Identifying stability of polymerase in master mixes used in PCR and repair possibilities for the degraded reagents
More details
Hide details
Department of Biochemistry, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, Poland
Submission date: 2014-05-02
Acceptance date: 2014-07-17
Online publication date: 2014-08-15
Publication date: 2020-04-06
Corresponding author
Anna Cieślińska   

Department of Biochemistry, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, Oczapowskiego 1A, 10-719 Olsztyn, Poland. Tel.: +48 895233763/0694238064.
Pol. Ann. Med. 2014;21(2):82-85
The component of commercial available master mix most sensitive to unfavorable conditions is a polymerase. Available commercial polymerase chain reaction (PCR) master mixes are generally recommended for storage at –20°C, otherwise they may lose their activity.

The aim of the experiment was to verify if storing mixes in adverse and extreme conditions may influence the quality of a PCR product. In the second phase of the research, it was to indicate if inactive PCR reagents that have lost their activity, may recover their enzymatic properties.

Material and methods:
Five different commercially available master mixes were incubated in unfavorable conditions. After the PCR, an electrophoresis was carried out and the obtained product was an evidence of a proper PCR reaction.

Results and discussion:
Total degradation of mixes was caused by their incubation at room temperature for 28 days and incubation at 100°C for 60 minutes. Addition of polymerases to the degraded mixes (incubation at room temperature for 28 days) resulted in a regeneration of all of five mixes. In the case of polymerases incubated at 1008C for 60 minutes, regeneration was effective only in two of the five mixes.

Our research confirms that PCR master mix is characterized by high resistance to varied conditions as well as in some cases can be repaired after degradation.

On behalf of all authors, the corresponding author states that there is no conflict of interest.
Kong H, Kucera RB, Jack WE. Characterization of a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis. J Biol Chem. 1993;268(3):1965–1975.
Mullis KB. The unusual origin of the polymerase chain reaction. Sci Am. 1990;262:56–61. 64–65.
Ralser M, Querfurth R, Warnatz HJ, Lehrach H, Yaspo M-L, Krobitsch S. An efficient and economic enhancer mix for PCR. Biochem Biophys Res Commun. 2006;347:747–751.
Chien A, Edgar DB, Trela JM. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol. 1976;127:1550–1557.
Deepthi KY, Ramesh M. Cloning, isolation, and characterization of Taq DNA polymerase using pET expression system. Int J Life Sci Pharma Rev. 2013;2(1):267–283.
Lawyer FC, Stoffel S, Saiki RK, Myambo K, Drummond R, Gelfand DH. Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus. J Biol Chem. 1989;264(11):6427–6437.
Mishra N, Kumar A. Cloning and characterization of isolated Taq DNA polymerase gene from phage. Bioscan Int Q J Life Sci. 2010;5(1):07–11.
Kim Y, Eom SH, Wang J, Lee D-S, Won Suh S, Steitz TA. Crystal structure of Thermus aquaticus DNA polymerase. Nature. 2002;376:612–616.
Eom SH, Wang J, Steitz JTA. Structure of Taq polymerase with DNA at the polymerase active site. Nature. 1996;382:278–281.
Gibbs MD, Reeves RA, Mandelman D, Mi Q, Lee J, Bergquist PL. Molecular diversity and catalytic activity of Thermus DNA polymerases. Extremophiles. 2009;13:817–826.
Kornberg A, Baker TA. DNA replication. 2nd ed. New York: WH Freeman and Company; 1992.
Villbrandt B, Sagner G, Schomburg D. Investigations on the thermostability and function of truncated Thermus aquaticus DNA polymerase fragments. Protein Eng. 1997;10(11):1281–1288.
Eisenthal R, Peterson ME, Daniel RM, Danson MJ. The thermal behaviour of enzyme activity: implications for biotechnology. Trends Biotechnol. 2006;24(7):289–292.
Summit M, Scott B, Nielson K, Mathur E, Baross J. Pressure enhances thermal stability of DNA polymerase from three thermophilic organisms. Extremophiles. 1998;2(3):339–345.
Journals System - logo
Scroll to top