Optimization of single strand DNA incorporation reaction by Moloney murine leukaemia virus reverse transcriptase

Yoshiyuki Ohtsubo, Haruna Sasaki, Yuji Nagata, Masataka Tsuda

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

In this study, we investigated CIS reaction (clamping-mediated incorporation of single-stranded DNA with concomitant DNA syntheses) of Moloney murine leukaemia virus reverse transcriptase (MMLV-RT), and established a set of conditions with which single-stranded DNA is ligated to a G-tailed model substrate DNA at efficiencies close to 100%. Prior to the CIS reaction, a target blunt-end DNA was 3' G-tailed by MMLV-RT in the presence of a tailing enhancer, deoxycytidine. In the CIS reaction, the G-tail reacted with a single-stranded DNA carrying a stretch of Cs on its 3' end (termed as GAO for guide adaptor oligonucleotide), and MMLV-RT performed DNA polymerization, starting from the 3' overhang, using the GAO as a template. We could append a given nucleotide sequence of as long as 72 nucleotides, which would be sufficient for various NGS-sequencing platforms. The high efficiency and the unique features of this MMLV-RT activity that enables the labelling of each DNA molecule with a unique degenerate sequence as a molecular identifier has many potential uses in biotechnology.

Original languageEnglish
Pages (from-to)477-487
Number of pages11
JournalDNA Research
Volume25
Issue number5
DOIs
Publication statusPublished - 2018

Keywords

  • Adaptor ligation
  • DNA polymerase
  • Reverse transcriptase
  • Template-independent DNA addition

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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