title: Enhanced detection of RNA by MMLV reverse transcriptase coupled with thermostable DNA polymerase and DNA/RNA helicase.
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, K4polL329Afrom 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.
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Okano
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Katano
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Misato
authors.initials: M
authors.lastname: Baba
authors.affiliation: Department of Bioscience, School of Science and Technology, Kwansei-Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Fujiwara
authors.affiliation: Department of Bioscience, School of Science and Technology, Kwansei-Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan.
authors.forename: Ryota
authors.initials: R
authors.lastname: Hidese
authors.affiliation: Department of Bioscience, School of Science and Technology, Kwansei-Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan.
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Fujiwara
authors.affiliation: Department of Developmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594-1101, Japan.
authors.forename: Itaru
authors.initials: I
authors.lastname: Yanagihara
authors.affiliation: Kainos Laboratories, Inc., 38-18, Hongo 2-chome, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Hayashi
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kojima
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Teisuke
authors.initials: T
authors.lastname: Takita
authors.affiliation: 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.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yasukawa
mesh_terms: D001483:Base Sequence; D004265:DNA Helicases / Q000235:genetics; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D015870:Gene Expression; D016965:Gram-Negative Anaerobic Straight, Curved, and Helical Rods / Q000201:enzymology / Q000235:genetics; D008979:Moloney murine leukemia virus / Q000201:enzymology / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D015202:Protein Engineering; D012313:RNA / Q000032:analysis* / Q000235:genetics*; D020365:RNA Helicases / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D012313:RNA; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases; D020365:RNA Helicases
keywords: DNA polymerase; Helicase; Reverse transcriptase; Taguchi’s method; cDNA synthesis