Figure 4.

Methylation-sensitive restriction endonuclease SacII digest of genomic DNA, 'hot-stop' PCR and subsequent BstUI digest to discriminate between the maternal and paternal alleles. A Schematic representation of the DNA methylation analysis using methyl-sensitive restriction endonuclease SacII and 'hot-stop' PCR in the H19 DMD containing the P2 repeat. The recognition site of SacII, 'hot-stop' PCR primers and the BstUI recognition sites are indicated. B Scanned agarose gel by infrared fluorescent detection. Lane 1 is an undigested 464 bp product that is 29 bp longer than the digested longer allele since there is a second BstUI restriction site. Lanes 2 and 3 show 137 bp fragments from foetal DNA indicating paternal hypermethylation and maternal hypomethylation at the CTCF P2 binding site. Lanes 4 and 5 are the 137 bp DNA fragments from newborn tissues and lanes 6 and 7 (137 bp and 435 bp fragments, the later having the alternative paternal allele methylated) are amplified DNA fragments from adult tissues. Also in lanes 6 and 7 paternal hypermethylation and maternal hypomethylation of the CTCF P2 binding sites are demonstrated. In lane 8 a control result of a 1:1 mix of DNA from respective homozygous CC and TT individuals for the AY044827.1:g.32530.C>T polymorphism is shown.