Effects of lateral spacing on enzymatic on-chip DNA polymerization

Abstract

Enzymatic on-chip DNA polymerization can be utilized to elongate surface-bound primers with DNA polymerase and to enhance the signal in the detection of target DNAs on the solid support. In order to investigate the steric effect of the enzymatic reaction on the solid support, we compared the efficiency of on-chip DNA polymerization on a high-density surface with that on a spacing-controlled surface. The spacing-controlled, 9-acid dendron-coated surface exhibited approximately 8-fold higher efficiency of on-chip DNA polymerization compared with the high-density surface. The increase in fluorescence intensity during the on-chip DNA polymerization could be fit to an exponential equation, and the saturation level of the 9-acid dendron slide was 7 times higher than that of the high-density slide. The on-chip DNA polymerization was employed to measure the transcription level of nine genes related to epithelial-to-mesenchymal transition in hepatocellular carcinoma cells. Compared to the high-density surface, the dendron-coated surface exhibited a lower detection limit in the on-chip DNA polymerization and higher correlation with transcription levels as determined by quantitative real-time PCR. Our results suggest that control of the lateral spacing of DNA strands on the solid support should significantly enhance the accessibility of DNA polymerase and the efficiency of the on-chip DNA polymerization. (C) 2010 Elsevier B.V. All rights reserved.