Using proteases to control restriction enzyme activity
Proteases are enzymes which hydrolyze protein enzymes, eliminating their activity. The present invention exploits the hydrolyzing activity of proteases including proteinase K, endoproteinase LysC and/or trypsin to control the activity of restriction enzymes and/or eliminate or reduce production of unwanted DNA or RNA fragments (known as star activity).
1. A process of inhibiting or preventing star activity by NcoI or BamHI comprising:
digesting an oligomer using a reaction mixture including NcoI or BamHI, buffer and oligomer;
including in the reaction mixture or adding proteinase K, endoproteinase LysC and/or trypsin in order to inhibit or prevent star activity by NcoI or BamHI; and
terminating the digestion by adding a stopping solution including a loading dye.
2. The process of claim 1 wherein the oligomer is lambda DNA.
3. The process of claim 1 wherein the buffer is 20 mM Tris-HCl, pH 7.4, 40 mM KoAc, 10 mM MgCl 2 , 0.1 mM CaCl 2 ; or 20 mM Tris-HCl, pH 7.5, 40 mM NaCl, 10 mM MgCl 2 , 0.1 mM CaCl 2 .
4. The process of claim 1 wherein the stopping solution includes 2.5% Ficoll®-400, 11 mM EDTA (pH 8.0), 3.3 mM Tris-HCL, 0.017% SDS, 0.015% bromophenol blue.
5. The process of claim 4 wherein the stopping solution is added.
6. A process of determining the optimal relative concentrations to inhibit or prevent star activity by Ncol or BamHI, or Ncol or BamHI and a proteinase comprising:
(i) combining Ncol or BamHI, the proteinase, a buffer and an oligomer in order to achieve a starting concentration of each;
(ii) determining the star activity of Ncol or BamHI after a reaction time, and if there is any, diluting the reaction mixture and repeating the reaction for said reaction time;
(iii) repeating step (ii) until there is no star activity; and
(iv) determining the concentrations of Ncol or BamHI and proteinase at which star activity ceases.
7. The process of claim 6 wherein the proteinase are combined simultaneously with the restriction endonuclease.
8. The process of claim 6 wherein the proteinase is proteinase K, endoproteinase LysC or trypsin.
9. The process of claim 6 wherein the oligomer is lambda DNA.
10. The process of claim 6 wherein the buffer is: 20 mM Tris-HCl, pH 7.4, 40 mM KoAc, 10 mM MgCl2, 0.1 mM CaCl2; or 20 mM Tris-HCl, pH 7.5, 40 mM NaCl, 10 mM MgCl2, 0.1 mM CaCl2; and, 20 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 0.1 mM CaCl2.
11. The process of claim 6 wherein Ncol or BamHI activity is terminated by addition of STOP solution.
12. The process of claim 1 wherein the buffer is 20 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 , 0.1 mM CaCl 2 .