Methods and compositions for amplification of nucleic acids
View Patent ↗The present invention provides methods, compositions, and kits for storing and enhancing the activity of polymerases and particularly thermostable polymerases. The methods comprise mixing a thermostable polymerase with at least one zwitterionic or ylide surfactant that has at least one PEO group. In another aspect the polymerase is mixed with a blocker such as PLURONIC® or TETRONIC® or an amine N-oxide derivative thereof. The thermostable polymerase may be reversibly inactivated by treatment with 2-(Methylsulfonyl)ethyl 4-nitrophenyl carbonate. Compositions and kits for performing the process according to the invention are also provided.
1. A composition comprising a thermostable polymerase that has been reversibly modified with a polymerase-modifying reagent of the following structure:
wherein Y=N-hydroxysuccinimide (NHS), p-nitrophenol (pNP) or imidazole;
X=CONMe 2 , MeSO 2 , CN, NO 2 , CO 2 Me, (CH 2 ) 2 SMe, N(CH 3 )CHO, or CH 2 C(O)CH 3 ;
and a zwitterionic amine oxide.
2. The composition of claim 1 , wherein the zwitterionic amine oxide has at least one polyethylene oxide (PEO) group.
3. The composition of claim 1 , wherein the zwitterionic amine oxide has a fatty alkyl group.
4. The composition of claim 1 , wherein the zwitterionic amine oxide has the following structure:
where Q=—O Θ ; or —X—Y, where X=C 1 -C 3 alkyl and Y=CO 2 Θ , SO 3 Θ ;
m+n=2-200;
R1=C 1 -C 30 alkyl
R2=CH 3 , CH 2 CH 3 ; x=1, 2
R3=H, CH 3 , or CH 2 OH.
5. The composition of claim 4 wherein m+n is 5 to 50.
6. The composition of claim 1 wherein the zwitterionic amine oxide is a tetrafunctional, ethoxylated and propoxylated ethylenediamine block copolymer surfactant modified to an amine oxide.
7. The composition of claim 1 , wherein the zwitterionic amine oxide has the following structure:
wherein m≧1 and n≧1 and wherein PEO=polyethylene oxide and PPO=polypropylene oxide.
8. The composition of claim 1 , wherein the polymerase-modifying reagent is 2-(methylsulfonyl)ethyl 4-nitrophenyl carbonate (MSEC).
9. The composition of claim 1 wherein the thermostable polymerase is Taq DNA polymerase.
10. The composition of claim 1 , wherein the zwitterionic amine oxide has the following structure:
wherein m≧1 and n≧1 and wherein PEO=polyethylene oxide and PPO=polypropylene oxide;
wherein the polymerase modifying reagent is 2-(methylsulfonyl)ethyl 4-nitrophenyl carbonate (MSEC); and
wherein the thermostable polymerase is Taq DNA polymerase.