PyroPhage® 3173 DNA Polymerase, Exonuclease Minus (Exo-)

Superior DNA amplification

The thermostability of PyroPhage 3173 DNA Polymerase makes it useful for thermocycling methods, including PCR. PyroPhage 3173 DNA Polymerase also has an efficient reverse transcription activity. The enzyme can perform single-tube, single enzyme reverse-transcription PCR of RNA transcripts. The Exo Minus mutant lacks the potent 3’-5’ exonuclease (proofreading) activity of the Wild Type enzyme.

 

RT-PCR capabilities.

The Exo Minus enzyme can be used for single-tube, single-enzyme reverse transcription PCR (RT-PCR), as shown in Figure 1.

Figure 1. Single-tube, single-enzyme RT-PCR with PyroPhage 3173 DNA Polymerase (Exo Minus). Mouse liver RNA was used as a template with actin-specific primers. A single band was produced at the expected size of 283 bp.

 

PyroPhage 3173 DNA Polymerase Properties

Table 1. Established characteristics of PyroPhage 3173 DNA Polymerase
	Exo Minus
3’-5’ exonuclease	none
5’-3’ exonuclease	none
Extension from nicks	strong
Thermostability (T½ @95° in PCR buffer)	10 min.
Km dNTPs	40 µM
Km DNA	5.3 nM
Processivity	47 nt
Fidelity	1.5 × 104
3’ ends of amplicons	single base A and G overhangs

Usage Information
Certain primer/template sets are more effectively amplified by PyroPhage 3173 DNA Polymerase. Amplification of other apparently similar primer/template sets can be problematic. The basis of this inconsistency is not understood. Because of this variability, no predictions can be made on the effectiveness of PyroPhage 3173 DNA Polymerase in amplifying a given primer/template set. Variability may be significantly reduced by use of alternative primer sets or common PCR additives, such as betaine or ectoine.

Published Paper

Michael J. Moser, Robert A. DiFrancesco, Krishne Gowda, Audrey J. Klingele, Darby R. Sugar, Stacy Stocki, David A. Mead, Thomas W. Schoenfeld (2012). "Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme." PLoS ONE 7(6): e38371. doi:10.1371/journal.pone.0038371

 

Legal Information
The PyroPhage DNA Polymerases and methods of using the PyroPhage DNA Polymerases are covered by pending patents assigned to Lucigen Corporation. Lucigen does not encourage or support the unauthorized or unlicensed use of the PCR process, reverse transcription PCR process or isothermal DNA synthesis. It is the sole responsibility of the buyer to ensure that use of the product does not infringe the patent rights of third parties.

References

1. Arezi, B., Xing, W., Sorge, J. A., Hogrefe, H. H. (2003). Amplification efficiency of thermostable DNA polymerases. Anal Biochem. 321(2), 226.
2. Hogrefe HH, Cline J, Lovejoy AE, Nielson KB. (2001). DNA polymerases from hyperthermophiles. Methods Enzymol. 334, 91.

Activity Determination. One unit catalyzes the incorporation of 10 nmol of dNTP into acid-insoluble material in 30 minutes at 70°C in 20 mM Tris-HCl (pH 8.8), 10 mM (NH₄ )₂S0₄, 10 mM KCl, 2 mM MgS0₄, 0.1% Triton X-100, 200 µM dGTP, dATP, dTTP, dCTP (a mix of unlabeled and [33P] dCTP), and 30 µg of Activated Calf Thymus DNA.

Purity. The Exo Minus enzyme is tested to be free of contaminating DNA, endonuclease, and nicking activities. The Exo Minus enzyme is also tested to be free of exonuclease activity, and is greater than 95% pure by SDS-PAGE.

Concentration. 5 units/µl.

Storage Buffer: 10 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.1% Triton X-100, 0.1 mM EDTA, 1mM DTT, and 50% glycerol.

PyroPhage 3173 2X PCR Buffer: 40 mM Tris-HCl, 20 mM (NH₄)₂SO₄, 20 mM KCl, 4 mM MgSO₄, 0.2% Triton X-100, thermoprotectant, pH 8.8 @ 25°C.

Absence of Endonuclease
PyroPhage 3173 DNA Polymerase, Exo– is determined to be free of detectable endonuclease or nicking activity. Supercoiled plasmid DNA is incubated with enzyme for 16 hours at 70°C. Agarose gel electrophoresis shows no alteration in mobility consistent with endonuclease or nicking activity.
Absence of Exonuclease
PyroPhage 3173 DNA Polymerase Exo– is tested to be free of contaminating exonuclease activity by incubating Hind III-digested lambda DNA with enzyme at 70°C for 16 hours. Agarose gel electrophoresis shows no alteration in mobility consistent with exonuclease activity.
Absence of Ribonuclease
PyroPhage 3173 DNA Polymerase, Exo– is tested to be free of contaminating RNAse activity by incubating with a fluorogenic RNAse substrate for 1 hour at 37°C. No increase in assay fluorescence above background was detected.
Functional Assay
PyroPhage 3173 DNA Polymerase, Exo– is tested for performance in PCR using the PyroPhage 3173 PCR Buffer to amplify a 2 kb region of the endA gene from 2 X 106 molecules (10 ng) of E. coli gDNA. The resulting PCR product is visualized on an ethidium bromide-stained agarose gel.