Freeze-Dried Composition

Abstract

The invention relates to the use of a polysaccharide having at least four saccharide units, such as stachyose, as a glass-forming agent for the freeze-drying of a reaction mixture comprising an enzyme. In particular, the enzyme is a polymerase useful in a nucleic acid amplification reaction such as a Polymerase Chain Reaction.Compositions comprising such polysaccharides as well as methods for preparing them, kits containing them and methods for using them form further aspects of the invention.

What is claimed is: 
     
         1 . A dried composition haying a cake three-dimensional structure which is resistant to collapse in an environment of 75% humidity, the composition comprising:
 (i) a polymerase that is useful in a nucleic acid amplification reaction; and   (ii) a glass-forming agent comprising a polysaccharide having at least 4 saccharide units,   wherein the dried composition is free of gelatin and is in a freeze-dried form, and the polymerase is an isothermal polymerase or a thermostable polymerase.   
     
     
         2 . The composition of  claim 1 , wherein the composition is free of stabilizer polysaccharide. 
     
     
         3 . The composition of  claim 1 , wherein the composition is free of cross-linked polysaccharide. 
     
     
         4 . The composition of  claim 1 , wherein the composition further comprises a buffer selected from the group consisting of Iris, Trizma, HEPES, tricine, and bicine. 
     
     
         5 . The composition according to  claim 1 , wherein the polysaccharide has 4-6 saccharide units. 
     
     
         6 . The composition according to  claim 1 , wherein the polysaccharide is selected from stachyose, verbascose and lycopose. 
     
     
         7 . The dried composition of  claim 1 , which further comprises at least one of the following:
 (i) a metal salt;   (ii) nucleotides which may be fluorescently labelled;   (iii) a blocking compound;   (iv) an anti-oxidant and/or anti-maillard reagent;   (v) a primer useful in an amplification reaction which is optionally labelled with a fluorescent label;   (vi) a probe useful in detection of an amplification reaction which is optionally labelled with one or more fluorescent labels;   (vii) a fluorescent dye;   (viii) an anti-Taq antibody that binds to the active site of the polymerase and inactivates it at ambient temperature, but denatures and dissociates from the enzyme at elevated temperatures;   (ix) a pyrophosphate salt and a pyrophosphatase enzyme;   (x) a nucleic acid hat is able to act as an internal control for an amplification reaction; or   (xi) a primer that is able to amplify the nucleic acid of item (x).   
     
     
         8 . The dried composition of  claim 7 , which is in the form of a freeze-dried cake which comprises two or more distinct layers, and wherein at least one of said further reagents is comprised within one distinct layer within the freeze-dried cake. 
     
     
         9 . The dried composition of  claim 1 , which further comprises a polymeric compound that is able to stabilize a freeze-dried cake, wherein the polymeric compound is selected from the group consisting of polyethylene glycol, polyvinylpyrrolidine, and polysaccharides. 
     
     
         10 . A kit comprising the dried composition of  claim 1 , and a second composition comprising a reagent for use in a reaction with said polymerase. 
     
     
         11 . The kit of  claim 10 , wherein the second composition is in freeze-dried form. 
     
     
         12 . The kit of  claim 10 , wherein said second composition comprises stachyose and a reagent mixture for use in a reaction with said polymerase. 
     
     
         13 . A method for preparing the dried composition of  claim 1 , wherein the method comprises forming a mixture by combining the polymerase and the glass-forming agent and optionally one or more further components, and freeze-drying the mixture to form the dried composition. 
     
     
         14 . The method of  claim 13 , wherein the polymerase is a polymerase solution free of glycerol. 
     
     
         15 . The method of  claim 14 , wherein the isothermal polymerase solution further comprises at least one of a detergent, an antioxidant and an anti-reducing agent. 
     
     
         16 . The method of  claim 15 , wherein the freeze-drying further comprises:
 (i) freezing a first portion of the mixture to form a first frozen layer;   (ii) freezing an additional portion of the mixture to form an additional frozen layer;   (iii) optionally repeating step (ii) until all components of the composition are present in distinct frozen layers; and   (iv) freeze-drying the frozen layers to form the dried composition.   
     
     
         17 . The method of  claim 13 , further comprising dividing the mixture amongst multiple reaction vessels so that each vessel contains sufficient reagents to carry out a single reaction using the polymerase, and wherein the freeze-drying further comprises freeze-drying the divided mixture within said reaction vessels. 
     
     
         18 . A method for carrying out a chemical or biochemical reaction using a polymerase, the method comprising forming a reaction mixture by adding a liquid and optionally one or more additional components for carrying out the reaction to the dried composition of  claim 1 , and subjecting the reaction mixture to suitable reaction conditions. 
     
     
         19 . The method of  claim 18 , wherein the chemical or biochemical reaction is a polymerase chain reaction. 
     
     
         20 . A method for enhancing the stability of a composition comprising a polymerase, said method comprising freeze-drying a mixture comprising the polymerase with a glass forming agent, wherein the mixture is free of gelatine. 
     
     
         21 . A composition of  claim 1 , further comprising an anti-maillard reagent, wherein the anti-maillard reagent optionally comprises threonine. 
     
     
         22 . The composition of  claim 1 , wherein the isothermal polymerase is selected from  M-MuLV reverse transcriptase, AMV reverse transcriptase, and Tth reverse transcriptase. 
     
     
         23 . The composition of  claim 1 , wherein the isothermal polymerase is an isothermal polymerase for an isothermal amplification reaction selected from transcription mediated amplification, nucleic acid sequence-based amplification, signal mediated amplification of RNA technology, strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification of DNA (LAMP), isothermal multiple displacement amplification, helicase-dependent amplification, single primer isothermal amplification, circular helicase-dependent amplification, and whole genome amplification.