In SituStrand Displacement Amplification

Abstract

In situamplification of nucleic acids has provided a means to analyze for the presence of single copy DNA (single copy genes or viral infection) and expression of low abundance RNAs. To date, PCR has been used extensively(1) but nonetheless presents some significant technical challenges that can be difficult to overcome. In particular, the thermal cycling of cells and tissues is generally detrimental to cell morphology and tissue integrity and can limit the success of the procedure and the efficacy of the results. To circumvent this issuein situamplification of nucleic acids can be accomplished using an isothermal amplification method such as strand displacement amplification (SDA) (2) The basic procedure involves cellular fixation/permeabilization followed by amplification which proceeds at a constant moderate temperature for a short period of time followed by the detection of amplicon. As an example of the effects of temperature on cellular integrity, peripheral blood mononuclear cells (PBMCs) can be analyzed by measuring forward and side scatter using fluorescence activated cell sorting (FACS) to differentiate among monocytes, lymphocytes and granulocytes. This differentiation is retained after exposure of the PBMCs to an SDA temperature profile, but is abolished after exposure to thermal cycling (personal communication, Becton Dickinson, Immunocytometry Systems). Furthermore, specific cell surface epitopes are retained after an SDA thermal profile as demonstrated by FACS analysis of cell surface antigens.