Methylation-Specific Oligonucleotide Microarray: A New Potential for High-Throughput Methylation Analysis

Here Gitan et al. (2001) reported a microarray- based method for the assessment of the methylation status of DNA sequences. Genomic target DNA is modified by bisulfite treatment, and a PCR amplification of the CpG island is carried out. The amplification products are then labelled with fluorescent dyes, one for unmethylated and another for methylated sequences, and hybridized to the oligo probes fixed in the microarray. The authors evaluated the feasibilty of this development by assessing the methylation status of the estrogen receptor (ER) CpG island.

[...] Test DNA samples are bisulfite-modified, PCR-amplified products that contain pools of DNA fragments with altered nucleotide sequences due to their differential methylation status. As shown, the unmethylated allele of a given DNA sequence is expected to have the unmethylated cytosine of the test CpG sites converted to thymine, whereas these CpG sequences remain unchanged in the methylated allele. Target DNA is then hybridized to arrayed oligonucleotide probes specifically designed to discriminate between converted and unconverted nucleotides at these CpG sites

Schematic outline of the MSO assay (Gitan et al./Genome Res)

[...] As with other oligonucleotide microarrays, cross-hybridization between imperfect-match probes and targets was observed in our initial study using the MSO assay. To overcome this, we have found that selecting the optimal sequence composition for each oligonucleotide probe is critical in the assay. The specificity of a probe drops greatly when it contains more than four consecutive T or G residues. In addition, some probes may have inherently diminished hybridization signals, probably due to decreased duplex stability of targets and probes. Through careful data analysis, we have noticed that cross-reactivity might also increase when oligonucleotide probes are designed to query methylation differences in one single CpG site. This issue is easily overcome by designing probes to include two or more CpG sites. This design consideration may limit our ability to detect methylation changes in single CpG sites. Nonetheless, it is usually not necessary to assess the overall methylation status of a given CpG island by analyzing every CpG site within the locus [...].

Gitan RS. et al. 2001. Genome Res 12:158-164
epigenetics