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DNA Res. 2018 Oct 1;25(5):477-487. doi: 10.1093/dnares/dsy018.

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

Author information

1
Department of Molecular and Chemical Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan.

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.

PMID:
29897438
PMCID:
PMC6191310
DOI:
10.1093/dnares/dsy018
[Indexed for MEDLINE]
Free PMC Article

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