Enhanced detection of RNA by MMLV reverse transcriptase coupled with thermostable DNA polymerase and DNA/RNA helicase.
Enzyme Microb Technol
; 96: 111-120, 2017 Jan.
Article
em En
| MEDLINE
| ID: mdl-27871370
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, K4polL329A 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.
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Base de dados:
MEDLINE
Assunto principal:
RNA
/
DNA Complementar
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article