Abstract
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.
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