Department of Molecular and Chemical Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan.
DNA Res. 2018 Oct 1;25(5):477-487. doi: 10.1093/dnares/dsy018.
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.
在这项研究中，我们研究了莫洛尼鼠白血病病毒逆转录酶（MMLV-RT）的 CIS 反应（单链 DNA 的夹合介导掺入以及伴随的 DNA 合成），并建立了一套条件，使单链 DNA 以接近 100%的效率连接到 G 尾模型底物 DNA 上。在 CIS 反应之前，在尾化增强剂脱氧胞苷存在的情况下，MMLV-RT 将靶标平头末端 DNA 3' G 尾化。在 CIS 反应中，G 尾与携带其 3' 端上一段 Cs 的单链 DNA 反应（称为引导适配体寡核苷酸 GAO），并且 MMLV-RT 从 3' 突出开始进行 DNA 聚合，以 GAO 为模板。我们可以添加长达 72 个核苷酸的给定核苷酸序列，这对于各种 NGS 测序平台来说已经足够了。这种 MMLV-RT 活性的高效率和独特功能使其能够将每个 DNA 分子标记为独特的简并序列作为分子标识符，这在生物技术中有许多潜在的用途。
