SUBTRACTIVE HYBRIDIZATION STRATEGY USING PARAMAGNETIC OLIGO(DT) BEADSAND PCR

Subtractive hybridization has been widely used for the identification of differential expressed genes. Here we describe a simple, sensitive strategy of subtractive hybridization that involves binding the driver poly(A)(+) RNA pool to paramagnetic Dynabends(R) Oligo (dT)25. After hybridization with target cDNA, the molecules common to both pools are removed. Tire subtracted cDNA is then amplified with PCR and used for library screening. Using this method we have identified Sour cDNA clo nes that represent developmentally regulated transcripts in the centra l nervous system of the tobacco hornworm Manduca sexta. All four trans cripts are of low abundance, comprising only 0.001%-0.5% of the poly(A )(+) RNA pool.