Specific Genomic Location Analysis
The following four 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. Upon PCR, uracil hydrogen bonds to adenine which will then hydrogen bond to thymine. Therefore, the concept is that unmethylated cytosines will become thymines and methylated cytosines will remain cytosines in the genomics sequence.
Methylation Specific PCR
This process uses two different PCR primer sets that are specific to the methylation status of each cytosine associated with the sequence were the primers will anneal. This is the least expensive method of determining methylation, however it will only indicate the methylation status of CpG's where the primers anneal.
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. A cytosine in the sequencing result would indicate a cytosine methylation while a thymine indicates absence of cytosine methylation. This method is more expensive than methylation specific PCR, however it is more comprehensive since it measures the methylation status of each CpG within the analyzed sequence.
This process is similar to bisulfite sequencing in that it will determine the methylation status at each CpG within a sequence. It does have several advantages over bisulfite sequencing. 1) Methylation status of each CpG is based on a very large population of sequences while bisulfite sequencing results are representative of a random sampling of only ten sequences. 2) Pyrosequencing is slightly less expensive than bisulfite sequencing per region investigated. For this process we utilize the Qiagen PyroMark Pyrosequencer instrumentation and software.
Genome Wide Methylation Analysis
Currently we are using two procedures. Methyl-sensitive cut counting (MSCC) developed by Ball et al. 2009*, generates a library of tags derived from all genomic locations that can be cut by the methylation sensitive restriction endonucleases like HpaII (CCmGG). For example, approximately 1,417,432 unique HpaII sites (greater than 40 bases from adjacent HpaII site, sequence present only once in genome) across the human genome (hg18) can be profiled using this method. Digestion of a parallel sample with the non-methylation sensitive MspI (CCGG) establishes the percent methylation when compared to HpaII which generates the unmethylated fraction of CpG's. High throughput sequencing of the generated tags is performed using the Illumina Genome Analyzer.
We also offer the Methyl CpG Binding Domain - Isolated Genome Sequencing (MiGS) method developed by Serre et al. 2010**. This method allows for the isolation of sheared DNA fragments that have methylated CpG's using the methyl CpG binding domain of the MBD2 protein. Samples are subjected to shearing using a sonicator. The sequences which contain methylated CpG's are enriched. With some modifications, these fragments are sequenced using the capabilities of the high throughput sequencing Illumina Genome Analyzer. Sample signal counts at each genomic location are compared between a control and treatment to determine changes in methylation.
*Ball MP, Li JB, Gao Y, Lee J, LeProust EM, Park I, Xie B, Daley GQ and Church GM. Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells. 2009, Nature Biotechnology 27, 361 - 368.
**Serre D, Lee BH and Ting AH. MBD-isolated Genome Sequencing provides a high-throughput and comprehensive survey of DNA methylation in the human genome. 2010. Nucleics Acids Research 38:2, 391-399.