Very low rate Y-chromosome mosaicism (1:5,400) detectable by a novel probe enzyme combination

DYZ1 is a repetitive DNA family located on the long arm of the Y chromosome and is the major component of the Q-positive region. DYZ1 consists of about 3,000 copies of a 3.4 kb repeat unit which mainly consists of a tandem array of pentanucleotides, TTCCA. Because of this large number of repeats, DYZ1 has been used as a probe in Southern hybridization for sensitive and rapid detection of the Y chromosome. In cases of XX/XY mosaicism, however, autosomal sequences having homology to DYZ1 hinder the detection of the Y chromosome, especially when the ratio of the Y-bearing cells is low. To solve this problem and improve the detection limit, we have sought the optimum hybridization condition by changing several variables. These variables include the length of probes, the methods of probe labeling, the endonucleases used to digest the genomic DNA and the hybridization buffer. Here we show that the StuI digestion of genomic DNA in combination with the nick translated DYZ1 probe significantly improves the detection limit of the Y-chromosome bearing cells. The presence of Y-chromosome bearing cells was detectable against a background of 5,400-fold female DNA.