Dried compositions containing flap endonuclease
View Patent ↗There is disclosed a composition of an aqueous solution comprising, consisting or consisting essentially of a flap endonuclease, a bulking agent and an organic buffer, wherein the aqueous solution has an inorganic salt concentration of 5 mM or less and wherein the composition is substantially free of glycerol.
1. A composition of an aqueous solution comprising a flap endonuclease, a bulking agent, and a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer or a tris(hydroxymethyl)aminomethane (Tris) buffer, wherein the aqueous solution has an inorganic salt concentration of 5 mM or less and wherein the composition is substantially free of glycerol.
2. The composition of claim 1 wherein the flap endonuclease is a Cleavase.
3. The composition of claim 2 , wherein the aqueous solution further comprises one or more of: at least one detection probe, at least one probe oligonucleotide, at least one invader probe, at least one signalling probe at least one signalling probe containing a flap region, at least one FRET probe, and at least one target capture probe.
4. The composition of claim 3 , wherein the aqueous solution comprises at least one signal probe and at least one invader probe, wherein the at least one signal probe and the at least one invader probe are capable of annealing to a target nucleic acid to form a three-dimensional structure that can be recognized by the flap endonuclease.
5. The composition of claim 1 , further comprising at least one polymerase selected from the group consisting of: a hot-start polymerase, Taq DNA polymerase, Taq polymerase wherein the polymerase is bound by an antibody that specifically blocks polymerase activity of the polymerase, a chemically modified recombinant Taq DNA polymerase wherein the chemical modification inhibits polymerase activity of the polymerase, a reverse transcriptase, an AMV reverse transcriptase, and an MMLP reverse transcriptase; wherein the polymerase is present at a concentration of about 0.1 U/μl to about 4.0 U/μl, about 0.10 U/μl to about 0.2 U/μl, about 0.14 U/μl, about 0.146 U/μl, or about 0.1686 U/μl.
6. The composition of claim 1 , wherein the bulking agent is trehalose, wherein the trehalose is present at a concentration of about 0.2 M to about 0.5 M, about 0.2 M to about 0.4 M, about 100 mM to about 300 mM , about 300 mM, about 0.36 M, or about 0.47 M.
7. The composition claim 1 , wherein the inorganic salt is: (a) sodium chloride, wherein the sodium chloride is present at a mass per microliter concentration of less than 1 mM, about 0.029 μg/μl to about 0.35 μg/μl, about 0.32 μg/μl, 0.292 μg/μl or less, 0.234 μg/μl or less, 0.175 μg/μl or less, 0.146 μg/μl or less, 0.117 μg/μl or less, 0.088 μg/μl or less, 0.058 μg/μl or less, 0.029 μg/μl or less, or 0.015 μg/μl or less; and/or (b) potassium chloride, wherein the potassium chloride is present at a mass per microliter of less than 1 mM, about 0.373 μg/μl to about 0.019 μg/μl, 0.373 μg/μl or less, 0.298 μg/μl or less, 0.224 μg/μl or less, 0.186 μg/μl or less, 0.149 μg/μl or less, 0.112 μg/μl or less, 0.075 μg/μl or less, 0.037 μg/μl or less, or 0.019 μg/μl or less.
8. The composition of claim 7 , wherein the aqueous solution does not contain sodium chloride.
9. The composition of claim 1 , wherein the aqueous solution contains less than 1 mM magnesium ions or less than 0.1 mM magnesium ions.
10. The composition of claim 1 , wherein the aqueous solution further comprises one or more of dATP, dGTP, dCTP, dTTP, and dTUP; wherein the dATP, dGTP, and/or dCTP, if present, are each at a concentration of about 0.1 mM to about 0.4 mM, about 0.29 to about 0.46 mM, about 0.3 mM to about 0.4 mM, or about 0.375 mM; the dTTP, if present, is at a concentration of about 0.1 mM to about 0.4 mM, about 0.2 to about 0.37 mM, about 0.3 mM to about 0.4 mM, or about 0.284 mM; and the dUTP, if present, is at a concentration of about 0.1 mM to about 0.4 mM, about 0.125 to about 0.234 mM, about 0.3 mM to about 04 mM, or about 0.182 mM.
11. The composition of claim 1 , wherein the flap endonuclease is present in the aqueous solution at about 0.020 μg/μl to about 0.040 μg/μl, about 0.030 μg/μl to about 0.04 μg/μl, or about 0.030 μg/μl to about 0.035 μg/μ1.
12. The composition of claim 1 , wherein the buffer is the MOPS buffer and present at a concentration of 10-20 mM, 12.5-20 mM, 12.5-15 mM, 15 mM, or 12.5 mM.
13. The composition of claim 1 , wherein the buffer is the Tris buffer and present at a concentration of 5-60 mM, 10-30 mM, 40-60 mM, 50 mM, 20 mM, or 10 mM.
14. The composition of claim 1 , wherein the composition contains a globular protein.
15. The composition of claim 14 , wherein the globular protein is selected from the group consisting of: bovine serum albumin (BSA), non-acetylated BSA, and ultrapure nonacetylated BSA.
16. The composition of claim 1 , wherein the flap endonuclease is a Cleavase® enzyme, the bulking agent is trehalose, and the buffer is the MOPS buffer, wherein the Cleavase® is present at about 0.030-0.035 μg/μl, the trehalose is present at about 0.3-0.36 M, the MOPS buffer is present at about 12.5-15 mM, and wherein the composition further comprises dNTPs at a concentration of about 0.3-0.38 mM each and bovine serum albumin at a concentration of about 0.5 μg/μl.
17. The composition of claim 1 , wherein the aqueous solution further comprises alpha cyclodextrin.
18. A dried form of the composition of claim 1 .
19. A method for preparing a dried composition for use in performing a nucleic acid based assay, the method comprising the steps of: (i) freezing an aqueous solution of claim 1 , thereby forming a frozen form of the aqueous solution; and (ii) exposing the frozen form from step (i) to lyophilization conditions, thereby forming a dried composition.
20. Use of the dried composition according to claim 19 , wherein the dried composition is combined with a reconstitution solution for performing a nucleic acid based assay.