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Modification of primer design facilitates the use of differential display.

The method of differential display developed by Liang and Pardee (10) has gained a large audience and is now the method of choice to quickly identify and isolate differentially expressed genes in several experimental systems. The method is powerful because of its inherent simplicity, and it allows the simultaneous comparison of upand down-regulated genes with small amounts of raw material. Nevertheless, several vulnerable spots have been identified in recent years. Most criticisms concerned false positives generated by the polymerase chain reaction (PCR) step (9). In addition, the identification of differentially expressed genes remains tedious and limited by the fact that the amplified cDNA fragment generally corresponds to the trailer region of mRNA. Additional difficulties are: (i) the contamination of bands recovered from the display gel by heterogeneous sequences (1) and (ii) the strong bias in favor of the abundant mRNAs (2). For these reasons, many authors have attempted to improve the method, either through different primer designs (19,20) or by adapting techniques for faster confirmation of differential gene expression (13,19) and developing methodologies for easier identification of true positives (3,11). The use of longer primers instead of the original 10-mers has been proposed to increase the specificity and reproducibility of the method (4,10,20) by favoring more specific hybridization in the PCR. This technique integrates the principles of differential display with those of RNA fingerprinting by arbitrarily primed PCR (18). PCR is performed in two steps: (i) a few initial low-stringency cycles allowing minor mispairing between the arbitrary primer and the cDNA template, followed by (ii) several high-stringency PCR cycles favoring perfect matches with the anchored primers. Inspired by these improvements, we developed a novel primer strategy aimed at facilitating re-amplification, analysis and cloning of the cDNA fragments (Figure 1A) in which the main feature is the use of a universal re-amplification primer set. The primers are modified with the addition of a constant region at either end of the product; this is obtained by extending the 5′ terminus of the oligo(dT) primers (DLP) and the arbitrary primer (XAp) (Life Technologies, Basel, Switzerland) with about one half of the T7 promoter (Figure 1A, f) and one half of the SP6 RNA polymerase promoter (Figure 1A, a), respectively. Universal re-amplification primers harboring the complete cognate