The “two-hit” theory of tumor suppressor gene (TSG) inactivation predicts that loss of function is a result of two separate genetic events, one affecting each allele (1 ). One hit is commonly a deletion of part of, or the entire allele sequence and can be detected by loss of heterozygosity (LOH) analysis in which “normal” DNA is compared with tumor DNA at various loci. At heterozygous loci, the two alleles in normal DNA are observed as separate bands; if an allele has been lost in a tumor, one of the bands is absent in the tumor DNA. Such deletions are termed LOH and indicate a likely site of a TSG. When LOH occurs, it is predicted that the retained allele is nonfunctional either because of a point mutation or a microdeletion. The next step in testing the authenticity of the putative suppressor gene is to look for small genetic alterations in the retained allele.