Allele-specific extension on microarray for DNA methylation analysis

Aberrant DNA methylation of CpG site in the gene promoter region has been confirmed to be closely associated with carcinogenesis. In this present study, a new method based on the allele-specific extension on microarray technique for detecting changes of DNA methylation in cancer was developed. The target gene regions were amplified from the bisulfite treated genomic DNA (gDNA) with modified primers and treated with exonuclease to generate single-strand targets. Allele-specific extension of the immobilized primers took place along a stretch of target sequence with the presence of DNA polymerase and Cy5-labeled dGTP. To control the false positive signals, the hybridization condition, DNA polymerase, extension time and primers design were optimized. Two breast tumor-related genes (P16 and E-cadherin) were analyzed with this present method successfully and all the results were compatible with that of traditional methylation-specific PCR. The experiments results demonstrated that this DNA microarray-based method could be applied as a high throughput tool for methylation status analysis of the cancer-related genes, which could be widely used in cancer diagnosis or the detection of recurrence.