Freeze-dried composition
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.
1. A dried composition having 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) stachyose;
wherein the dried composition is free of gelatin and is in a freeze-dried form, and the polymerase is a thermostable polymerase.
2. The dried composition of claim 1 wherein the polymerase is a reverse-transcriptase or is chemically modified or blocked so that it is inactivated until subject to an initial heat activation step.
3. The dried composition of claim 1 which is for carrying out a polymerase chain reaction and which further comprises at least one of the following further reagents:
(i) a buffer;
(ii) a metal salt;
(iii) nucleotides which may be fluorescently labelled;
(iv) a blocking compound;
(v) an anti-oxidant and/or anti-maillard reagent;
(vi) a primer useful in a polymerase chain reaction which is optionally labelled with a fluorescent label;
(vii) a probe useful in detection of a polymerase chain reaction which is optionally labelled with one or more fluorescent labels;
(viii) a fluorescent dye;
(ix) 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;
(x) a pyrophosphate salt and a pyrophosphatase enzyme;
(xi) a nucleic acid that is able to act as an internal control for an amplification reaction;
(xii) a primer that is able to amplify the nucleic acid of item (xi); or
(xiii) an isothermal polymerase.
4. The dried composition of claim 3 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.
5. 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.
6. A kit comprising the dried composition of claim 1 and a second composition comprising a reagent for use in a reaction with said polymerase.
7. The kit of claim 6 wherein the second composition is in freeze-dried form.
8. The kit of claim 7 wherein said second composition comprises stachyose and a reagent mixture for use in a reaction with said polymerase.
9. A method for preparing the dried composition of claim 1 , wherein the method comprises forming a solution by combining the polymerase and stachyose and optionally one or more further components, and freeze-drying the solution.
10. The method of claim 9 wherein the polymerase is in the form of a solution from which glycerol has been substantially removed.
11. The method of claim 10 wherein at least one of a detergent, an antioxidant or an anti-reducing agent is added to the polymerase after removal of glycerol and before combination with stachyose.
12. The method of claim 9 which comprises one or more further components and wherein the method comprises (i) freezing a first solution of stachyose and either one or more of said further components or the polymerase to form a first frozen layer, (ii) adding a second solution of stachyose and a different one of said further components or the polymerase and freezing the second solution into a second frozen layer, (iii) optionally repeating step (ii) until all components of the composition are present in distinct frozen layers, and (v) freeze-drying the resultant structure.
13. The method of claim 9 wherein the solution is divided amongst multiple reaction vessels so that each vessel contains sufficient reagents to carry out a single reaction using the polymerase, and freeze-dried within said vessels.
14. A method for carrying out a chemical or biochemical reaction using a polymerase, said method comprising forming a reaction mixture by addition of a liquid and any additional components necessary for carrying out said reaction to the dried composition of claim 1 , and subjecting the thus formed solution to suitable reaction conditions.
15. The method of claim 14 wherein the chemical or biochemical reaction is a polymerase chain reaction.
16. A method for enhancing the stability of a composition comprising a polymerase, said method comprising freeze-drying a mixture comprising the polymerase with stachyose as a glass forming agent, wherein the mixture is free of gelatine.