Taq DNA polymerase variants with increased reverse transcriptase activity
Taq DNA polymerase mutants exhibiting reverse transcriptase activity compared to wild type polymerase were engineered, characterized, and selected via polymerase chain reactions visualized via electrophoresis on agarose gels. Initial screening was followed up with probe-based qualitative, real-time PCR (qPCR) with a typical reverse transcription cycling protocol to detect specified ribonucleic acid (RNA) target sequences. The engineered variants can render robust cDNA from RNA target substrates and amplify that cDNA under standard reaction conditions without the assistance of added reverse transcriptase enzymes.
1. A mutant Taq polymerase comprising at least 90% amino acid sequence identity with the type Taq polymerase as shown in SEQ ID NO: 2 and comprising the mutation E9K relative to SEQ ID NO:2.
2. The mutant Taq DNA polymerase of claim 1 wherein the mutant Taq polymerase is capable of reverse transcribing mRNA to generate cDNA.
3. The mutant Taq DNA polymerase of claim 2 wherein the reverse transcribing mRNA to generate cDNA takes place at 38 or more amplification cycles.
4. The mutant Taq DNA polymerase of claim 2 wherein the reverse transcribing mRNA to generate cDNA takes place at a greater extent than performed by wild type Taq DNA polymerase under the same conditions.
5. A reverse transcription PCR process comprising: providing a reverse transcription PCR mixture including an RNA target, target sequence primers and the mutant Taq DNA polymerase of claim 1 , subjecting the reverse transcription PCR mixture to PCR cycling conditions to generate cDNA and detecting the quantity of cDNA generated to quantitate the RNA target.
6. The method of claim 5 wherein the detection of the quantity of cDNA is by determining a quantity of reporter signal generated by an intercalating dye or by cleavage of a labeled target probe.
7. A reverse transcription qPCR process comprising: providing a reverse transcription qPCR mixture including an RNA target, target sequence primers, an intercalating dye or a labeled target probe which fluoresces when the label is cleaved from the probe by exonuclease activity, and the mutant Taq DNA polymerase of claim 1 , providing a de-annealing temperature for a sufficient time to de-anneal primers from targets; providing a number of cycles of an annealing temperature followed by an extension temperature and determining the quantity of reporter signal generated by the intercalating dye or cleavage of the labeled target probe to quantify the amount of RNA target sequence present in the qPCR mixture.
8. The qPCR process of claim 7 wherein there are 38 or more cycles.
9. The qPCR process of claim 7 wherein the reporter signal is fluorescent.
10. The qPCR process of claim 7 wherein the intercalating dye is SYBR Green or EvaGreen.