HomeResearch LabsGuan LabGuan LabTemplate switching of reverse transcriptaseTemplate switching of reverse transcriptase

单细胞 RNA-Seq (scRNA-Seq) 使人们对单个细胞中的基因表达和调控有了前所未有的了解。许多 scRNA-Seq 方法依赖于莫洛尼鼠白血病病毒 (MMLV) 型逆转录酶的模板转换特性。模板转换被认为是在一个连续的过程中发生的，该过程涉及将三个突出的核苷酸 (+CCC) 非模板化添加到新生 cDNA 的 3\'-末端，然后可以退火到模板转换寡核苷酸的匹配 rGrGrG 3\'-末端(TSO)，允许逆转录酶 (RT) 切换模板并继续复制 TSO 序列。在这项研究中，我们对 RNA 模板的模板转换偏差进行了详细分析，特别是序列的作用和其 5\' 端（封端与非封端）在这些偏差中的作用。我们的调查结果确认 5\'-m7G 帽的存在提高了模板切换效率。我们还分析了在没有 TSO 的情况下非模板添加的组成，并观察到 ​​RNA 模板的 5\'-末端影响 RT 的末端转移酶活性。此外，我们发现设计与最常见的非模板添加配对的新 TSO 对提高模板切换效率几乎没有作用。我们的结果提供的证据表明，与目前对模板切换过程的理解相反，非模板添加和模板切换是并发和竞争的过程。

< p>逆转录非模板添加和模板转换之间的并发和竞争性质说明ase.HomeResearch LabsGardner labGardner lab projectsTechnology development and enzyme discoveryTechnology development and enzyme discovery

New England Biolabs 拥有丰富的创新基础研究历史，这些研究赋予了现代分子生物学以力量，并加速了新方法和新技术的开发。我们的实验室不断发现新的酶，了解它们的工作原理，然后将它们应用到新技术中以获得更深入的生物学见解。具体来说，我们正在开发功能宏基因组学和生物信息学管道，以更广泛、更快速地发现新酶。此外，下一代测序 (NGS) 是一种了解酶及其在细胞中的作用方式的强大技术。最近，我们发布了一种新的全基因组 NGS 方法，称为 RADAR-seq，用于分析细胞中的 DNA 复制和修复。目前，我们正在使用 RADAR-seq 了解古细菌 DNA 聚合酶如何合成前导链和滞后链，以及了解 DNA 损伤和在细胞中修复。

我们不断开发新的检测方法以了解酶和将它们应用到新技术中。图为以单分子分辨率研究 DNA 解旋酶的策略（来自 Kelly M. Schermerhorn、Nathan Tanner、Zvi Kelman 和 Andrew F. Gardner。(2016) 高温古细菌 MCM 解旋酶的高温单分子动力学分析。核酸第 44 (18) 号决议：8764-8771。PMID：27382065)

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