Hotel Estoril Eden, Monte Estoril,
Portugal
5-8 October 2005

 

 
 

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Developing DNA Methylation Array Technology

Natalie Thorne
Cambridge University, UK

DNA methylation plays an important role in regulation of gene transcription and is strongly implicated in cancer development.  Current methods to detect DNA methylation lack a practical high-throughput genome-wide profiling method.  This is limiting the identification of potential new DNA methylation markers that predict or promote neoplastic progression.

We have developed and validated a method called Microarray Methylation Assessment of a Single Sample (MMASS) to compare the methylated with unmethylated CpG islands from a single source using an annotated 12K CpG islands microarray and two colour competitive hybridisation.  An existing approach, Differential Methylation Hybridisation (DMH), compares methylation between two samples.  While another common approach is to assess the abundance of methylated CpG islands within a single sample, potentially limiting the detection of unmethylated CpG islands. MMASS should provide a useful approach to assessing multiple independent samples and enable profiling of methylation patterns in normal tissue where a reference sample for DMH may be problematic.

We used various approaches to validate and compare the performance of MMASS methods. In particular, we have made use of unmethylated mitochondrial genes; we designed and used methylated and unmethylated spiked sequences for which there is a probe within the arrays and we validated the methylation status of a subset of genes using combined bisulphite restriction analysis (COBRA). 

Results on the analysis, comparison and assessment of four methods will be presented, including details of our approach for normalising the inherently non-symmetric log-ratio data resulting from this kind of methylation profiling.  Investigations into sequence features such as probe length, repeat element content and number of methylation sensitive restriction sites will also be discussed.