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通过高分辨率熔解曲线分析进行核酸解链温度测量的评估。

Assessment for Melting Temperature Measurement of Nucleic Acid by HRM.

作者信息

Wang Jing, Pan Xiaoming, Liang Xingguo

机构信息

College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.

出版信息

J Anal Methods Chem. 2016;2016:5318935. doi: 10.1155/2016/5318935. Epub 2016 Oct 19.

DOI:10.1155/2016/5318935
PMID:27833775
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5090098/
Abstract

High resolution melting (HRM), with a high sensitivity to distinguish the nucleic acid species with small variations, has been widely applied in the mutation scanning, methylation analysis, and genotyping. For the aim of extending HRM for the evaluation of thermal stability of nucleic acid secondary structures on sequence dependence, we investigated effects of the dye of EvaGreen, metal ions, and impurities (such as dNTPs) on melting temperature ( ) measurement by HRM. The accuracy of HRM was assessed as compared with UV melting method, and little difference between the two methods was found when the DNA was higher than 40°C. Both insufficiency and excessiveness of EvaGreen were found to give rise to a little bit higher , showing that the proportion of dye should be considered for precise measurement of nucleic acids. Finally, HRM method was also successfully used to measure s of DNA triplex, hairpin, and RNA duplex. In conclusion, HRM can be applied in the evaluation of thermal stability of nucleic acid (DNA or RNA) or secondary structural elements (even when dNTPs are present).

摘要

高分辨率熔解曲线分析（HRM）对区分微小差异的核酸种类具有高灵敏度，已广泛应用于突变扫描、甲基化分析和基因分型。为了扩展HRM用于评估核酸二级结构热稳定性的序列依赖性，我们研究了EvaGreen染料、金属离子和杂质（如dNTPs）对HRM测量熔解温度（ ）的影响。与紫外熔解方法相比评估了HRM的准确性，当DNA 高于40°C时，发现两种方法之间差异很小。发现EvaGreen不足和过量都会导致 略高，表明为了精确测量核酸的 应考虑染料的比例。最后，HRM方法也成功用于测量DNA三链体、发夹结构和RNA双链体的 。总之，HRM可应用于评估核酸（DNA或RNA）或二级结构元件的热稳定性（即使存在dNTPs时）。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbf8/5090098/6ce46cd1c0fc/JAMC2016-5318935.006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbf8/5090098/bf5ceeaad81a/JAMC2016-5318935.001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbf8/5090098/9a5b979ac8d7/JAMC2016-5318935.002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbf8/5090098/8d1cb149bb02/JAMC2016-5318935.003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbf8/5090098/3c2422e344ea/JAMC2016-5318935.004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbf8/5090098/926cca94efb4/JAMC2016-5318935.005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbf8/5090098/6ce46cd1c0fc/JAMC2016-5318935.006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbf8/5090098/bf5ceeaad81a/JAMC2016-5318935.001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbf8/5090098/9a5b979ac8d7/JAMC2016-5318935.002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbf8/5090098/8d1cb149bb02/JAMC2016-5318935.003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbf8/5090098/3c2422e344ea/JAMC2016-5318935.004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbf8/5090098/926cca94efb4/JAMC2016-5318935.005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbf8/5090098/6ce46cd1c0fc/JAMC2016-5318935.006.jpg

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