Differential Display-PCR.

This method uses PCR to amplify and display many cDNAs derived from the mRNAs of a given cell or tissue type. The method relies on two different types of synthetic oligonucleotides: anchored antisense primers and arbitrary sense primers. A typical anchored primer is complementary to approx. 13 nucleotides of the poly(A) tail of mRNA and the adjacent two nucleotides of the transcribed sequence. Anchored primers therefore anneal to the junction between the poly(A) tail and the 3'-untranslated region of mRNA templates, from where they can prime synthesis of first-strand cDNA. A second primer, an arbitrary sequence of approx. 10 nucleotides, is then added to the reaction mixture, and double-stranded cDNAs are produced by conventional PCR, carried out at low stringency. The products of the amplification reaction are separated by electrophoresis through a denaturing polyacrylamide gel and visualized by autoradiography. By comparing the banding patterns of cDNA products derived from two different cell types, or from the same cell type grown under different conditions, it is sometimes possible to identify the products of differentially expressed genes. Bands of interest can then be recovered from the gel, amplified further, and cloned and/or used as probes to screen northern blots, cDNA libraries, etc. To reduce the chance of contamination with exogenous DNAs, prepare and use a special set of reagents and solutions for PCR only. Bake all glassware for 6 hours at 150°C and autoclave all plasticware.