Formulations and methods for stabilizing PCR reagents
Described herein are stabilized polymerase compositions comprising a polymerase and an polymerase stabilizing agent, such as a non-detergent zwitterionic stabilizer or a cationic ester disclosed, for use in nucleic acid amplification or nucleic acid sequencing. Compositions are provided for the stabilization of one or more polymerases in a single stabilized liquid formulation. Also disclosed are methods for making and using stabilized polymerase compositions and kits for nucleic acid amplification and sequencing comprising the stabilized polymerase compositions provided.
1. A liquid composition comprising a polymerase and a storage stabilizing agent, wherein the storage stabilizing agent comprises at least one cationic ester compound selected from the group consisting of:
[i] 1-(2-ethoxy-2-oxoethyl)-3-hydroxy-1-methylpiperidium bromide,
[ii] N-(2-cyanoethyl)-2-ethoxy-N,N-dimethyl-2-oxoethanaminium bromide,
[iii] 2-ethoxy-N,N-diethyl-N-methyl-2-oxoethanaminium bromide,
[iv] N-(2-ethoxy-2-oxoethyl)-N,N-dimethylcyclohexanaminium bromide, and
[v] 2-ethoxy-N-(2-(2-hydroxyethoxy)ethyl)-N,N-dimethyl-2-oxoethanaminium bromide.
2. The composition of claim 1 , wherein the composition comprises a disaccharide, a trisaccharide, or a combination of a disaccharide and a trisaccharide.
3. The composition of claim 2 , wherein the disaccharide is sucrose, or the trisaccharide is melezitose or raffinose.
4. The composition of claim 1 , wherein the composition comprises a polymer.
5. The composition of claim 4 , wherein the polymer is polyvinyl alcohol (PVA).
6. The composition of claim 1 , further comprising BSA.
7. The composition of claim 1 , further comprising a buffering agent.
8. The composition of claim 7 , wherein the buffering agent is selected from the group consisting of Tris, MOPS, HEPES, TAPS, Bicine, Tricine, TES, PIPES, MES, and combinations thereof.
9. The composition of claim 1 , wherein the polymerase retains the same activity when stored at room temperature for at least two weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 1 year compared to the activity of the polymerase when stored at a temperature of −20° C. for the same time period.
10. The composition of claim 1 , further comprising one or more additional reagents selected from the group consisting of a buffering agent, reducing agent, a non-ionic detergent, a salt, at least one dNTP, a primer, and combinations thereof.
11. The composition of claim 1 , comprising about 0.05-5M alanyl-glutamine, about 0.5-10% sucrose, about 0.5-10% melezitose, about 0.1-1% PVA, and about 1-25 mg/ml 2-ethoxy-N-(2(2-hydroxyethoxy)ethyl)-N,N-dimethyl-2-oxoethanaminium bromide.