Detection of DNA methylation by hyperbranched rolling circle amplification and DNA microarray

Aberrant DNA methylation of CpG sites has been confirmed to be closely associated with carcinogenesis. Based on the hyperbranched rolling circle amplification (HRCA) and microarray techniques, a new method for qualitative detection of methylation was developed. In the present study, padlock probes hybridize the sample DNA at the methylation site to form a probe-DNA complex which is ligated and digested simultaneously by methylation specific enzymes. Only at the methylated CpG site is the padlock probe ligated successfully to form a circle template for the HRCA reaction. Utilizing the method of 3-dimensional polyacrylamide gel-based microarray, the HRCA product will be immobilized on the slide to form a DNA microarray, which can universally hybridize the Cy3-labeled oligonucleotide probe to detect the methylation status of CpG sites. To control the false positive signals, DNA ligase and temperature of ligation/digestion are optimized. Methylation status of four CpG sites located in P15, E-cadherin, hMLH1 and MGMT genes were analyzed successfully with this method and all the results were compatible with that of methylation-specific PCR. Our research proves that this method is simple and inexpensive, and could be applied as a high-throughput tool to qualitatively determine the methylation status of CpG sites.

Padlock probes hybridize the sample DNA at the methylation site to form a probe-DNA complex which is ligated and digested simultaneously by methylation specific enzymes. Only at the methylated CpG site the padlock probe is ligated successfully to form a circle template for the HRCA reaction, and then HRCA product will be immobilized on the slide to form a DNA microarray, which can universally hybridize the Cy3-labeled oligonucleotide probe to detect the methylation status of CpG sites.Figure optionsDownload as PowerPoint slide