Steric Factors Controlling the Surface Hybridization of PCR Amplified Sequences

This study elucidated the hybridization behavior of surfacebound oligonucleotides to their longer PCR-amplified targets. The screen-printed gold surface of disposable electrodes was the platform onto which thiol-tethered oligonucleotides (21-mer) were immobilized by chemisorption. As a model case, 600-bp amplicons were studied. Surface hybridization was monitored by means of an enzyme-linked assay with electrochemical detection. Use of different surface-tethered probe sequences over a wide range of surface densities was explored to achieve the highest duplex yield. Both the surface coverage by the probe and its relative position on the target strand were found to control the efficiency of capture of the target sequence. Interfacial hybridization occurred with the highest efficiency for a probe coverage of 2.9 1012 molecules/cm2 and when the 3¢ end of the amplicon was involved. An unusual (bell-shaped) response/amplicon concentration profile was additionally found. It was hypothesised that when the amount of solution-phase target is relatively high, random collisions make reannealing of the 600-bp strands favored over formation of the surfacetethered probe-amplicon complex. This paper also describes a strategy to enhance the sensitivity of enzymelinked hybridization assays. Such a strategy relies on formation, around the long target sequence, of dendriticlike structures, which could offer multiple anchoring points for the enzyme conjugate. The results shown in this work might have great significance for the practical application of hybridization to oligonucleotide chips.