General Protocol for Loss of Heterozygosity Detection

Because of their wide distribution in the genome and polymorphic length in a given population, microsatellite sequences (MS) are suitable as markers of deletion at specific chromosomal loci. As the loss of one marker allele – but the retention of the other one (loss of heterozygosity, LOH) – is rather frequent in both sporadic and inherited cancers, LOH has been extensively adopted to identify potential locations of tumour suppressor genes to establish clonal evolution of tumour cell populations and is currently used in a clinical setting as an indicator for prognosis and response to therapy. This chapter provides two methodological approaches for LOH assessing in FFPE samples: a basic method involving singleplex amplification of two microsatellite markers mapping at the 9p21 locus (CDKN2A), with subsequent PAGE run and silver stain detection of PCR products; a singleplex PCR amplification of the same two markers coupled with capillary electrophoresis analysis of PCR products. A rationale for electropherogram interpretation is also supplied.