Process of probe qPCR using Taq DNA polymerase mutants
Disclosed are Taq DNA polymerase mutants which exhibit enhanced efficiency in qPCR compared to the wild type Taq DNA polymerase. Such mutants include: V62S, V64S, A70F, F73A, A77F, P253G, E255K, D257R, A259F, A271F, L288S, E289K, S357I, L361S, L376S, P382G, T385I, G418P, R419D, E421K, L461S, A472F, E497K, L498S, E524K, D551R, R556D, S679l, L789S, E189K/E507K/E742K (See Sequence Listing Guide for the mutants' amino acid and protein sequences).
1. A qPCR process comprising:
providing a qPCR mixture including a target nucleic acid, target nucleic acid primers, a mutant Taq DNA polymerase and an intercalating dye or a labeled target probe which fluoresces when the label is cleaved from the probe by exonuclease activity, wherein the mutant Taq DNA polymerase comprises the amino acid mutation A70F and the amino acid sequence of SEQ ID NO: 8, with the exception that the mutant Taq DNA polymerase does not include the 6-membered histidine tag at the C-terminus and the six immediately preceding Glycine and Serine amino acids included in SEQ ID NO: 8, providing a de-annealing temperature for a sufficient time to de-anneal primers from targets;
providing a number of cycles of an annealing temperature for a sufficient time to anneal primers to targets followed by an extension temperature held for about one second; and
measuring a reporter signal generated to quantify the amount of target sequence present in the qPCR mixture.
2. The qPCR process of claim 1 wherein the de-annealing temperature is 95° C. for 30 seconds; the annealing temperature is 95° C. for 4 seconds and the extension temperature is 60° C.
3. The qPCR process of claim 1 wherein there are 40 cycles.
4. The qPCR process of claim 1 wherein the reporter signal is fluorescent.
5. The qPCR process of claim 1 wherein the reporter signal is from the labeled probe or the intercalating dye.
6. The qPCR process of claim 5 wherein the intercalating dye is SYBR Green or EvaGreen.