Inactive X chromosome has the highest concentration of unmethylated Hha I sites.

A procedure enabling the highly sensitive detection of accessible restriction endonuclease sites on metaphase chromosomes is described. The procedure is based on the following: (i) a terminal deoxynucleotidyltransferase is used to add a biotinylated nucleotide (Bio-11-dUTP) tail to the 3' hydroxyl terminus generated by the action of a restriction enzyme and (ii) the biotinylated oligonucleotide is detected by a peroxidase-based immunocytochemical method. When used with the 5-methylcytosine-sensitive enzyme Hha I, it gives rise to a pattern close to R and T banding on autosomes. In addition, the staining of one X chromosome in females appears very unusual by its pattern and its strong intensity. This procedure, as applied on a case with a polysomy X chromosome, provides direct evidence of an overall hypomethylation of the inactive X chromosomes.