Slab Gel Electrophoresis

From the very beginning of recombinant DNA technology, slab gel analysis has been a favorite technique to separate DNA fragments. In epigenetic analysis, it is used in combination with restriction enzymes (MSREs) sensitive to 5mC for RFLP/Southern blot analyses, or with specific sequencing protocols such as bisulfite and hydrazine/permanganate sequencing. More recently, slab gel analysis has often been replaced by separation techniques that are more sensitive, less labor intensive, and automatable. In recent years, bisulfite sequencing has been used as the standard method to detect DNA methylation at CpG islands. Genomic DNA first reacted with bisulfite converts unmethylated cytosine to uracil while leaving 5mC unchanged (Figure 6.1). The conversion to uracil is detected with specifically designed PCR primers. Methylation-specific restriction enzymes employed in combination with PCR provide a very sensitive, commonly used technique that requires much less DNA than traditional Southern blot analysis. A widely used combination of MSREs for detection of methylated cytosines is MspI/HpaII isoschizomers, although other restriction enzymes afford alternatives to one or both of these. For instance, detection of the methylation state of the FMR1 gene promoter used analysis of EcoRI and EagI digests of DNA from fragile X patients to distinguish the normal genotype, the premutation, and the full mutation.83 Bisulfite sequencing is straightforward and efficient and in the event that a site is only partially methylated, it has the added advantage of enabling the determination of the proportion of cells that is methylated. Bisulfite sequencing has been combined with other methodologies such as "combined bisulfite restriction analysis and amplification,'' abbreviated COBRA.84 COBRA is relatively easy to use, quantitatively accurate, and compatible with paraffin sections. Another method that uses bisulfite sequencing is ''methylation-sensitive single nucleotide primer extension,'' abbreviated MS-SNuPE. This approach uses very small amounts of DNA, can be used with microdissected material, and avoids the use of restriction enzymes.

The MS-SNuPE technique has also been adapted to semiautomatic detection of DNA methylation at CpG islands.77

Was this article helpful?

0 0

Post a comment