DNA polymerases as useful reagents for biotechnology - the history of developmental research in the field.

Sonoko Ishino,Yoshizumi Ishino

doi:10.3389/fmicb.2014.00465

Abstract

DNA polymerase is a ubiquitous enzyme that synthesizes complementary DNA strands according to the template DNA in living cells. Multiple enzymes have been identified from each organism, and the shared functions of these enzymes have been investigated. In addition to their fundamental role in maintaining genome integrity during replication and repair, DNA polymerases are widely used for DNA manipulation in vitro, including DNA cloning, sequencing, labeling, mutagenesis, and other purposes. The fundamental ability of DNA polymerases to synthesize a deoxyribonucleotide chain is conserved. However, the more specific properties, including processivity, fidelity (synthesis accuracy), and substrate nucleotide selectivity, differ among the enzymes. The distinctive properties of each DNA polymerase may lead to the potential development of unique reagents, and therefore searching for novel DNA polymerase has been one of the major focuses in this research field. In addition, protein engineering techniques to create mutant or artificial DNA polymerases have been successfully developing powerful DNA polymerases, suitable for specific purposes among the many kinds of DNA manipulations. Thermostable DNA polymerases are especially important for PCR-related techniques in molecular biology. In this review, we summarize the history of the research on developing thermostable DNA polymerases as reagents for genetic manipulation and discuss the future of this research field.

Highlights

DNA polymerase is a ubiquitous enzyme that synthesizes complementary DNA strands according to the template DNA in living cells
It was imagined that a heat-stable DNA polymerase that is not inactivated at the denaturation step from double-stranded to single-stranded DNA would transform this method of gene amplification to a practical technology
Tth polymerase is that it has a distinct reverse transcriptase (RT) activity, and a single-tube RT-PCR method was developed with this enzyme

Summary

Sonoko Ishino and Yoshizumi Ishino*

DNA polymerase from Thermus thermophilus (Tth polymerase) was developed as a commercial product in the early age of the PCR, but a scientific report was only an abstract of ASBMB in 1974 from the Mitsubishi-Kasei Institute of Life Sciences, Japan, where this enzyme was originally identified. The amount of the recombinant Taq polymerase produced in E. coli cells was very low, probably because of the low expression of the T. aquaticus gene, which has a high GC content (70%), the protein quality was improved, as compared to the native Taq polymerase (Lawyer et al, 1989). We successfully constructed an efficient overproduction system by changing the codons around the N-terminal region from the original gene to either the AT-type at the third letter or the optimal codons for E. coli These manipulations improved the production of Taq polymerase more than 10-fold, as compared with the production of AmpliTaq (Ishino et al, 1994).

Ishino and Ishino

Findings

Aquifex pyrophilus