对RNA聚合酶和逆转录酶忠度的离散测量揭示了进化的调整
Vladimir Potapov1, Stanislas Krudup1,2, Sean Maguire1
1New England Biolabs, Inc., Ipswich, Massachusetts 01938, USA.
概括
使用单分子测序,Roll-Seq精确地测量了RNA聚合酶和逆转录酶的忠实性. 这种方法可以区分RNA聚合酶与逆转录酶的错误,从而提高了酶表征的准确性.
科学领域:
- 分子生物学分子生物学
- 生物化学 生物化学
- 遗传学 是一个遗传学.
背景情况:
- 由于复杂的样本准备,评估RNA聚合酶和逆转录酶的忠实性是具有挑战性的.
- 现有的方法经常引入错误,使准确的忠实度确定变得复杂.
研究的目的:
- 开发一种新的测序方法,Roll-Seq,用于同时测量RNA聚合酶和逆转录酶的忠实性.
- 通过尽量减少样本操纵和错误引入来克服当前方法的局限性.
主要方法:
- Roll-Seq使用太平洋生物科学公司的单分子实时测序与圆形RNA模板.
- 高滚动循环活动的逆转录酶产生长的协体cDNA.
- 错误来源的区别在于它们存在于单个或所有 concatemers.
主要成果:
- 罗尔-塞克精确测量了T7 RNA聚合酶,Induro RT,R2-RT和LINE-1 RT的忠实度.
- 与第一链相比,LINE-1 RT在第二链合成过程中显示出2.5倍低的保真度.
- 所有测试的逆转录酶在第二链合成过程中都表现出更高的删除和插入率.
- 结构化的RNA模板影响了RNA聚合酶和逆转录酶的忠实性.
结论:
- Roll-Seq提供了一种准确和精确的方法来确定RNA聚合酶和逆转录酶的忠实性.
- 该方法可以作为酶表征的补充工具或用于酶忠实性的选方法.
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