21] In situ hybridization of metaphase and prometaphase chromosomes

Abstract

Publisher Summary This chapter presents in-situ hybridization of metaphase and prometaphase chromosomes. In-situ hybridization has been used with probes cloned into plasmids and phage, genomic sequences and cDNA. The longer the probe, the more labeling is seen at a specific site. However, a longer probe is more likely to contain repetitive sequences. In-situ hybridization is done in conjunction with somatic cell hybrid studies. To ensure an unbiased assay, grains are scored by a person who does not know the chromosome location. Genes with multiple copies are hybridized in a manner similar to single copy probes. Statistical analysis of the data reveals the number of sites. The identification of a specific member of a gene family is often verified with Southern filter analysis of somatic cell hybrids. The importance of having excellent metaphase chromosomes before beginning in-situ hybridization cannot be overemphasized. All the steps involved in this procedure only exaggerate the problems with poorly prepared slides. Despite the difficulties encountered with in-situ hybridization, the data obtained yield localization to single chromosome bands. More localization that is precise can be obtained by using in-situ hybridization with cells containing defined chromosomal aberrations.