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 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.