In situ hybridization to metaphase chromosomes and interphase nuclei

In situ hybridization is used to determine the chromosomal map location and the relative order of genes and DNA sequences within a chromosomal band. It can also be used to detect aneuploidy, gene amplification, and subtle chromosomal rearrangements. Fluorescence in situ hybridization (FISH), probably the most widely used method, is described in the first basic protocol. Two support protocols are provided to amplify weak fluorescent signals obtained in FISH. Nonisotopic probes can also be detected by enzymatic reactions using horseradish peroxidase or alkaline phosphatase, as described in alternate protocols. Nonisotopic labeling of DNA probes by nick translation is described in a support protocol. The order of closely spaced FISH probes along chromosomes in interphase nuclei can be determined. A basic protocol for isotopic in situ hybridization (IISH) with (3)H is provided followed by a support protocol for preparation of autoradiographic emulsion.