Stabilization of nucleic acid amplification cocktails

Abstract

The present invention provides a cocktail of reagents for nucleic acid amplification that are stabilized by inclusion of a reversible inhibitor of undesirable reactions. Such cocktail of reagents eliminates the requirement for separate preparation and quality control of each reagent used in a reaction. Methods to prepare stabilized cocktails and to use stabilized cocktails also are included. The stabilized cocktail compositions also can include reagents to release nucleic acid from cells and to label the nucleic acid, allowing detection of nucleic acid in a sample with a single reagent addition step. The invention also provides kits for performing the above methods.

We claim:  
     
         1 . A composition for performing nucleic acid amplification that avoids undesirable reactions between the individual reagents, comprising one or more reagents necessary to perform nucleic acid amplification and an inhibitory concentration of a reversible inhibitor(s) of the undesirable reaction, wherein: 
 a) the nucleic acid amplification is selected from the group consisting of ligase chain reaction (LCR), transcription mediated amplification (TMA) reaction, nucleic acid sequence based amplification (NASBA) reaction and strand displacement amplification (SDA) reaction; or    b) the inhibitor inhibits nucleic acid amplification; or    c) the inhibitor is not a lipid; or    d) the inhibitor does not compartmentalize the composition.    
     
     
         2 . A method of amplifying a nucleic acid, comprising: 
 adding a nucleic acid template to be amplified to the composition of  claim 1 , and optionally a diluent in sufficient amounts to lower the inhibitor concentration to such that it does not substantially inhibit the amplification reaction; and    incubating the mixture under conditions sufficient to achieve amplification.    
     
     
         3 . A method for preparing a composition for performing nucleic acid amplification that avoids undesirable reactions between individual reagents, comprising mixing one or more reagents necessary to perform nucleic acid amplification with a reversible inhibitor(s) of the undesirable reaction, wherein the inhibitor is added to the composition at a concentration that is inhibitory to the undesirable reaction but at a concentration which will be non-inhibitory when the composition is later diluted for nucleic acid amplification, and wherein: 
 a) the nucleic acid amplification is selected from the group consisting of ligase chain reaction (LCR), transcription mediated amplification (TMA) reaction, nucleic acid sequence based amplification (NASBA) reaction and strand displacement amplification (SDA) reaction; or    b) the inhibitor inhibits nucleic acid amplification; or    c) the inhibitor is not a lipid; or    d) the inhibitor does not compartmentalize the composition.    
     
     
         4 . The composition of  claim 1 , which comprises one or more nucleic acid polymerase(s) or ligase(s), one or more nucleoside triphosphate(s), one or more nucleic acid primer(s) and an amplification buffer.  
     
     
         5 . The composition of  claim 1 , which comprises all the reagents necessary to perform a nucleic acid amplification reaction.  
     
     
         6 . The composition of  claim 1 , wherein said inhibitor is a nucleic acid binding ligand.  
     
     
         7 . The composition of  claim 6 , wherein said nucleic acid binding ligand is an intercalator compound.  
     
     
         8 . The composition of  claim 7 , wherein said intercalator compound is monoadduct forming.  
     
     
         9 . The composition of  claim 8 , wherein said intercalator compound is a furocoumarin or a phenanthridine.  
     
     
         10 . The composition of  claim 9 , wherein said furocoumarin is 4′-aminomethyltrioxsalen (AMT).  
     
     
         11 . The composition of  claim 9 , wherein said furocoumarin is an angelicin derivative.  
     
     
         12 . The composition of  claim 6 , wherein said binding ligand is a non-intercalating compound.  
     
     
         13 . The composition of  claim 12 , wherein said non-intercalating compound is selected from the group consisting of benzimides, netropsins and distamycins.  
     
     
         14 . The method of  claim 3 , wherein said nucleic acid amplification is a transcription based amplification reaction and said inhibitor(s) is a phosphate ion.  
     
     
         15 . The method of  claim 3 , wherein said nucleic acid amplification is a ligase chain reaction and said inhibitor(s) is a phosphate ion at a concentration of 1.25 mM.  
     
     
         16 . The method of  claim 3 , wherein said inhibitor is a nucleic acid binding ligand.  
     
     
         17 . The method of  claim 16 , wherein said nucleic acid binding ligand is an intercalator compound.  
     
     
         18 . The method of  claim 17 , wherein said intercalator compound is 4′-aminomethyltrioxsalen (AMT).  
     
     
         19 . The method of  claim 16 , wherein said nucleic acid binding ligand is a non-intercalator compound.  
     
     
         20 . A kit comprising a vial containing the composition of  claim 1 .  
     
     
         21 . The kit of  claim 20 , wherein the composition comprises one or more nucleic acid polymerase or ligase, one or more nucleoside triphosphate(s), one or more nucleic acid primer(s) and an amplification buffer.  
     
     
         22 . The kit of  claim 20 , wherein said inhibitor is a nucleic acid binding ligand.