EQUALIZING CDNA SUBTRACTION BASED ON SELECTIVE SUPPRESSION OF POLYMERASE CHAIN-REACTION - CLONING OF JURKAT CELL TRANSCRIPTS INDUCED BY PHYTOHEMAGLUTININ AND PHORBOL 12-MYRISTATE 13-ACETATE

The major drawback of subtractive cDNA libraries is that the original disproportion in concentrations of different types of transcripts is p reserved. This usually makes the isolation of specific rare transcript s extremely difficult. To overcome this difficulty, we propose a strat egy that introduces the equalization of concentrations (normalization) of specific transcripts during the subtractive process. This makes po ssible obtaining both rare and highly abundant transcripts in the resu lting subtracted library. This technique has been applied for isolatio n of transcripts activated upon induction of Jurkat cells by phytohema glutinin and phorbol 12-myristate 13-acetate. Six novel up-regulated s equences belonging to a low-abundance class of transcripts have been o btained. (C) 1996 Academic Press, Inc.