...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Applied and Environmental Microbiology >Sensitive, Efficient Quantitation of 13C-Enriched Nucleic Acids via Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry for Applications in Stable Isotope Probing
【24h】

Sensitive, Efficient Quantitation of 13C-Enriched Nucleic Acids via Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry for Applications in Stable Isotope Probing

机译：通过高效液相色谱-串联质谱对稳定同位素探测中的应用进行灵敏，高效的13C富集核酸定量

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Stable isotope probing (SIP) of nucleic acids is a powerful tool for studying the functional traits of microbial populations within complex communities, but SIP involves a number of technical challenges. Many of the difficulties in DNA-SIP and RNA-SIP experiments can be effectively overcome with an efficient, sensitive method for quantitating the isotopic enrichment of nucleic acids. Here, we present a sensitive method for quantitating 13C enrichment of nucleic acids, requiring a few nanograms of sample, and we demonstrate its utility in typical DNA-SIP and RNA-SIP experiments. All five nucleobases (adenine, guanine, cytosine, thymine, and uracil) were separated and detected by using ultrahigh-performance liquid chromatography–tandem mass spectrometry. We detected all isotopic species in samples with as low as 1.5 atom% 13C above natural abundance, using 1-ng loadings. Quantitation was used to characterize the isotopic enrichment kinetics of cellulose- and lignin-based microcosm experiments and to optimize the recovery of enriched nucleic acids. Application of our method will minimize the quantity of expensive isotopically labeled substrates required and reduce the risk of failed experiments due to insufficient recovery of labeled nucleic acids for sequencing library preparation.
机译：核酸的稳定同位素探测（SIP）是研究复杂社区内微生物种群功能特征的强大工具，但是SIP涉及许多技术挑战。 DNA-SIP和RNA-SIP实验中的许多困难可以通过有效，灵敏的定量核酸同位素富集的方法来克服。在这里，我们提出了一种定量13C富集核酸的灵敏方法，需要几纳克的样品，并且我们证明了其在典型的DNA-SIP和RNA-SIP实验中的效用。使用超高效液相色谱-串联质谱法分离并检测了所有五个核碱基（腺嘌呤，鸟嘌呤，胞嘧啶，胸腺嘧啶和尿嘧啶）。我们使用1-ng装载量检测到样品中所有同位素物种的样品都比自然丰度高出1.5原子％13C。定量用于表征基于纤维素和木质素的微观世界实验的同位素富集动力学，并优化富集核酸的回收率。我们方法的应用将最大程度地减少所需的昂贵同位素标记底物的数量，并降低由于无法回收用于测序文库制备的标记核酸而导致实验失败的风险。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号