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

被引：6

作者：

Ohtsubo, Yoshiyuki ^{[1
]}

Sasaki, Haruna ^{[1
]}

Nagata, Yuji ^{[1
]}

Tsuda, Masataka ^{[1
]}

机构：

[1] Tohoku Univ, Grad Sch Life Sci, Dept Mol & Chem Life Sci, Sendai, Miyagi 9808577, Japan

来源：

DNA RESEARCH | 2018年 / 25卷 / 05期

关键词：

adaptor ligation; template-independent DNA addition; DNA polymerase; reverse transcriptase; CLAMP; SITE;

D O I：

10.1093/dnares/dsy018

中图分类号：

Q3 [遗传学];

学科分类号：

071007 ; 090102 ;

摘要：

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.

引用

页码：477 / 487

页数：11

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