Figure 2a: Methylation Specific Enzyme Digestion. Methylation sensitive restriction enzymes (HpaII and HhaI) cleave DNA on their recognition sites {100bp away of the highly polymorphic (CAG)n repeat in exon1 of the HUMARA gene}. If random X-Inactivation occurs then PCR products from both alleles are detected. Conversely, in the case of skewed X-Inactivation, cleavage of non-methylated allele abolishes primer annealing site and leads to preferential or isolated amplification of the methylated allele on the inactive X-chromosome.