An innovative variation of fluorescence in situ hybridization (FISH) allows preparation of single-stranded chromatids for determination of chromosomal orientation and strand direction of DNA sequences (1). Standard fluorescent in situ hybridization methods rely on nucleotide base pairing between a labeled probe and the complementary chromosomal target sequence. Both probe and target DNAs must be functionally single-stranded at the start of the procedure. Single-stranded probes can be constructed or prepared, but the chromosomal DNA must be made single-stranded by denaturation. Because both strands are present in both chromatids of each chromosome, even single-stranded probes will hybridize to both chromatids of the target chromosome.