Epigenetic studies are aimed to find the changes in gene expression and regulation mechanisms which are not in accordance with the genome sequence. These changes are usually heritable in nature. The studies consider the mechanism which causes the phenotypic effects from gene expression during the differentiation and development processes.
There are mainly four processes involved in epigenetics namely DNA methylation, Chromatin remodeling, Histone modification and RNA interference.
DNA methylation is analyzed using various techniques such as
• Bisulfite sequencing - MethyLight, Pyrosequencing, Illumina
• Direct hybridization
• Microarray based methods
The choice of the suitable method depends on the number of samples and number of sites being interrogated.
1. Bulsulfite sequencing
Bisulfite sequencing uses bisulfite to treat DNA to determine the methylation pattern. Methylation usually occurs at the cytosine residues to form 5' methyl cytosine. These residues repress the process of transcription. When bisulfite is added, it causes the cytosine residues to be converted into uracil. However, Bisulfite does not act on 5'methyl cytosine leaving the residues intact.
The treatment with bilsulfite causes specific changes in the DNA sequence which reflects the methylation status of Cytosine residues. The analysis is thus useful to differentiate the single nucleotide polymorphism resulting from the bisulfite treatment of DNA.
In this process, one of the alleles of the gene is methylated while the other remains unmethylated. The changes between the alleles after the denaturation are analyzed using methylation specific or non methylation specific PCR methods.
Non methylation specific PCR procedures
• Direct sequencing:
Bisulfite specific primer sequences are employed for sequencing the genome. This will result in amplification of both methylated and non methylated DNA sequences. The resulting amplified sequences display the unmethylated cytosines as thymine residues. The antisense strand has adenines as corresponding residues. The
Pyrosequencing is used to analyze the bisulfite treated DNA sequence. The technique calculates the ratio of the C:T residues by comparing the number of individual residues incorporated while extending the sequence. Separate analysis of paternal and maternal alleles is possible though this method by the use of allele specific primers. Analysis of genomic imprinting is done through this technique.
• Methylation-sensitive single-strand conformation analysis (MS-SSCA)
It is based on single strand conformation polymorphism analysis (SSCA) for studying single nucleotide polymorphism (SNP). The SSCA method lacks sensitivity with single nucleotide differences. But the bisulfite treatment results in large number of cytosine residues being converted to thymine residues and this increases the sensitivity of the process. Because of this, the method is not appropriate for studying single methylation sites.
• High resolution melting analysis (HRM)
High-resolution melting analysis (HRM) is used to find the single nucleotide polymorphism. The method is based on the technique of real time PCR. The extent of DNA methyaltion is calculated from the C:T ratio as determined by changes in temperature and subsequent release of the fluorescent dye.
• Methylation-sensitive single-nucleotide primer extension (MS-SnuPE)
The bisulfite treated DNA is annealed to bisulfite specific primers up to the base pair before the gene sequence of interest. The primer is extended and the ratio of C: T is determined.
The process employs a wide range of methods including radioactive labeling of ddNTPs, fluorescence based methods, pyrosequencing etc to determine the ratio. Ion pair- Reverse phase HPLC and MALDI-TOF ( Matrix assisted laser desorption ionization/time of flight) methods are also widely employed to analyze the extended sequences and to calculate the C:T ratio.
Methylation specific PCR (MSP) procedures
The MSP method identifies the methylated region discriminately and amplifies the selected regions. Methylation specific primers are used which anneal specifically to methylated sequences. Unmethylated DNA sequence specific primers can be employed to determine the C: T ratio. The ability of the primer to amplify the sequence is used to determine the extent of methylation. The method is best suited for determining the methylation status at a locus.
• MethyLight method
This method is also based on MSP procedure, but gives a more accurate analysis of methylation using real time PCR technique. Methylated sequence specific primers and fluorescence probes are employed in this method.
• Melting curve analysis (Mc-MSP)
The bisulfite treated DNA is amplified and then annealed with both methylated and non methylated specific primers. The ratio of the products is compared by analyzing the melting curve. Real time quantification is possible and the method is more sensitive in detecting low amounts of methylation in the DNA sequences.
2. Direct hybridization
Direct hybridization to CpG island arrays are used to analyze the extent of methylation either with radioactive or fluorescent probes. This is again based on the principle of melting curve analysis.
3. Microarray methods
Oligonucelotide microarrays are designed for CpG sites. One of the sequences are complementary to the methylated genome sequence while the other is complementary to the unmethylated sequence. Bisulphate specific oligonucleotide arrays are used.
Illumina allows detection of methylation in large number of samples up to 96. By employing the microarray technique it is possible to generate genome wide data on methylation. Although the method is suitable for detecting polymorphism, this requires extensive design of primers. Restriction enzyme based enrichment methods are used for large scale analysis.
Other prominent methods used include
• MeDIP, or methylated DNA immunoprecipitation, an antibody-mediated methyl-specific fractionation;
• HELP, or HpaII tiny fragment enrichment by ligation-mediated PCR
• McrBC fractionation, an enzyme that cuts most methylated DNA.
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