Microarray-based method to analyze methylation status of E-cadherin gene in leukemia.

BACKGROUND

Aberrant DNA methylation of the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. Recently, several studies have indicated the aberrant methylation of E-cadherin gene could be a potential marker for leukemic patients.

METHOD

We used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated, but not methylated, cytosine into thymine within CpG islands of interest. The amplified product containing a pool of DNA fragments with altered nucleotide sequences was then hybridized with an oligonucleotide-based microarray. Five sets of oligonucleotide probes were designed to detect the methylation patterns of E-cadherin gene CpG islands in leukemia samples. The results were further validated by methylation-specific PCR (MSP).

RESULTS

We found that all leukemia samples were methylated at different levels within the target sequences. The specific regions (the CpG sites #16-19 and #20-22) were revealed as hotspots for methylation in leukemic patients. These results showed that the microarray assay could successfully detect methylation changes of E-cadherin gene in leukemia quantitatively.

CONCLUSION

The oligonucleotide-based microarray can be a quick and reliable tool to map methylation status in CpG islands. This established microarray could be potentially useful for clinical researches and diagnosis.