Title
A repeat protein-based DNA polymerase inhibitor for an efficient and accurate gene amplification by PCR†
Author
Da-Eun Hwang, Yong-Keol Shin, Palinda Ruvan Munashingha, So-Yeon Park, Yeon-Soo Seo, Hak-Sung Kim
Year
2016
Journal
Biotechnology and Bioengineering
Abstract
A polymerase chain reaction (PCR) using a thermostable DNA polymerase is the most widely applied method in many areas of research, including life sciences, biotechnology, and medical sciences. However, a conventional PCR incurs an amplification of undesired genes mainly owing to non-specifically annealed primers and the formation of a primer-dimer complex. Herein, we present the development of a Taq DNA polymerase-specific repebody, which is a small-sized protein binder composed of leucine rich repeat (LRR) modules, as a thermolabile inhibitor for a precise and accurate gene amplification by PCR. We selected a repebody that specifically binds to the DNA polymerase through a phage display, and increased its affinity to up to 10 nM through a modular evolution approach. The repebody was shown to effectively inhibit DNA polymerase activity at low temperature and undergo thermal denaturation at high temperature, leading to a rapid and full recovery of the polymerase activity, during the initial denaturation step of the PCR. The performance and utility of the repebody was demonstrated through an accurate and efficient amplification of a target gene without nonspecific gene products in both conventional and real-time PCRs. The repebody is expected to be effectively utilized as a thermolabile inhibitor in a PCR. This article is protected by copyright. All rights reserved
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Protein denaturation, Thermodynamics, Biochemistry