Effect of primer purity on the banding patterns of differential display polymerase chain reaction.
Differential display polymerase chain reaction (DD-PCR) is a novel method for identification of differentially expressed genes by comparative display of arbitrarily amplified cDNA subsets. The basic principle of the DD-PCR is to systematically amplify messenger RNAs and then distribute their 3' termini on a denaturing polyacrylamide gel. Although this technology has been successfully applied in a large number of studies, few novel genes of interest have been identified, suggesting that the method needs further improvement. We discovered that primer purity is crucial. We show that by purifying primers using reverse-phase high-performance liquid chromatography, sampling of differentially expressed genes can be greatly enhanced, and relevant genes can be isolated. Using these purified primers in DD-PCR, when compared with the unpurified primers, it should be possible to identify threefold to fourfold differences in expression or differential expression in a fraction of the cell population.
Wen
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Biology Department, Microchemical Core Facility, San Diego State University, San Diego, CA 92182-4614, USA. lwen@sciences.sdsu.edu