Dna amplification apparatus and method

Abstract

The invention relates to a method for the amplification of DNA and apparatus for such amplification. The method comprises steps typically found in amplification methods but utilises an optical procedure to detect denaturation of DNA or attainment of the desired denaturation temperature. The apparatus ( 1 ) comprises a temperature controllable chamber ( 2 ) including a rotor ( 3 ) for holding a plurality of reaction vessels (see 7 for example) for reaction mixtures including DNA, a drive means for the rotor, a heater ( 8 ) within the chamber for transiently supplying infrared energy to the reaction vessels, and an optical system ( 12 - 16 ) for determining denaturation of at least a reference DNA or for detecting attainment of a desired denaturation temperature in at least a reference reaction vessel.

1 . A method for the amplification of DNA, the method comprising the steps of: 
 i) forming a reaction mixture comprising said DNA, an oligonucleotide primer complementary to at least one strand of said DNA, nucleotides, and a thermostable DNA polymerase;    ii) heating said mixture to denature said DNA with optical detection of denaturation or the desired denaturation temperate;    iii) on detecting denaturation of said DNA or attainment of the denaturation temperature, allowing said mixture to cool to a temperature at which primer anneals to its complementary td;    iv) incubating said mixture at a temperature which allows synthesis of a DNA strand complementary to the strand to which said at least one primer anneals; and    v) repeating steps (ii) to (iv) until the desired level of amplification is attained.    
     
     
         2 . The method according to  claim 1 , wherein said optical detection of denaturation is by measuring the fluorescence emitted by an intercalating fluorophore present in a tube containing a reference DNA or in at least one of the reaction mixtures.  
     
     
         3 . The method according to  claim 2 , wherein said reference DNA has a similar melting temperature to that of the DNA being amplified.  
     
     
         4 . The method according to  claim 1 , wherein said optical detection of denaturation utilises a thermochromic liquid crystal.  
     
     
         5 . The method according to  claim 4 , wherein thermochromic liquid crystal has a clearing point or a color transition point within the range of 92-95° C.  
     
     
         6 . The method according to  claim 1 , wherein in step (iii) cooling is aided by supplying a cooling agent to the environment of said reaction mixtures.  
     
     
         7 . The method according to  claim 1 , wherein said annealing temperature is within the range of 50-65° C.  
     
     
         8 . The method according to  claim 1 , wherein said steps (ii) to (iv) are repeated at least twenty times  
     
     
         9 . The method according to  claim 1 , wherein said amplification of DNA is linear or expomential.  
     
     
         10 . Apparatus for the amplification of DNA in a reaction mixture, the apparatus comprising: 
 a temperature controllable chamber including a rotor for holding a plurality of reaction vessels for reaction mixtures including DNA;    a drive means for said rotor;    a heater within said chamber for transiently supplying infrared energy to said reaction vessels; and    an optical system for determining denaturation of at least a reference DNA or for detecting attainment of a desired denaturation temperature in at least a reference reaction vessel.    
     
     
         11 . The apparatus according to  claim 10 , wherein said temperature control of said apparatus chamber is effected by providing a heater linked to a temperature sensor.  
     
     
         12 . The apparatus according to  claim 10 , wherein said apparatus further includes a fan for circulating heated air within said chamber.  
     
     
         13 . The apparatus according to  claim 10 , wherein said apparatus includes a cooling system.  
     
     
         14 . The apparatus according to  claim 10 , wherein said rotor is a flat disc with an annular ring forming an outward portion thereof which is angled upwardly and has apertures therein for holding a plurality of reaction vessels.  
     
     
         15 . The apparatus according to  claim 10 , wherein said drive means is selected from a direct-coupled AC motor, a DC motor, or an AC motor that drives said rotor via a gearbox or pulleys.  
     
     
         16 . The apparatus according to  claim 10 , wherein said infrared heater is capable of delivering at least 100 watts.  
     
     
         17 . The apparatus according to  claim 10 , wherein said infrared heater is located at the bottom of said apparatus chamber.  
     
     
         18 . The apparatus according to  claim 10 , wherein said infrared heater is mounted on ceramic insulators fixed to a metal reflector plate located at the bottom of said apparatus chamber.  
     
     
         19 . The apparatus according to  claim 10 , wherein said optical system comprises a light source and detector.  
     
     
         20 . The apparatus according to  claim 10 , wherein said light source is selected from an LED, a laser light source, or a halogen lamp.  
     
     
         21 . The apparatus according to  claim 10 , wherein fluorescence emitted by a fluorophore included in said optical system is filtered and measured by a photomultiplier tube, CCD array, photodiode, or CCD camera.  
     
     
         22 . The apparatus according to  claim 10 , which further includes a computer for control of said apparatus.  
     
     
         23 . The apparatus according to  claim 22 , wherein said computer is used for control of any one of or any combination of rotor start up and speed, the annealing and polymerisation temperatures and the times thereof, operation of said infrared heater during denaturation of DNA, processing of data generated by said optical system, rotor braking, and cooling of said device chamber.  
     
     
         24 . Use of apparatus according to any one of  claims 10  to  23  for the amplification of DNA by the method of  claim 1.