Systematic Analysis of Intrinsic Factors Affecting Differential Display

Abstract

Differential display (DD) is a widely used method for identifying differentially expressed genes. To improve further the efficiency and reproducibility of the method, this report systematically examines four critical parameters of standard DD-PCR. Specifically, the study determined the optimal annealing temperature, elongation time, dNTP concentration, and arbitrary primer concentration. By using a thermal cycler that was capable of displaying a temperature gradient across a PCR plate, it was possible to determine (in a single experiment) the effect of different annealing temperatures. The optimal annealing temperature for a 13-mer arbitrary primer fell within a broad range of 40°C–50°C. Elongation times over a range of 30–120 s worked best. The optimal concentration for dNTPs was within a very broad range of 2–50 μM, with higher amounts allowing for greater pipetting accuracy. The most favorable concentration for the arbitrary primer was also within a broad range of 0.1–2.0 μM. A primer concentration below this range greatly reduced the efficiency of the amplification process. In conclusion, the experimental findings delineated the best possible DD conditions for a more reliable assessment of differential gene expression.