Method Mechanism Additional information Limitations Assays
Enrichment of differences in Methylation
Bisulfite conversion Converts unmethylated cytosines to uracil (and eventually thymine following PCR amplification), while leaving methylated cytosines unconverted. PCR product may be analyzed by direct sequencing or mass spectrometry to quantify cytosine methylation -Conversion needs to be complete for accurate results.
-Labor intensive & high cost prohibits genome-wide application. Partially overcome by combining with MS-PCR, microarrays & high-throughput sequencing. 		Pyrosequencing
 MassARRAY
Methylation-Sensitive Restriction Digestion Assays Uses restriction enzyme that identifies methylated DNA. Often used in initial genome-wide screening Limited to the analysis of CpG sites located within the enzyme recognition site HELP, McrBC
Immunoprecipitation-Based Enrichment Assays Uses antibodies that recognize methylated CpG to enrich methylated DNA In combination with DNA microarrays (ChIP-chip) & massively parallel sequencing (ChIP-Seq), has made genome-wide analysis easier Lack of specificity in low CpG-dense regions due to noise MeDip

Analysis of Methylation

High Throughput Interrogation
Bead arrays Uses two primers each recognizing methylated Vs unmethylated cytosine   Provides less coverage than other array-based methods Illumina
Short oligonucleotide arrays Millions of 25-mer probes per chip in a tiling design Good specificity Decreased sensitivity, noise Affymetrix
Long oligonucleotide arrays 60-mer probes, can analyze test and control DNA on the same array Better balance between sensitivity, specificity and noise   Nimblegen, Agilent
Massively Parallel sequencing Involves direct sequencing of enriched DNA -Best-possible resolution and quantitative methylation information (using bisulfite converted DNA)
-Data can be re-analyzed if needed 		  Fluorescent nucleotide-based system (Illumina), MethylC-seq, SMRT (Pacific Biosciences)
PCR – Polymerase chain reaction MS-PCR – Methylation specific PCR HELP - HpaII tiny fragment enrichment by ligation-mediated PCR McrBC - an endonuclease which cleaves DNA containing methylcytosine on one or both strands MeDIP - Methylated DNA immunoprecipitation SMRT - Single molecule real time sequencing
Table 1: Common Assays for Methylation Analysis.