Methylated DNA Immunoprecipitation - Limitations of MeDIP

Limitations of MeDIP

Limitations to take note when using MeDIP are typical experimental factors. This includes the quality and cross-reactivity of 5mC antibodies used in the procedure. Furthermore, DNA detection methods (i.e. array hybridization and high-throughput sequencing) typically involve well established limitations. Particularly for array-based procedures, as mentioned above, sequences being analyzed are limited to the specific array design used.

Most typical limitations to high-throughput, next generation sequencing apply. The problem of alignment accuracy to repetitive regions in the genome will result in less accurate analysis of methylation in those regions. Also, as was mentioned above, short reads (e.g. 36-50bp from an Illumina Genome Analyzer) represent a part of a sheared fragment when aligned to the genome; therefore, the exact methylation site can fall anywhere within a window that is a function of the fragment size. In this respect, bisulfite sequencing has much higher resolution (down to a single CpG site; single nucleotide level). However, this level of resolution may not be required for most applications, as the methylation status of CpG sites within < 1000 bp has been shown to be significantly correlated.