Methylation

The following types of methylation analyses are based on the treatment of genomic DNA with sodium bisulfite. This process deaminates unmethylated cytosine residues to uracil leaving methylated cytosine residues unchanged. After PCR, uracil bonds to adenine which will then bond to thymine. The basic concept is that the unmethylated cytosines will become thymines and methylated cytosines will remain cytosines in the genomic sequence. Check out the recommended guidelines on bisulfite sequencing experiments by the NIH.

Applications

Methylation Specific PCR/QPCR

This process uses two different PCR primer sets that are specific to the methylation status of each cytosine associated with the primer sequence. This is the least expensive method of determining methylation for small scale applications and it will only indicate the methylation status of CpG's where the primers anneal.

Bisulfite Sanger Sequencing

This process aims at capturing a given sequence usually 500 to 600 bases in length using PCR. Individual sequences are cloned into a vector and sequenced. Usually ten (but not limited to ten) cloned sequences are used to represent the methylation status for each sample. This method is more expensive than methylation specific PCR, yet more comprehensive, since it measures the methylation status of each CpG within the analyzed sequence.

Genome Wide Methylation Analysis

This can be done using either whole genome Infinium arrays or NGS as both can offer large scale methylation discovery. Whole genome or reduced libraries can be sequenced on the Illumina HiSeq 2500 platform or scanned using the Illumina 450k Beadchip for methlyation profiling.