DOUBLE AND TRIPLE IN-SITU CHROMOSOMAL LABELING OF HUMAN SPERMATOZOA BY PRINS

The PRimed IN Situ (PRINS) labeling method allows rapid, specific dete ction of human chromosomes in situ. We have adapted the PRINS protocol to mature human sperm in combination with a 3 M NaOH protocol for sim ultaneous in situ decondensation and denaturation of sperm nuclei. Usi ng fluorochrome-labeled dNTPs in a sequential PRINS reaction, the dire ct detection of two or three distinct chromosomes must be performed wi thin a timespan of 3 h. The method was tested with primers specific fo r chromosomes 8, 9, 12, 13, 16, 18, and 21 and the X. The frequencies of disomy ranged from 0.11% to 0.34%. Chromosome-specific primers have been defined for most of the human chromosomes, including some that a re indistinguishable by fluorescence in situ hybridization (FISH) with centromeric probes. Consequently, this new strategy constitutes a rap id and efficient alternative to FISH for detecting nondisjunction in h uman sperm.