Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/88372
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Type: Journal article
Title: Differential effect of three base modifications on DNA thermostability revealed by high resolution melting
Author: Rodriguez Lopez, C.
Lloyd, A.J.
Leonard, K.
Wilkinson, M.J.
Citation: Analytical Chemistry, 2012; 84(17):7336-7342
Publisher: American Chemical Society
Issue Date: 2012
ISSN: 0003-2700
1520-6882
Statement of
Responsibility: 
Carlos M. Rodríguez López, Amanda J. Lloyd, Kate Leonard and Mike J. Wilkinson
Abstract: High resolution melting (HRM) can detect and quantify the presence of 5-methylcytosine (5mC) in DNA samples, but the ability of HRM to diagnose other DNA modifications remains unexplored. The DNA bases N6-methyladenine and 5-hydroxymethylcytosine occur across almostall phyla. While their function remains controversial, their presence perturbs DNA structure. Such modifications could affect gene regulation, chromatin condensation and DNA packaging. Here, we reveal that DNA containing N6-methyladenine or 5-hydroxymethylcytosine exhibits reduced thermal stability compared to cytosine-methylated DNA. These thermo-stability changes are sufficiently divergent to allow detection and quantification by HRM analysis. Thus, we report that HRM distinguishes between sequence-identical DNA differing only in the modification type of one base. This approach is also able to distinguish between two DNA fragments carrying both N6-methyladenine and 5-methylcytosine but differing only in the distance separating the modified bases. This finding provides scope for the development of new methods to characterize DNA chemically and to allow for low cost screening of mutant populations of genes involved in base modification. More fundamentally, contrast between the thermostabilizing effects of 5mC on dsDNA compared with the destabilizing effects of N6-methyladenine (m6A) and 5-hydroxymethylcytosine (5hmC) raises the intriguing possibility of an antagonistic relationship between modification types with functional significance.
Keywords: Cytosine; 5-Methylcytosine; Adenine; DNA; Cluster Analysis; Nucleic Acid Denaturation; Phase Transition; Principal Component Analysis; Transition Temperature
Rights: © 2012 American Chemical Society
RMID: 0030007033
DOI: 10.1021/ac301459x
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