Purification and Cloning of Differential Display Products

摘要

Differential display was first described by Liang and Pardee as a way to identify and clone eukaryotic, differentially expressed genes. The main advantage of this method is its ability to display rapidly and simultaneously the expression of mRNA fromvariant cells having the same genetic background. The strategy of the method consists of the following steps: (i) reverse transcription using a set of anchored primers, (ii) polymerase chain reaction (PCR) in the presence of [α-~(35)S]dATP using a set of arbitrary and anchored primers, (iii) electrophoretic separation of the PCR products, (iv) re-amplifica-tion of products that are differentially expressed and (v) cloning these cDNAs for further identification. Although powerful and straightforward, one ma-jor drawback of the method is the fact that most of the PCR products obtained after purification contain contaminating DNA sequences. Therefore, a method is needed to determine whether the correct cDNA has been isolated. Another drawback is that the re-ampli-fication of a purified cDNA often yields very low levels, making it technically difficult to clone.