Enhanced detection of RNA by MMLV reverse transcriptase coupled with thermostable DNA polymerase and DNA/RNA helicase

Hiroyuki Okano; Yuta Katano; Misato Baba; Ayako Fujiwara; Ryota Hidese; Shinsuke Fujiwara; Itaru Yanagihara; Tsukasa Hayashi; Kenji Kojima; Teisuke Takita; Kiyoshi Yasukawa

doi:10.1016/j.enzmictec.2016.10.003

Enhanced detection of RNA by MMLV reverse transcriptase coupled with thermostable DNA polymerase and DNA/RNA helicase

Enzyme Microb Technol. 2017 Jan:96:111-120. doi: 10.1016/j.enzmictec.2016.10.003. Epub 2016 Oct 11.

Authors

Affiliations

¹ Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
² Department of Bioscience, School of Science and Technology, Kwansei-Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan.
³ Department of Developmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594-1101, Japan.
⁴ Kainos Laboratories, Inc., 38-18, Hongo 2-chome, Bunkyo-ku, Tokyo 113-0033, Japan.
⁵ Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. Electronic address: yasukawa@kais.kyoto-u.ac.jp.

PMID: 27871370
DOI: 10.1016/j.enzmictec.2016.10.003

Abstract

Detection of mRNA is a valuable method for monitoring the specific gene expression. In this study, we devised a novel cDNA synthesis method using three enzymes, the genetically engineered thermostable variant of reverse transcriptase (RT), MM4 (E286R/E302K/L435R/D524A) from Moloney murine leukemia virus (MMLV), the genetically engineered variant of family A DNA polymerase with RT activity, K4pol_L329A from thermophilic Thermotoga petrophila K4, and the DNA/RNA helicase Tk-EshA from a hyperthermophilic archaeon Thermococcus kodakarensis. By optimizing assay conditions for three enzymes using Taguchi's method, 100 to 1000-fold higher sensitivity was achieved for cDNA synthesis than conventional assay condition using only RT. Our results suggest that DNA polymerase with RT activity and DNA/RNA helicase are useful to increase the sensitivity of cDNA synthesis.

Keywords: DNA polymerase; Helicase; Reverse transcriptase; Taguchi’s method; cDNA synthesis.

MeSH terms

Base Sequence
DNA Helicases / genetics
DNA, Complementary / biosynthesis*
DNA, Complementary / genetics*
DNA-Directed DNA Polymerase / genetics
DNA-Directed DNA Polymerase / metabolism
Enzyme Stability
Gene Expression
Gram-Negative Anaerobic Straight, Curved, and Helical Rods / enzymology
Gram-Negative Anaerobic Straight, Curved, and Helical Rods / genetics
Moloney murine leukemia virus / enzymology
Moloney murine leukemia virus / genetics
Oligonucleotide Array Sequence Analysis
Protein Engineering
RNA / analysis*
RNA / genetics*
RNA Helicases / genetics
RNA-Directed DNA Polymerase / genetics
RNA-Directed DNA Polymerase / metabolism
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Temperature
Thermococcus / enzymology
Thermococcus / genetics

Substances

DNA, Complementary
Recombinant Proteins
RNA
RNA-Directed DNA Polymerase
DNA-Directed DNA Polymerase
DNA Helicases
RNA Helicases