Nucleic acid amplification and testing
Methods for amplifying a target nucleic acid by self-sustained amplification methods are described. The methods are designed, in particular, to be carried out without use of specialised lab facilities or instruments. Compositions, lyophilised formulations, and kits for carrying out the methods are also described.
1. A universal lyophilised formulation comprising a polysaccharide, a low molecular weight saccharide, and enzyme activities required for carrying out a self-sustained amplification reaction, wherein the enzyme activities are provided by one or more enzymes, and wherein the formulation does not include cofactors or substrates required for amplification of a target nucleic acid using the enzyme activities.
2. A formulation according to claim 1 , which does not include salt.
3. A formulation according to claim 1 or 2 , wherein the polysaccharide is a dextran or a dextran derivative.
4. A formulation according to any of claims 1 to 3 , wherein the low molecular weight saccharide is trehalose, sucrose, or maltose.
5. A lyophilised formulation comprising a polysaccharide, a low molecular weight saccharide, and enzyme activities required for carrying out a self-sustained amplification reaction, wherein the enzyme activities are provided by one or more enzymes, and wherein the formulation does not include a buffering agent.
6. A formulation according to claim 5 , wherein the polysaccharide is a dextran or a dextran derivative.
7. A formulation according to claim 5 or 6 , wherein the low molecular weight saccharide is trehalose, sucrose, or maltose.
8. A formulation according to claim 5 , wherein the enzyme activities comprise an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, a DNA/RNA duplex-specific ribonuclease, and a DNA-dependent RNA polymerase.
9. A formulation according to claim 5 , which does not include substrates or cofactors for the enzyme activities.
10. A formulation according to claim 9 , which does not include salt.
11. A kit comprising a plurality of different, separate lyophilised formulations wherein each lyophilised formulation is a lyophilised formulation according to claim 1 or 5 .
12. A universal lyophilised formulation comprising a polysaccharide, a low molecular weight saccharide, and enzyme activities required for carrying out a self-sustained amplification reaction, wherein the enzyme activities are provided by one or more enzymes, and wherein the formulation does not include cofactors or substrates required for amplification of a target nucleic acid using the enzyme activities, and wherein the formulation does not include a buffering agent, or the buffering agent is present at a concentration of less than 1 mM.
13. A universal lyophilized formulation according to claim 1 , wherein the enzyme activities comprise an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, a DNA/RNA duplex-specific ribonuclease, and a DNA-dependent RNA polymerase.
14. A universal lyophilized formulation according to claim 12 , wherein the enzyme activities comprise an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, a DNA/RNA duplex-specific ribonuclease, and a DNA-dependent RNA polymerase.