Patent CA-3086320-A1 (CA)
Title: Improvements in or relating to amplification of nucleic acids
Inventor: SHEN DAIWEI
IPC: C12Q1/6848
Filed: 2019-01-02
Abstract: CA 03086320 2020-06-18 (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) CORRECTED VERSION (19) World Intellectual Property Organization 1 11111 1011111 11 111111 010 11111111111 101110111 0111 100 111011 10 1111 11111111111 1111 1111 International Bureau (10) International Publication Number (43) International Publication Date WO 2019/135074 A8 11 July 2019 (11.07.2019) WIPO I PCT (51) International Patent Classification: don Riverside, London Greater London SE1 2AQ (GB). C12Q 1/6848 (2018.01) PEREZ, Victor; LumiraDx UK Ltd, 3 More LondonRiver-side, London Greater London SE1 2AQ (GB). PROVINS, (21) International Application Number: Jarrod; LumiraDx UK Ltd, 3 More London Ri PCT/GB2019/050005 verside, Lon-don Greater London SE1 2AQ (GB). (22) International Filing Date: (74) Agent: LIPSCOMBE, Martin et al.; Nash Matthews LLP, 02 Janumy 2019 (02.01.2019) 24 Hills Road, Cambridge Cambridgeshire CB2 1JP (GB). (25) Filing Language: English (81) Designated States (unless otherwise indicated, for every (26) Publication Language: English kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (30) Priority Data: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, 1800109.9 04 Janumy 2018 (04.01.2018) GB DZ, EC, EE, EG, ES, FL GB, GD, GE, GH, GM GT, HN, (71) Applicant: LUMIRADX UK LTD [GB/GB]; 3 More Lon-HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, don Riverside, London SE1 2AQ (GB). KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, My MZ, NA, NG, NL NO, NZ, (72) Inventors: SHEN, Daiwei; LumiraDx UK Ltd, 3 More OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, London Riverside, London Greater London SE1 2AQ (GB). KRAYNACK, Bryan; LumiraDx UK Ltd, 3 More Lon-(54) Title: IMPROVEMENTS IN OR RELATING TO AMPLEFICATION OF NUCLEIC ACEDS qSTAR, Example Temperature Profile 64 _______________________________________________ ______________________ 63 ______________________________________________ Initiation Phase: 62 --------------- I 1\ 11 A_ 11 k Polymerase Active r 51 ' = ' ______________________ 112 60 ' = ' , Exponential Phase: 1 59 I I I- I- = ! Nicking Active a 58 I I I I I!rihi41. 41\ '1 r1,1_11 57 _______________________________________________ 56 _______________________________________________ FIG. 2 54 0 1 2 3 ______ 4 5 Time [min) (57) Abstract: Disclosed is a method of performing a non-isothermal nucleic acid amplification reaction, the method comprising the steps of: (a)mixing a target sequence with one or more complementary single stranded primers in conditions which permit a hybridisation = event in which the primers hybridise to the target, which hybridisation event, directly or indirectly, leads to the formation of a duplex = structure comprising two nicking sites disposed at or near opposite ends of the duplex; and performing an amplification process by; (b)using a nicking enzyme to cause a nick at each of said nicking sites in the strands of the duplex; (c)using a polymerase to extend the nicked strands to as to form newly synthesised nucleic acid, which extension with the polymerase recreates nicking sites; (d)repeating steps (b) and (c) as desired so as to cause the production of multiple copies of the newly synthesised nucleic acid; characterised in that Nre h the temperature at which the method is performed is non-isothermM, and subject to shuttling, a plurMity of times, between an upper 0 temperature and a lower temperature during the amplification process of steps (b)-(d), wherein at the upper temperature, one of said polymerase or nicking enzyme is more active than the other of said enzymes, such that there is a disparity in the activity of the enzymes, re) and at the lower temperature the disparity in the activity of the enzymes is reduced or reversed. [Continued on next page] CA 03086320 2020-06-18 WO 2019/135074 A8 I11111 NMI II lIllIl 11111 11111 011MI I II 111 11111 11111 11111 11111 110 ll RIME ll 1111 SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): AREPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Published: ¨ with international search report (Art. 21(3)) ¨ with sequence listing part of description (Rule 5.2(a)) (48) Date of publication of this corrected version: 2'7February 2020 (2'7.02.2020) (15) Information about Correction: see Notice of 2'7February 2020 (2'7.02.2020)