Differential Display PCR Fragments as Probes for cDNA Cloning

Abstract

Differential display of expressed mRNAs (DDRT-PCR) (Liang and Pardee 1992) is rapidly becoming the principal method for comparing gene expression in different cells or tissues. Differentially expressed bands can be excised from the DDRT-PCR gels, reamplified and purified (see Chap. XXVI). The next step is to identify the genes (mRNAs) that correspond to the differentially displayed bands. As a first attempt, the DNA fragment is sequenced, as described in Chap. XXXIII in this volume, followed by searches in the DNA sequence databases, using the DNA sequence as query sequence. If the gene is known, this will identify the differentially expressed gene; however, unless if human samples have been analyzed, the chance that the gene is known is rather small since relatively few nonhuman genes have been sequenced. If human samples have been analyzed, there is a high probability that the gene is represented in the databases as an expressed sequence tag (EST), and this may then identify the gene (see Chap. XXXIII). If the sequence is not present in the database, or if it is a nonhuman sample, it is necessary to clone the corresponding cDNA and sequence parts of the coding region and then repeat the search in the database with coding region sequence as query sequence(s).