Cation chelator hot start

Abstract

The invention is in the field of regulation of enzymatic activity in nucleic acid modifying reactions. It describes a method of regulating enzymatic activity by adding chelating agents to the reaction composition and exploits the fact that both the binding of divalent cations to these chelating agents and the pH of commonly used buffers is temperature dependent. PCR experiments that are hampered by non-specific side products can be regulated such that the target sequence is amplified in a more specific manner.

1 . A kit, comprising:
 (i) a DNA polymerase suitable for nucleic acid amplification, optionally a reverse transcriptase, and   (ii) a buffer comprising: (a) Tris, (b) magnesium divalent cation, and (c) EGTA, wherein when in an amplification reaction mixture, the magnesium divalent cation is at a concentration between 0.01 and 20 mM, and EGTA is at a concentration between 0.1 and 20 mM,   wherein the nucleic acid amplification is not an isothermal nucleic acid amplification.   
     
     
         2 . The kit of  claim 1 , wherein the DNA polymerase is a DNA polymerase from an organism of genus  Thermus, Aquifex, Thermotoga, Thermocridis, Hydrogenobacter, Thermosynchecoccus, Thermoanaerobacter, Pyrococcales, Thermococcus , or  Sulfolobus.    
     
     
         3 . The kit of  claim 1 , wherein the DNA polymerase is a DNA polymerase from an organism organism selected from  Aquifex  aeolicus,  Aquifex pyogenes, Thermus thermophilus, Thermus aquaticus, Thermotoga neopolitana, Thermus pacificus, Thermus eggertssonii  and  Thermotoga maritima.    
     
     
         4 . The kit of  claim 1 , wherein the DNA polymerase is  Thermus aquaticus  (Taq) DNA polymerase. 
     
     
         5 . The kit of  claim 1 , wherein the kit comprises a reverse transcriptase. 
     
     
         6 . The kit of  claim 5 , wherein the reverse transcriptase is a viral reverse transcriptase. 
     
     
         7 . The kit of  claim 6 , wherein the viral reverse transcriptase (RT) is Moloney murine leukemia virus (MMLV) RT, avian myeloblastosis virus (AMV) RT, human immunodeficiency virus (HIV) RT, or equine infectious anemia virus (EIAV) RT. 
     
     
         8 . The kit of  claim 1 , wherein when in an amplification reaction mixture, the magnesium divalent cation is at a concentration between 0.1 and 10 mM. 
     
     
         9 . The kit of  claim 1 , wherein when in an amplification reaction mixture, EGTA is at a concentration between 0.5 and 10 mM. 
     
     
         10 . The kit of  claim 1 , wherein when in an amplification reaction mixture, EGTA is at a concentration between 1 and 8 mM. 
     
     
         11 . The kit of  claim 1 , further comprising dNTPs.