US 11,655,496 B2
Amplification of nucleic acids
Daiwei Shen, London (GB); Bryan Kraynack, London (GB); Victor Perez, London (GB); and Jarrod Provins, London (GB)
Assigned to LumiraDx UK Ltd., London (GB)
Appl. No. 16/956,552
Filed by LUMIRADX UK LTD, London (GB)
PCT Filed Jan. 2, 2019, PCT No. PCT/GB2019/050005
§ 371(c)(1), (2) Date Jun. 19, 2020,
PCT Pub. No. WO2019/135074, PCT Pub. Date Jul. 11, 2019.
Claims priority of application No. 1800109 (GB), filed on Jan. 4, 2018.
Prior Publication US 2021/0292826 A1, Sep. 23, 2021
Int. Cl. C12Q 1/6848 (2018.01); C12Q 1/6851 (2018.01); C12Q 1/6853 (2018.01)
CPC C12Q 1/6848 (2013.01) [C12Q 1/6851 (2013.01); C12Q 1/6853 (2013.01); C12Q 2521/101 (2013.01); C12Q 2521/107 (2013.01); C12Q 2521/307 (2013.01); C12Q 2525/113 (2013.01); C12Q 2525/131 (2013.01); C12Q 2525/301 (2013.01); C12Q 2527/101 (2013.01); C12Q 2537/137 (2013.01)] 26 Claims
1. 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 hybridization event in which the one or more primers hybridizes to the target, which hybridization 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 so as to form newly synthesized nucleic acid, wherein extension with the polymerase recreates the nicking sites; and
d) repeating steps (b) and (c) as desired so as to cause the production of multiple copies of the newly synthesized nucleic acid;
wherein the temperature at which the method is performed is non-isothermal, and subject to shuttling, a plurality of times, between an upper temperature and a lower temperature during the amplification process of steps (b)-(d), and
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, and at the lower temperature the disparity in the activity of the enzymes is reduced or reversed.