Kits for in vitro RNA transcription and other polynucleotide synthetic reactions

Abstract

The present invention relates to compositions and methods for increasing the yields of polynucleotide synthetic reactions. In particular, it relates to an improved reaction mixture for use in in vitro RNA trancription and in various other enzymatic reactions in which a polynucleotide is synthesised. The reaction mixture uses high concentrations of total nucleotides, in the order of 12 mM to 40 mM, i.e. levels that were previously thought to be inhibitory. Other useful modifications are also disclosed.

What is claimed is:  
     
         1 . A reaction mixture comprising: 
 (a) a total nucleotide concentration of greater than 12 mM;    (b) a molar concentration of Mg ++  that is subsaturating with respect to the molar total nucleotide concentration; and    (c) inorganic pyrophosphatase.    
     
     
         2 . The reaction mixture of  claim 1 , wherein the molar concentration of Mg ++  is not more than 10% greater than the total molar nucleotide concentration.  
     
     
         3 . The reaction mixture of  claim 1 , wherein the molar concentration of Mg ++  is equal to or less than the total molar nucleotide concentration.  
     
     
         4 . The reaction mixture of  claim 3 , wherein the molar concentration of Mg ++  is less than the molar total nucleotide concentration.  
     
     
         5 . The reaction mixture of  claim 1 , further defined as comprising a nucleotide concentration of between about 16 mM and 40 mM.  
     
     
         6 . The reaction mixture of  claim 6 , wherein the nucleotide concentration is between about 20 mM and 40 mM.  
     
     
         7 . The reaction mixture of  claim 1 , wherein at least one nucleotide is present in a form other than as a compound with Na + , Li + , K + , Ba ++ , or NH 4   + .  
     
     
         8 . The reaction mixture of  claim 7 , wherein the nucleotides present are added in the form of a Mg ++ -nucleotide.  
     
     
         9 . The reaction mixture of  claim 7 , wherein the nucleotides present are added in the form of a Tris-nucleotide.  
     
     
         10 . A reaction mixture comprising a free Mg ++  concentration that is equal to or less than 2×10 −4  M.  
     
     
         11 . A-reaction mixture for use in producing a poly-nucleotide, wherein the nucleotides present are added in a form other than as a compound with Na + , Li + , K + , Ba ++ , or NH 4   + .  
     
     
         12 . The reaction mixture of  claim 11 , wherein the nucleotides present are added in the form of a Mg ++ -nucleotide.  
     
     
         13 . The-reaction mixture of  claim 11 , wherein the nucleotides present are added in the form of a Tris-nucleotide.  
     
     
         14 . The reaction mixture of claims  1 ,  10  or  11  further comprising an RNA polymerase enzyme.  
     
     
         15 . The reaction mixture of  claim 14 , wherein the RNA polymerase enzyme is a bacteriophage RNA polymerase.  
     
     
         16 . The reaction mixture of  claim 15 , wherein the RNA polymerase used is T7 RNA polymerase, or T3 RNA polymerase, or SP6 RNA polymerase.  
     
     
         17 . The reaction mixture of  claim 11 , further comprising an DNA polymerase enzyme.  
     
     
         18 . The reaction mixture of  claim 17 , wherein the DNA polymerase used is Taq polymerase, or T7 DNA polymerase, or T4 DNA polymerase, or.Klenow polymerase.  
     
     
         19 . The reaction mixture of claims  1 ,  10  or  11 , wherein the inorganic pyrophosphatase is a thermostable pyrophosphatase.  
     
     
         20 . A method for preparing a polynucleotide, comprising: 
 (a) preparing a reaction mixture in accordance with claims  1 ,  10  or  11 ;    (b) adding to the mixture a polynucleotide template and polymerase enzyme; and    (c) incubating the reaction mixture.    
     
     
         21 . The method of  claim 20 , wherein: 
 (a) the inorganic pyrophosphatase is a thermostable pyrophosphatase; and    (b) the amplification synthesis of the polynucleotide is directed by the use of two primers that hybridize to spatially separated sequences within the template and where the product of each primer provides a template for the other.    
     
     
         22 . The method of  claim 20 , wherein the amplification synthesis of the polynucleotide uses both DNA and RNA polymerases in alternate cycles of DNA and RNA synthesis.  
     
     
         23 . The method of  claim 20 , wherein the RNA template contains recognition sequences for QB replicase.  
     
     
         24 . A method for preparing a polynucleotide, comprising: 
 (a) preparing a reaction mixture in accordance with claims  1 ,  10  or  11 ;    (b) adding two oligonucleotides and a ligase enzyme; and    (c) incubating the reaction mixture.    
     
     
         25 . The method of  claim 23  wherein the amplification synthesis of the polynucleotide comprises a ligase chain reaction.  
     
     
         26 . The method of  claim 23 , wherein the concentration of ATP is greater than the concentration of Mg ++ .  
     
     
         27 . The methods of claims  20  or  24 , wherein the polynucleotide produced is a.polyribonucleotide.  
     
     
         28 . The methods of claims  20  or  24  wherein the polynucleotide produced is a polydeoxyribonucleotide.  
     
     
         29 . A kit for producing a polynucleotide, the kit comprising: 
 (a) a suitably aliquoted reaction mixture in accordance with claims  1 ,  10  or  11 , or a mixture which, when admixed, diluted or brought into solution, will provide such a reaction mixture; and    (b) a suitably aliquoted polymerase enzyme.