Multiplex detection of CpG methylation using microarray combining with target-selection-padlock probe

BackgroundMicroarray technology combining with bisulfite-PCR offers a high-throughput approach for detection of DNA methylation. However, the use of microarray-based DNA methylation analysis has been limited by the low throughput of sample preparation due to the difficulty in simultaneous amplification of multiple targets.MethodsA set of target-selection-padlock probes was designed to capture the target sequences containing the queried CpG sites from bisulfite-treated genomic DNA. Then all targets were simultaneously amplified by a pair of common primers. The methylation status of multiple targets was detected by single base extension (SBE) on oligonucleotide microarray based on polyacrylic acid-covered surface.ResultsThis assay has been successfully applied to analyze promoter methylation of 8 tumor suppressor genes in 12 colorectal cancer samples and 2 normal control samples. The target-selection-padlock probe exhibited both high specificity and high efficiency for the parallel amplification of multiple genes. The accurate and high-throughput detection for DNA methylation was achieved by a combination of target-selection-padlock probes and microarray.ConclusionsThe present study provides a robust and accurate assay for DNA methylation status of multiple genes. This method may be useful for a large-scale screen of DNA methylation in cancer cell lines and clinical samples.