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differential PCR display

A method for identification of mRNAs produced under specific physiological conditions. Total cellular RNA is reverse-transcribed, and the resultant cDNAs are used as templates for PCR. The 3'-primer has a poly(T) sequence that directs it to the poly(A) tail of mRNA, and is 5'-ended with two bases that make this primer more selective. (The two bases may be varied to achieve different selections.) The 5'-primer is short and of arbitrary sequence, and is intended to allow, under controlled non-stringent reannealing conditions of temperature, concentration, time, etc., amplification of a manageable number of specific cDNAs (between 50 and 100 bands) that can be electrophoretically separated on a DNA sequencing gel. The ladder of cDNAs can be displayed side-by-side with the cDNAs derived from cells in a different physiological state. This allows the identification of unique cDNAs that can be extracted, amplified by PCR, sequenced and identified. In a variant version called RNA fingerprinting arbitrary primer PCR (RAP-PCR), no poly(T) primer is used; one primer can serve for each of the two transcribed strands and the non-stringent reannealing conditions allow a fit to a manageable number of sites on the cDNA template.Liang, P. and Pardee, A. (1992) Science 257, 967-971; McClelland, M., Mathieu-Daude, F. and Welsh, J. (1995) Trends Genet. 11, 242-246

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