Differential mRNA fingerprinting by preferential amplification of coding sequences

The need for rapid identification of differentially expressed genes will pe rsist even after the complete human genomic sequence becomes available. The most popular method for identifying differentially expressed genes acquire s expressed sequence tags (ESTs) from the extreme 3' non-coding end of mRNA s. Such ESTs have limitations for downstream applications. We have develope d a method, termed preferential amplification of coding sequences (PACS), t hat was applied to identify differentially expressed coding sequence tags ( dCSTs) between osteoblasts and osteosarcoma cells. PACS was achieved by PCR with a set of primers to anchor at sequences complementary to AUG sequence s in mRNAs and another set of primers to anchor at a PCR-amplifiable distan ce from AUG sequences. An initial screen identified 103 candidate dCSTs aft er screening similar to 15% of the expressed genes between the two cell typ es. Of these sequences, 27 represent CSTs of known genes and two are from 3 '-ESTs of known mRNAs. Thus, PACS identified CSTs similar to 13.5 times mor e often than it identified 3' ESTs, attesting to the objective of the metho d. Since many of the dCSTs represent known genes, their identity and potent ial relevance to osteosarcoma could be immediately hypothesized. Differenti al expression of many of the dCSTs was further demonstrated by northern blo tting or RT-PCR. Since PACS is not dependent on the existence of a poly A t ail on an mRNA, it should have application to identify dCSTs for both proka ryotic and eukaryotic organisms. Additionally, PACS should aid in the ident ification of cell-specific or tissue-specific genes and bidirectional acqui sition of cDNA sequence enabling rapid retrieval of full-length cDNA sequen ce of novel genes. (C) 2000 Elsevier Science B.V. All rights reserved.