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Methylation Profile Methods
There are numerous other methods that utilize restriction enzymes for methylation profiling. These include:
- Methylation-Sensitive Cut Counting Assay (MSCC) involves analyzing untreated and bisulfite treated DNA by assessing differential migration of single-stranded DNA containing the CpG sites of interest through non-denaturing gels. The C to T content will vary with methylation status (1,2).
- Methylation specific PCR (MSP) involves analyzing untreated and bisulfite treated DNA using two sets of PCR primer pairs that target the unaltered, methylated sequence and the converted, unmethylated sequence (3,4).
- Quantitative Analysis by methylation-sensitive PCR (qAMP) involves using methylation sensitive enzymes to fragment genomic DNA for quantitative analysis by real-time PCR (5).
- Restriction Landmark Genome Scanning (RLGS) is a method that uses 2-dimensional gel electrophoresis to separate DNA fragments generated with methylation-sensitive restriction enzymes (6,7).
- Combined Bisulfite Restriction Analysis (COBRA) involves digesting PCR amplicons from untreated, and bisulfite treated DNA with methylation-sensitive or -insensitive restriction enzymes. The resulting DNA fragments are electroblotted, hybridized to radiolabeled oligonucleotides and quantitated by densiometry (8).
- High-Throughput Genome-wide methylation profiling and analysis, can be accomplished by CpG Island microarrays. CpG islands are targeted using oligonucleotide adaptors. These are prepared by two rounds of a combination of methylation-sensitive and methylation-insensitive nuclease digests, PCR amplicons are fluorochrome labeling, for microarray of sample and control genomic DNAs (9).
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