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Introduction
Discoverase™ dHPLC DNA Polymerase is an enzyme mixture composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D polymerase (1,2). Pyrococcus species GB-D polymerase possesses a proofreading ability by virtue of its 3’ to 5’ exonuclease activity(3). Mixture of the proofreading enzyme with Taq DNA polymerase at an optimized ratio increases fidelity approximately eight times over that of Taq DNA polymerase alone and allows amplification of simple and complex DNA templates. The enzyme mixture is provided with an optimized buffer that improves enzyme fidelity.
The Discoverase™ dHPLC DNA Polymerase enzyme mixture and buffer formulation have been optimized for use with denaturing high-performance liquid chromatography (dHPLC) systems(4). They were developed and tested using the Transgenomic WAVE® System.Discoverase™ dHPLC DNA Polymerase is supplied at 1 unit per µl.
The Discoverase™ dHPLC DNA Polymerase enzyme mixture and buffer formulation have been optimized for use with denaturing high-performance liquid chromatography (dHPLC) systems(4). They were developed and tested using the Transgenomic WAVE® System.Discoverase™ dHPLC DNA Polymerase is supplied at 1 unit per µl.
| | Kit Size | |
|---|---|---|
| Component | 100 rxn | 500 rxn |
| Discoverase™ dHPLC DNA Polymerase | 100 µl | 500 µl |
| 5X Discoverase™ PCR Buffer | 1 ml | 5 × 1 ml |
| 50 mM Magnesium Sulfate (MgSO4) | 1 ml | 1 ml |
Protocol
The following procedure is suggested as a guideline and starting point when using Discoverase™ dHPLC DNA Polymerase in any PCR amplification. The reaction size may be altered if necessary.
1. Add the following components to a sterile microcentrifuge tube on ice:*
*For multiple reactions, prepare a master mix of components common to all reactions to minimize reagent loss and enable accurate pipetting.
2. Mix contents of the tubes.
3. Cap the tubes and centrifuge briefly to collect the contents.
4. The following cycling protocol is recommended as a starting point, and may need to be optimized for different thermal cyclers. Note that the optimal annealing temperature is typically 5°C below the Tm of the primers.
5. Maintain the reaction at 4°C after cycling. The samples can be stored at -20°C until use.
6. Proceed to dHPLC analysis
1. Add the following components to a sterile microcentrifuge tube on ice:*
| Component | Volume | Final Concentration |
|---|---|---|
| 5X Discoverase™ PCR Buffer | 10 µl | 1X |
| 10 mM dNTP mixture | 1 µl | 0.2 mM each |
| Primer mix (10 µM each) | 1 µl | 200 nM each |
| Template DNA x µl | (as required) | |
| Discoverase™ DNA Polymerase | 1 µl | 1.0 unit |
| Sterile, distilled water | to 50 µl | — |
*For multiple reactions, prepare a master mix of components common to all reactions to minimize reagent loss and enable accurate pipetting.
2. Mix contents of the tubes.
3. Cap the tubes and centrifuge briefly to collect the contents.
4. The following cycling protocol is recommended as a starting point, and may need to be optimized for different thermal cyclers. Note that the optimal annealing temperature is typically 5°C below the Tm of the primers.
| Step | Temp | Time | Cycling |
| Denature | 94°C | 30 s | 1 cycle |
| Denature Anneal Extend | 94°C Primer dependent 68°C | 15–30 s 30 s 1 min per kb | 30–35 cycles |
5. Maintain the reaction at 4°C after cycling. The samples can be stored at -20°C until use.
6. Proceed to dHPLC analysis
PCR Recommendations and Guidelines
- Keep all components, reaction mixes, and samples on ice. After preparation of the samples, transfer them immediately to a preheated thermal cycler (94°C) and start the amplification program.
- Thin-walled reaction tubes are recommended
- One microliter of enzyme (1 unit) is appropriate for most targets.
- The annealing temperature of the reaction will vary depending on the Tm of your primers. The optimal annealing temperature is typically 5°C below the Tm of the primers.
References
1. Innis, M.A., Myambo, K.B., Gelfand, D.H. and Brow, M.A.D. (1988) Proc. Natl. Acad. Sci. USA 85, 9436.
2. Barnes, W.M. (1994) Proc. Natl. Acad. Sci. USA 91, 2216.
3. Tindall, K.R. and Kunkel, T.A. (1988) Biochemistry 27, 6008.
4. Xiao, W. and Oefner, P.J. (2001) Denaturing high-performance liquid chromatography: A review. Hum Mutat. 17, 439–74.
2. Barnes, W.M. (1994) Proc. Natl. Acad. Sci. USA 91, 2216.
3. Tindall, K.R. and Kunkel, T.A. (1988) Biochemistry 27, 6008.
4. Xiao, W. and Oefner, P.J. (2001) Denaturing high-performance liquid chromatography: A review. Hum Mutat. 17, 439–74.
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