaction assay for rapid detection of foot-and mouth disease virus. JAVMA 220: 1636-1642 (2002).* US 2016/O115513 A1 Apr. 28, 2016 Gilbert et al. Typing of bovine viral diarrhea viruses directly from blood of persistently infected cattle by multiplex PCR. Journal of Clinical Microbiology 37:2020-2023 (1999).* International search report and written opinion dated Feb. 26, 2015 Related U.S. Application Data for PCT Application No. CN2014/094914. International search report and written opinion dated Nov. 26, 2014 (63) Continuation of application No. for PCT Application No. CN2013/090425. PCT/CN2014/094914, filed on Dec. 25, 2014, which is a continuation-in-part of application No. * cited by examiner PCT/CN2013/090425, filed on Dec. 25, 2013. Primary Examiner — Samuel Woolwine (74) Attorney, Agent, or Firm — Wilson, Sonsini, (51) Int. Cl. Goodrich & Rosati CI2O I/68 (2006.01) (57) ABSTRACT CI2P 19/34 (2006.01) The present disclosure provides methods and systems for CI2O 1/70 (2006.01) amplifying nucleic acid samples. In some aspects, the meth GOIN 2L/64 (2006.01) ods and systems described provided can be useful in con BOIL 7/00 (2006.01) ducting multiple nucleic acid amplification reactions in (52) U.S. Cl. parallel. In some embodiments, methods and systems pro CPC .................. CI2P 19/34 (2013.01); B0IL 7/52 vided herein can be useful in conducting reverse transcrip (2013.01); C12O 1/68 (2013.01): CI2O I/686 tion and DNA amplification in parallel. Moreover, in some (2013.01); C12O 1/6862 (2013.01); C12O aspects, the methods and systems described herein can be I/70 (2013.01); G0IN 2 1/64 (2013.01); BOIL useful in analysis of nucleic acid samples. In some embodi 2200/14 (2013.01); B0IL 2300/027 (2013.01) ments, methods and systems provided herein can be useful (58) Field of Classification Search for conducting multiple series of primer extension reactions, None which can aid in analysis of a nucleic acid sample. See application file for complete search history. 29 Claims, 46 Drawing Sheets U.S. Patent Jan. 17, 2017 Sheet 1 of 46 US 9,546,389 B2 107 13 s’ 1) . Input 1 08 N. : Devices / 11 \ ? - --------------------- x ------------------- F ^ Computer Network 9 Eiectronic O) Dispiay Computer : Interface Printer He-109 1. 1. U.S. Patent Jan. 17, 2017 Sheet 2 of 46 US 9,546,389 B2 zºo?sezedeezºzzcz_0zen91yiz?ol89wz_0, Þz'81-I goiaqueuoduopa?n?, %%****************** 10000G/', (0000G/ 1000'009 1000OGZ e3u80SelOn. U.S. Patent Jan. 17, 2017 Sheet 7 of 46 US 9,546,389 B2 00zl? 0000ZI? 00000ZIl zl?-. ºf'81-I eOueoSejon |OJ?UOOennebau,000'09 U.S. Patent Jan. 17, 2017 Sheet 8 of 46 US 9,546,389 B2 &sississŠs: xxxx ex wk YYYYYYYY ******** & - s & S S S S ww.xx xxxi-xxx-xxxx xxxx S U.S. Patent Jan. 17, 2017 Sheet 9 of 46 US 9,546,389 B2 07 z-Hd,--???|,000:09,?- ?000’009 )|--|:000’00”|--.--() 000ogy},,, ,000oseL-H.dº--’’...--- ?000 ,!?? 10000Sz?- -|000'00Z -||-. -:-::|000‘099%--::::- ~~!000'00|| -|000’09) e3ueOSeOn. U.S. Patent Jan. 17, 2017 Sheet 10 of 46 US 9,546,389 B2 qolaquæuoduooninae 00‘006 00’008 00'00/ eOue3SeOn U.S. Patent Jan. 17, 2017 Sheet 11 of 46 US 9,546,389 B2 Z-Hc] ?o?em?uauoduooninwa VZ(81-I 0$89#7Z 000'009 000'099 000‘00G 000'097 80UeOS8. 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U.S. Patent Jan. 17, 2017 Sheet 27 of 46 US 9,546,389 B2 U.S. Patent Jan. 17, 2017 Sheet 28 of 46 US 9,546,389 B2 s Sw &SSSSSXXXXX: Fig. 18 U.S. Patent Jan. 17, 2017 Sheet 29 of 46 US 9,546,389 B2 &ax- Maxxxxxxxxsssy Fig. 19A U.S. Patent Jan. 17, 2017 Sheet 30 of 46 US 9,546,389 B2 Fig. 19B U.S. Patent Jan. 17, 2017 Sheet 31 of 46 US 9,546,389 B2 Fig. 20 U.S. Patent Jan. 17, 2017 Sheet 32 of 46 US 9,546,389 B2 Fig. 21 U.S. Patent Jan. 17, 2017 Sheet 33 of 46 US 9,546,389 B2 six-st: Fig. 22A U.S. Patent Jan. 17, 2017 Sheet 34 of 46 US 9,546,389 B2 {...or sex Fig. 22B U.S. Patent Jan. 17, 2017 Sheet 35 of 46 US 9,546,389 B2 Fig. 23A Patent Jan. 17, 2017 Sheet 36 of 46 US 9,546,389 B2 assa&ss Saaaaaaaaaaaaaaaaaas Fig. 23B U.S. Patent Jan. 17, 2017 Sheet 37 of 46 US 9,546,389 B2 S. y:yyyyyyyyyyyyyyyyy six Fig. 23 U.S. Patent Jan. 17, 2017 Sheet 38 of 46 US 9,546,389 B2 Fig. 24A U.S. Patent Jan. 17, 2017 Sheet 39 of 46 US 9,546,389 B2 F. g. 24B U.S. Patent Jan. 17, 2017 Sheet 40 of 46 US 9,546,389 B2 ar www.www. ww.s- U.S. Patent Jan. 17, 2017 Sheet 41 of 46 US 9,546,389 B2 U.S. Patent Jan. 17, 2017 Sheet 42 of 46 US 9,546,389 B2 Arpiitication Fig. 26A U.S. Patent Jan. 17, 2017 Sheet 43 of 46 US 9,546,389 B2 . U.S. Patent Jan. 17, 2017 Sheet 44 of 46 US 9,546,389 B2 &xxx&saxxaaaaaaasaxas isssssssssssssessex's U.S. Patent Jan. 17, 2017 Sheet 45 of 46 US 9,546,389 B2 Fig. 28A U.S. Patent Jan. 17, 2017 Sheet 46 of 46 US 9,546,389 B2 Fig. 28B US 9,546,389 B2 1. 2 METHODS AND SYSTEMIS FOR NUCLEC polymerase, and (iii) a primer set for the target RNA, to ACIDAMPLIFICATION obtain a reaction mixture; and (b) Subjecting the reaction mixture in the reaction vessel to multiple cycles of a primer CROSS-REFERENCE extension reaction to generate amplified DNA product that is indicative of the presence of the target RNA, each cycle This application is a continuation of Patent Cooperation comprising (i) incubating the reaction mixture at a denatur Treaty Application No. PCT/CN2014/094914, filed on Dec. ing temperature for a denaturing duration that is less than or 25, 2014, which is a continuation-in-part of Patent Coop equal to 60 seconds, followed by (ii) incubating the reaction eration Treaty Application No. PCT/CN2013/090425, filed mixture at an elongation temperature for an elongation on Dec. 25, 2013, said applications are incorporated herein 10 duration that is less than or equal to 60 seconds, thereby by reference in their entireties for all purposes.
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