Patent SG-11201903369P-A (SG)
Title: Amplification of nucleic acids using exonuclease and strand displacement
Inventor: PATSOS GEORGIOS
IPC: C12Q1/68
Filed: 2017-10-16
Abstract: INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property C.--.` 1111111111111111111111111111111111111111111111111111111111111111111111111111111111 Organization International Bureau (10) International Publication Number 03 (43) International Publication Date ......P' WO 2018/069737 Al 19 April 2018 (19.04.2018) WIP0 1 PCT (51) International Patent Classification: DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, C12Q 1/68 (2018.01) HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, (21) International Application Number: KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, PCT/GB2017/053128 MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (22) International Filing Date: SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, 16 October 2017 (16.10.2017) TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every (26) Publication Language: English kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, (30) Priority Data: UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, 1617491.4 14 October 2016 (14.10.2016) GB TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FL FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (71) Applicant: REVOLUGEN LIMITED [GB/GB]; Unit MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, 4, Rossington Place, Graphite Way, Hadfield Derbyshire TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, SK13 1QG (GB). KM, ML, MR, NE, SN, TD, TG). (72) Inventors: PATSOS, Georgios; Unit 4, Rossington Place, Graphite Way, Hadfield Derbyshire SK13 1QG (GB). Declarations under Rule 4.17: MINTER, Stephen John; Unit 4, Rossington Place, — of inventorship (Rule 4.17(iv)) — Graphite Way, Hadfield Derbyshire SK13 1QG (GB). Published: Agent: MARKS & CLERK LLP; 1 New York Street, — with international search report (Art. 21(3)) (74) = Manchester Greater Manchester M1 4HD (GB). Designated States (unless otherwise indicated, for every (81) = _ kind of national protection available): AE, AG, AL, AM, — AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, _ CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, = Title: AMPLIFICATION OF NUCLEIC ACIDS USING EXONUCLEASE AND STRAND DISPLACEMENT (54) = (57) : There is provided a method of amplifying a nucleic acid se- = 101 quence. The method comprises providing an amplification mixture compris- 104 102 106 •—•—• 105 3' end sequence ing an exonuclease capable of digestion of a strand of a double stranded nude- 7 , primer A 11 ) / i 5 end primer sequence is acid molecule from the 5'-end towards the 3'-end, a strand displacing poly- I - .1 . = - = merase, a double stranded nucleic acid molecule comprising first and second I On ! I • i 103 /-/ J -- --- 5 .108= 4 : l nucleic acid strands, a first nucleic acid primer, and nucleotides as appropriate 0 os = ' Xexcnuclease action i 7 to provide for amplification of the first nucleic acid sequence to be amplified. i 02d 1 i — = I \ I : The method further comprises effecting the amplification reaction under con- = ---113 1 I H ditions permitting digestion, exonuclease digestion and strand displacement 112 .:../ 103d 1 1 polymerisation thereby producing a product mixture comprising an amplified = 106 110 1 amount of said first nucleic acid sequence. There is also provided a method ---- ---4,_ . . 102d : Polymerase and iwimeral of determining the presence or quantity of target nucleic acid sequence in a = = 1 I C biological sample using a double stranded probe having a fluorophore on one I Ti 112 I ''--113 its the denaturation and re-hybridisati 1 ! I strand and quencher on other and using 103d : 1 i 4.1.q•t• i on to detect the target. i 114 ll 115 — 11 en 106 ! 102d 106 117 I C:r . i 1 C 116 \ 108! ..1 103d ---.... 0 I I GC Fig. 3b Fig. 3c 11 © ei Fig. 3a C