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Novel Iodine-induced Cleavage Real-time PCR Assay for Accurate Quantification of Phosphorothioate Modified Sites in Bacterial DNA.
Scientific Reports ( IF 3.8 ) Pub Date : 2019-05-16 , DOI: 10.1038/s41598-019-44011-x Yi Chen 1 , Tao Zheng 1 , Jinli Li 1 , Jinjie Cui 2 , Zixin Deng 1 , Delin You 1 , Litao Yang 1, 2
Scientific Reports ( IF 3.8 ) Pub Date : 2019-05-16 , DOI: 10.1038/s41598-019-44011-x Yi Chen 1 , Tao Zheng 1 , Jinli Li 1 , Jinjie Cui 2 , Zixin Deng 1 , Delin You 1 , Litao Yang 1, 2
Affiliation
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DNA Phosphorothioate (PT), replacing a non-bridging phosphate oxygen atom with a sulfur atom, is one kind of common DNA modification in bacteria. Whole genome scale description of the location and frequency of PT modification is the key to understand its biological function. Herein we developed a novel method, named with iodine-induced cleavage quantitative real-time PCR (IC-qPCR), to evaluate the frequency of PT modification at a given site in bacterial DNA. The efficiency, dynamic range, sensitivity, reproducibility and accuracy of IC-qPCR were well tested and verified employing an E. coli B7A strain as example. The amplification efficiency of IC-qPCR assay ranged from 91% to 99% with a high correlation coefficient ≥0.99. The limit of quantification was determined as low as 10 copies per reaction for the 607710 and 1818096 sites, and 5 copies for the 302695 and 4120753 sites. Based on the developed IC-qPCR method, the modification frequency of four PTs in E. coli B7A was determined with high accuracy, and the results showed that the PT modification was partial and that the modification frequency varied among investigated PT sites. All these results showed that IC-qPCR was suitable for evaluating the PT modification, which would be helpful to further understand the biological function of PT modification.
中文翻译：
新型碘诱导裂解实时 PCR 测定可准确定量细菌 DNA 中硫代磷酸酯修饰位点。
DNA硫代磷酸酯（PT），用硫原子取代非桥联磷酸氧原子，是细菌中常见的一种DNA修饰。全基因组规模描述PT修饰的位置和频率是理解其生物学功能的关键。在此，我们开发了一种新方法，称为碘诱导裂解定量实时 PCR (IC-qPCR)，用于评估细菌 DNA 中给定位点的 PT 修饰频率。以大肠杆菌B7A菌株为例，对IC-qPCR的效率、动态范围、灵敏度、重现性和准确性进行了充分的测试和验证。 IC-qPCR检测的扩增效率为91%~99%，相关系数≥0.99。对于 607710 和 1818096 位点，定量限制低至每个反应 10 个拷贝，对于 302695 和 4120753 位点，每个反应的定量限制低至 5 个拷贝。基于所开发的IC-qPCR方法，高精度地测定了大肠杆菌B7A中四个PT的修饰频率，结果表明PT修饰是部分的，并且不同PT位点的修饰频率存在差异。这些结果表明IC-qPCR适合评价PT修饰，有助于进一步了解PT修饰的生物学功能。
更新日期：2019-05-16
中文翻译：
新型碘诱导裂解实时 PCR 测定可准确定量细菌 DNA 中硫代磷酸酯修饰位点。
DNA硫代磷酸酯（PT），用硫原子取代非桥联磷酸氧原子，是细菌中常见的一种DNA修饰。全基因组规模描述PT修饰的位置和频率是理解其生物学功能的关键。在此，我们开发了一种新方法，称为碘诱导裂解定量实时 PCR (IC-qPCR)，用于评估细菌 DNA 中给定位点的 PT 修饰频率。以大肠杆菌B7A菌株为例，对IC-qPCR的效率、动态范围、灵敏度、重现性和准确性进行了充分的测试和验证。 IC-qPCR检测的扩增效率为91%~99%，相关系数≥0.99。对于 607710 和 1818096 位点，定量限制低至每个反应 10 个拷贝，对于 302695 和 4120753 位点，每个反应的定量限制低至 5 个拷贝。基于所开发的IC-qPCR方法，高精度地测定了大肠杆菌B7A中四个PT的修饰频率，结果表明PT修饰是部分的，并且不同PT位点的修饰频率存在差异。这些结果表明IC-qPCR适合评价PT修饰，有助于进一步了解PT修饰的生物学功能。
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