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.


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
TOP

Ordering Information

Sku Name Size Price Qty
12607024 Discoverase™ dHPLC DNA Polymerase 500 reactions USD 1,102.00

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:*

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
TOP

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. 
TOP

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.
TOP
LT092