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...
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| Format: | Article |
| Language: | English |
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Wiley
2016-01-01
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| Series: | Journal of Analytical Methods in Chemistry |
| Online Access: | http://dx.doi.org/10.1155/2016/5318935 |
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| author | Jing Wang Xiaoming Pan Xingguo Liang |
| author_facet | Jing Wang Xiaoming Pan Xingguo Liang |
| author_sort | Jing Wang |
| collection | DOAJ |
| description | 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 DNA Tm was higher than 40°C. Both insufficiency and excessiveness of EvaGreen were found to give rise to a little bit higher Tm, showing that the proportion of dye should be considered for precise Tm measurement of nucleic acids. Finally, HRM method was also successfully used to measure Tms 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). |
| format | Article |
| id | doaj-art-499dc971bcf649f986492a7559fde6a2 |
| institution | Kabale University |
| issn | 2090-8865 2090-8873 |
| language | English |
| publishDate | 2016-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Analytical Methods in Chemistry |
| spelling | doaj-art-499dc971bcf649f986492a7559fde6a22025-02-03T06:13:08ZengWileyJournal of Analytical Methods in Chemistry2090-88652090-88732016-01-01201610.1155/2016/53189355318935Assessment for Melting Temperature Measurement of Nucleic Acid by HRMJing Wang0Xiaoming Pan1Xingguo Liang2College of Food Science and Engineering, Ocean University of China, Qingdao 266003, ChinaCollege of Food Science and Engineering, Ocean University of China, Qingdao 266003, ChinaCollege of Food Science and Engineering, Ocean University of China, Qingdao 266003, ChinaHigh 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 DNA Tm was higher than 40°C. Both insufficiency and excessiveness of EvaGreen were found to give rise to a little bit higher Tm, showing that the proportion of dye should be considered for precise Tm measurement of nucleic acids. Finally, HRM method was also successfully used to measure Tms 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).http://dx.doi.org/10.1155/2016/5318935 |
| spellingShingle | Jing Wang Xiaoming Pan Xingguo Liang Assessment for Melting Temperature Measurement of Nucleic Acid by HRM Journal of Analytical Methods in Chemistry |
| title | Assessment for Melting Temperature Measurement of Nucleic Acid by HRM |
| title_full | Assessment for Melting Temperature Measurement of Nucleic Acid by HRM |
| title_fullStr | Assessment for Melting Temperature Measurement of Nucleic Acid by HRM |
| title_full_unstemmed | Assessment for Melting Temperature Measurement of Nucleic Acid by HRM |
| title_short | Assessment for Melting Temperature Measurement of Nucleic Acid by HRM |
| title_sort | assessment for melting temperature measurement of nucleic acid by hrm |
| url | http://dx.doi.org/10.1155/2016/5318935 |
| work_keys_str_mv | AT jingwang assessmentformeltingtemperaturemeasurementofnucleicacidbyhrm AT xiaomingpan assessmentformeltingtemperaturemeasurementofnucleicacidbyhrm AT xingguoliang assessmentformeltingtemperaturemeasurementofnucleicacidbyhrm |