Analysis of solid-phase allele-specific primer extension characteristics on biochip in combination with modified primers and PicoGreen staining method

Abstract

DNA microarray technology offers potential for future high-throughput variation genotyping. Allele-specific primer extension procedure on microarray has been considered as an efficient method for single nucleotide polymorphism (SNP) genotyping. However, the high cost of the fluorescent-labeled dNTP used for signal detection in this method limits its application. In the present study, we evaluated the characteristics of solid-phase allele-specific primer extension, in terms of specificity and efficiency and demonstrated that compared to liquid-phase reaction, it requires lower annealing temperature, and higher template and Mg²⁺concentrations. The extension efficiency and specificity were though linked, behave diametric during the gradient change of template and Mg²⁺concentrations or annealing temperature. To obtain both optimal signal intensity and specificity, we introduced an artificial mismatched base at the third position from the primer 3′end, which enhanced the specificity significantly. The PicoGreen staining method, which could decrease the cost greatly, was then introduced to replace the fluorescent-labeled dNTP for signal detection.