Assessment for Melting Temperature Measurement of Nucleic Acid by HRM.
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 (Tm) 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 DNATmwas higher than 40°C. Both insufficiency and excessiveness of EvaGreen were found to give rise to a little bit higherTm, showing that the proportion of dye should be considered for preciseTmmeasurement of nucleic acids. Finally, HRM method was also successfully used to measureTms 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).
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
Jing
0000-0003-2267-4287
J
Wang
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
Xiaoming
0000-0001-8307-2736
X
Pan
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
Xingguo
0000-0001-7778-4593
X
Liang