Dried amplification compositions
The disclosure provides dried compositions providing reagents for nucleic acid amplification, which are essentially free of inorganic salts. Lack of inorganic salts increases stability of such compositions and decreases formation of byproducts. Salts required for use of enzymes in the composition are supplied on reconstitution.
1. A composition comprising an aqueous solution containing a reverse transcriptase, another polymerase, a bulking agent consisting essentially of trehalose at a concentration from about 0.16 M to about 0.32 M, a detergent, an organic buffer, from 0.1 mM to 0.3 mM of dATP, from 0.1 mM to 0.3 mM of dGTP, from 0.1 mM to 0.3 mM of dCTP, from 0.2 mM to 0.6 mM of dTTP or dUTP, RNase inhibitor and at least two oligonucleotides useful for performing a molecular assay, wherein the aqueous solution has a concentration of Mg 2+ of less than 0.1 mM and a concentration of K + of less than 1 mM.
2. The composition of claim 1 , wherein the at least two oligonucleotides are selected from the group consisting of: an amplification oligonucleotide, a detection probe oligonucleotide, a target capture probe oligonucleotide, an adaptor oligonucleotide, and combinations thereof.
3. The composition of claim 1 , wherein the aqueous solution comprises an inorganic salt concentration of 1 mM or less.
4. The composition of claim 1 , wherein the aqueous solution comprises a mass per microliter of inorganic salt from about 0.075 μg/μl to about 0.058 μg/μl and wherein the aqueous solution comprises a mass per microliter of chloride ions from about 0.071 μg/μl to about 0.018 μg/μl.
5. The composition of claim 3 , wherein the inorganic salt concentration of the aqueous solution is less than 1 mM sodium chloride.
6. The composition of claim 1 , wherein the other polymerase is present in the aqueous solution at a concentration from about 0.20 U/μl to about 0.72 U/μl in the aqueous solution.
7. The composition of claim 6 , wherein the other polymerase is present in the aqueous solution at a concentration selected from: 0.25 U/μl, 0.30 U/μl, 0.32 U/μl, 0.4 U/μl, 0.5 U/μl, 0.45 U/μl, and 0.72 U/μl.
8. The composition of claim 1 , wherein the reverse transcriptase is present in the aqueous solution at a concentration from about 0.1 U/μl to about 0.6 U/μl.
9. The composition of claim 1 , wherein the RNase inhibitor is at a concentration from about 0.12 U/μl to about 0.20 U/μl.
10. The composition of claim 1 , wherein the aqueous solution further comprises a chelating agent.
11. The composition of claim 10 , wherein the chelating agent is EDTA and is present in the aqueous solution at a concentration from 1.5 mM to 2.0 mM.
12. The composition of claim 1 containing no magnesium ions.
13. A dried composition made by drying a composition comprising an aqueous solution containing a reverse transcriptase, another polymerase, a bulking agent consisting essentially of trehalose at a concentration from about 0.16 M to about 0.32 M, a detergent, an organic buffer, from 0.1 mM to 0.3 mM of dATP, from 0.1 mM to 0.3 mM of dGTP, from 0.1 mM to 0.3 mM of dCTP, from 0.2 mM to 0.6 mM of dTTP or dUTP, RNase inhibitor and at least two oligonucleotides useful for performing a molecular assay, wherein the aqueous solution has a concentration of Mg 2+ of less than 0.1 mM and a concentration of K + of less than 1 mM.
14. A method of forming a mixture for use in performing a nucleic acid based amplification reaction, the method comprising combining a reconstitution solution and the dried composition of claim 13 , wherein the reconstitution solution comprises at least one inorganic salt.
15. The method of claim 14 , wherein the reconstitution solution comprises MgCl 2 at a concentration from about 3.8 mM to about 4.4 mM, KCl at a concentration from about 50 mM to about 80 mM, or both.
16. The method of claim 15 , wherein the reconstitution solution comprises, methyl paraben at a concentration from about 0.012% w/v to about 0.020% w/v, propyl paraben at a concentration from about 0.006% w/v to about 0.010% w/v, absolute ethanol at a concentration from about 0.20% v/v to about 0.30% v/v, or a combination thereof.
17. The method of claim 16 , wherein the concentration of the methyl paraben in the reconstitution solution is 0.016% w/v, the concentration of the propyl paraben in the reconstitution solution is 0.008% w/v and the concentration of the absolute ethanol is present in the reconstitution solution at about 0.26% v/v.