Identification of a DNA fragment which escaped methylation inactivation on the X chromosome by two-dimensional electrophoresis of human genomic DNA

Genomic DNA was digested with NotI and EcoRV and labelled at the NotI recognition sites. After first-dimension electrophoresis, DNA in the disc gel was further digested in situ with HinfI prior to second dimension electrophoresis. Approximately 2000 end-labelled fragments were visualized simultaneously. We have analyzed the two-dimensional preparations derived from six mother/father/child trios by using a computer assisted image analyzer. In the quantitative study, a separate fragment (spot 369) was observed in which male appeared to have a one-copy spot, while female always exhibited a spot twice this size. This could be interpreted as the result of a NotI site on the X chromosome that is not methylated on either X in females (escaped methylation inactivation on the X chromosome). We have cloned this DNA fragment to confirm that the DNA fragments really existing on the X chromosome. The cloned DNA fragment was mapped to Xp11.4 by FISH analysis. We have also sequenced the DNA fragment, but there was no identical sequence in the DNA data base.