IN-SITU REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION - APPLICATIONS FOR LIGHT AND ELECTRON-MICROSCOPY

Since its discovery in 1986 by Mullis, the polymerase chain reaction ( PCR) has been extensively developed by morphologists in order to overc ome the main limitation of in situ hybridization, the lack of sensitiv ity. In situ PCR combines the extreme sensitivity of PCR with the cell -localizing ability of in situ hybridization. The amplification of DNA (PCR) or a cDNA (RT-PCR) in cell or tissue sections has been develope d at light and electron microscopic levels. A successful PCR experimen t requires the careful optimization of several parameters depending on the tissue (or of cell types), and a compromise must be found between the fixation time, pretreatments and a good preservation of the morph ology. Other crucial factors (primer design, concentration in MgCl2, a nnealing and elongation temperatures during the amplification steps) a nd their influence on the specificity and sensitivity of in situ PCR o r RT-PCR are discussed. The necessity to run appropriate controls, esp ecially to assess the lack of diffusion of the amplified products, is stressed. Current applications and future trends are also presented. ( (C) Elsevier, Paris).