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. 2007 Sep 13;35(18):e119. doi: 10.1093/nar/gkm662

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© 2007 The Author(s)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — SYBR Green melt curve analysis to determine PCR success. SYBR Green PCR melt curve analysis and its compatibility with the MassCLEAVE™ assay was assessed. (A) The use of SYBR Green in the PCR reaction enables a rapid screening for a successful amplification. Here, typical results of a completely methylated (red line), a completely unmethylated (green lines) and a no template control (blue line) PCR of EN1 CpG island are shown. Rapid screening using SYBR Green PCR melt curve analysis also yields information on the global methylation state of the region interrogated (i.e. a temperature shift in the position of the peak in the melt curve, indicated by the two-headed arrow). (B) A comparison of a MassCLEAVE™ analysis with or without SYBR Green for EN1 CpG island (solid blue line or dashed black line, respectively) revealed similar results, indicating that SYBR Green does not influence the methylation calling in the assay. Similar studies for SCTR and INHBB CpG island regions are given as Supplementary Data files.