NUCLEIC ACID AMPLIFICATION PROCESSES
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) causing 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 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 the temperature at which the method is performed is non-isothermal, and subject to a reduction of at least 2° C. during the amplification process of steps (b)-(d).
1 . A method of performing a non-isothermal nucleic acid amplification method comprising:
(a) incubating a reaction mixture comprising a target nucleic acid and one or more complementary single stranded primers at a first temperature, T 1 , which permits a hybridisation event in which the one or more primers hybridize 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;
(b) cooling the temperature of the reaction mixture to a second temperature, T 2 , which is at least 2° C. lower than T 1 ;
(c) using a nicking enzyme to cause a nick at each of said nicking sites in the strands of the duplex;
(d) using a polymerase to extend the nicked strands so as to form newly synthesized nucleic acid, which extension with the polymerase creates nicking sites;
(e) repeating steps (c) and (d) so as to cause the production of multiple copies of the newly synthesized nucleic acid;
wherein after step (b), the temperature does not return to the first temperature.
2 .- 31 . (canceled)
32 . The method of claim 1 , wherein the cooling is by application of active cooling to the reaction mix.
33 . The method of claim 1 , wherein the cooling is achieved by passive means.
34 . The method of claim 1 , wherein the cooling is achieved by a combination of active and passive means.
35 . The method of claim 1 , wherein the reaction mix is cooled during step (b) by at least 15° C.
36 . The method of claim 1 , wherein the temperature T 1 in step (a) is in the range 55-62° C.
37 . The method of claim 1 , wherein steps (b)-(e) are performed substantially immediately after step (a), and wherein steps (a)-(e) are performed in the same reaction vessel or on the same solid support.
38 . The method of claim 1 , wherein step (c) is initiated by formation of the duplex structure which comprises two nicking sites disposed at or near opposite ends of the duplex.
39 . The method of claim 1 , further comprising a step of contacting a mixture obtained by performance of the method with a thermolabile enzyme which degrades nucleic acids, the mixture being contacted with the thermolabile enzyme at a temperature at which the thermolabile enzyme is substantially active.
40 . The method of claim 1 , further comprising a step of directly or indirectly detecting the newly synthesized nucleic acid.
41 . The method of claim 1 , wherein step (e) is performed while further cooling the reaction mix.
42 . The method of claim 41 , wherein the reaction mix is cooled during step (b) and/or step (e) by at least 15° C.
43 . The method of claim 41 , wherein the cooling is by application of active cooling to the reaction mix.
44 . The method of claim 41 , wherein the cooling is achieved by passive means.
45 . The method of claim 41 , wherein the cooling is achieved by a combination of active and passive means.
46 . The method of claim 41 , wherein the temperature T 1 in step (a) is in the range 55-62° C.
47 . The method of claim 41 , wherein steps (b)-(e) are performed substantially immediately after step (a), and wherein steps (a)-(e) are performed in the same reaction vessel or on the same solid support.
48 . The method of claim 41 , comprising use of a first polymerase and/or a first nicking enzyme having an optimum temperature, and a second polymerase and/or a second nicking enzyme having an optimum temperature, wherein the optimum temperature of the second polymerase and/or second nicking enzyme is lower than the optimum temperature of the respective first polymerase and/or first nicking enzyme.
49 . The method of claim 41 , further comprising the step of contacting a mixture obtained by performance of the method with a thermolabile enzyme which degrades nucleic acid, the mixture being contacted with the thermolabile enzyme at a temperature at which the thermolabile enzyme is substantially active.
50 . The method of claim 41 , further comprising a step of directly or indirectly detecting the newly synthesized nucleic acid.