A simple and efficient method for extraction of Taq DNA polymerase

Sique Chen,Linghui Jiang,Hongrui Cao,Xinli Sun,Xiujuan Zheng,Fangqian Liu

doi:10.1016/j.ejbt.2015.08.001

Sique Chen, Linghui Jiang + Show 4 more

Open Access

https://doi.org/10.1016/j.ejbt.2015.08.001

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Abstract

Thermostable DNA polymerase (Taq Pol Ι) from Thermus aquaticus has been widely used in PCR, which was usually extracted with Pluthero's method. The method used ammonium sulfate to precipitate the enzyme, and it saved effort and money but not time. Moreover, we found that 30–40% activity of Taq Pol I was lost at the ammonium sulfate precipitation step, and the product contained a small amount of DNA. We provided a novel, simplified and low-cost method to purify the Taq Pol Ι after overproduction of the enzyme in Escherichia coli, which used ethanol instead of ammonium sulfate to precipitate the enzyme. The precipitate can be directly dissolved in the storage buffer without dialysis. In addition, DNA and RNA contamination was removed with DNase I and RNase A before precipitation, and the extraction procedure was optimized. Our improvements increase recovery rate and specific activity of the enzyme, and save labor, time, and cost. Our method uses ethanol, DNase I, and RNase A to purify the Taq Pol Ι, and simplifies the operation, and increases the enzyme recovery rate and quality.

Highlights

PCR is a most widely-used technique in biology, which utilizes thermostable DNA polymerase from Thermus aquaticus (Taq) [1,2]
We found that 30–40% activity of Taq Pol I was lost at the ammonium sulfate precipitation step, and the product contained a small amount of DNA
The activity of fresh Taq Pol Ι was fine, but its activity was lost after two d of storage at -20°C

Summary

Introduction

PCR is a most widely-used technique in biology, which utilizes thermostable DNA polymerase from Thermus aquaticus (Taq) [1,2]. The gene that encoded the Taq DNA polymerase had been cloned and expressed in Escherichia coli efficiently [3]. This has greatly facilitated the production and reduced the price of the enzyme. Thermostable DNA polymerase (Taq Pol Ι) from Thermus aquaticus has been widely used in PCR, which was usually extracted with Pluthero's method. Results: We provided a novel, simplified and low-cost method to purify the Taq Pol Ι after overproduction of the enzyme in Escherichia coli, which used ethanol instead of ammonium sulfate to precipitate the enzyme. Conclusions: Our method uses ethanol, DNase I, and RNase A to purify the Taq Pol Ι, and simplifies the operation, and increases the enzyme recovery rate and quality

Methods

Results

Conclusion