Date of Award
DNA microarrays, Oligonucleotides -- Methylation, DNA -- Methylation, Microarray, Methylation, Oligonucleotide, Methylation-specific PCR, SBE-TAGs
DNA methylation is a key event regulating gene expression. DNA methylation analysis plays a pivotal role in unlocking association of epigenetic events with cancer. However, simultaneous evaluation of the methylation status of multiple genes is still a technical challenge. Microarray is a promising approach for high-throughput analysis of the methylation status at numerous CpG sites within multiple genes of interest. In this dissertation study, we conducted a systematic study to examine the use of microarray methods for methylation analysis. First, a robust universal microarray was established with more flexible in design and content, and potential cost saving over commercial arrays. In order to produce high quality microarray data, we optimized the attachment chemistry for the modified oligonucleotides, searched for the good combination of fluorescent dyes, and hybridization conditions. To improve the specificity of the microarray, we conducted a study to experimentally search for a set of highly discriminative tag Sequences. Second, SBE-TAGs microarray was successfully adapted from the SNP detection for methylation analysis of multiple genes. SBE-TAGs microarray performed quite well in multiplex methylation analysis of cell lines if a standard calibration curve method was used. 10 CpG sites of 9 tumor suppressor genes (MGMT, GATA4, HLTF, SOCS1, p16, RASSF2, CHFR, TPEF, and Reprimo) were selected for this study. Third, a novel method called CHZMA (Competing-Hybridization- Zipcode-MicroArray) was developed for methylation analysis of tumor tissue samples, which is based on two steps of hybridization to achieve the specific detection of methylation on microarray. On the basis of analysis of seven genes (MGMT, GATA4, HLTF, SOCS1, RASSF2, ER, 3-OST-2), we found that the CHZMA assay can robustly detect methylation of multiple genes in the samples containing as low as 10 of methylated DNA. With the strict control group test and statistical analysis, CHZMA can be a good high-throughput method in place of MSP for methy
Li, Xiaopeng, "Development of Oligonucleotide Microarray for High Throughput Dna Methylation Analysis" (2008). ETD Archive. 182.