Amplification of nucleic acids

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 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), 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.

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 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 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), 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; 
       wherein the temperature of the reaction mixture is held constant at the upper temperature for upper temperature dwell time and/or the reaction mixture is held constant at the lower temperature for a lower temperature dwell time. 
     
     
         2 - 31 . (canceled) 
     
     
         32 . The method according to  claim 1 , wherein the upper temperature dwell time and the lower temperature dwell time are the same. 
     
     
         33 . The method according to  claim 1 , wherein the upper temperature dwell time and the lower temperature dwell time are different. 
     
     
         34 . The method according to  claim 32 , wherein the upper temperature dwell time is less then the lower temperature dwell time. 
     
     
         35 . The method according to  claim 32 , wherein the upper temperature dwell time is greater than the lower temperature dwell time. 
     
     
         36 . The method according to  claim 1 , wherein the upper temperature dwell time and/or the lower temperature dwell time are varied during the plurality of temperature shuttles. 
     
     
         37 . The method according to  claim 1 , wherein the transition times between the upper temperature and the lower temperature and between the lower temperature and the upper temperature are substantially the same for the plurality of temperature shuttles. 
     
     
         38 . The method according to  claim 1 , wherein the transition time between the upper temperature and the lower temperature is varied across the plurality of temperature shuttles. 
     
     
         39 . The method according to  claim 1 , wherein the transition time between the lower temperature and the upper temperature is varied across the plurality of temperature shuttles. 
     
     
         40 . The method according to  claim 1 , wherein the upper temperature is in the range 50-68° C. 
     
     
         41 . The method according to  claim 1 , wherein the lower temperature is in the range 20.0-58.5° C. 
     
     
         42 . The method according to  claim 1 , wherein the temperature shuttling is performed continuously for a plurality of shuttles and over a period of at least two minutes. 
     
     
         43 . The method according to  claim 1 , wherein each of the plurality of temperature shuttles has a duration in the range 5-60 seconds. 
     
     
         44 . The method according to  claim 1 , wherein each of the plurality of temperature shuttles has a dwell time at the upper temperature in the range 1-10 seconds. 
     
     
         45 . The method according to  claim 1 , wherein each of the plurality of temperature shuttles has a dwell time at the lower temperature in the range 2-40 seconds. 
     
     
         46 . The method according to  claim 1 , wherein each of the plurality of temperature shuttles has a transition time between the lower temperature and the upper temperature in the range seconds. 
     
     
         47 . A method of determining the amount and/or concentration of a target polynucleotide in a sample, the method comprising the steps of:
 performing the amplification reaction of  claim 1  to amplify the target polynucleotide in the sample; and   detecting, in a quantitative manner, the direct or indirect product(s) of the amplification reaction, so as to allow a determination of the amount and/or concentration of the target polynucleotide in the sample.