Gerard J. Nuovo

11 – PCR in Situ Hybridization

This chapter describes a manual “hot-start” modification for PCR in situ hybridization that allows the detection of one target copy per cell with a single primer pair. The greatest impact of PCR in situ hybridization is in the area of human immunodeficiency virus type 1 related pathogenesis. One of the most perplexing issues regarding acquired immunodeficiency syndrome is that early studies stated that few of the T helper cells actually contain the virus, especially early in the disease process. A great deal of variation is seen in the different published protocols for PCR in situ hybridization. Much of this variation is based on methods that attempt to inhibit diffusion of the amplified product from the site of origin and to prevent drying of the amplifying solution. Much attention has focused on the ability to label the product directly in in situ PCR that eliminate the need for a hybridization step. The weakness of PCR in situ hybridization is related to the weaknesses of both PCR and in situ hybridization. Competing pathways in PCR can limit its specificity and sensitivity. Further, background signal from complexing of the probe and non-target molecules in in situ hybridization lead to false positive results.