Steric Factors Controlling the Surface Hybridization of PCR Amplified Sequences

Abstract

This study elucidated the hybridization behavior of surface-bound 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, approximately 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 approximately 2.9 x 10(12) 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 approximately 600-bp strands favored over formation of the surface-tethered probe-amplicon complex. This paper also describes a strategy to enhance the sensitivity of enzyme-linked hybridization assays. Such a strategy relies on formation, around the long target sequence, of dendritic-like 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.

A Highly Sensitive Electrochemical Platform for the Assay of Uracil-DNA Glycosylase Activity Combined with Enzymatic Amplification

“…The SA-ALP can convert its electro-inactive substrate 1-naphthyl phosphate into an electroactive derivative 1-naphthol, generating a measurable electrochemical signal. 18,19 In the presence of UDG, uracil bases were removed from the signal probe, resulting in a lower melting temperature of the substrate and liberating the biotinylated signal probe from the electrode. Consequently, the amount of the bound SA-ALP decreased, resulting in a weaker electrochemical signal.…”

Designs of Enterobacteriaceae Lac Z Gene DNA Gold Screen Printed Biosensors

“…DNA and particularly surface immobilized oligonucleotides are nowadays being used, among other applications, in the specific detection of pathogenic bacteria [2,3] by exploiting their unique nucleic acid sequence [4]. A number of these methods are based on the detection of the hybridization event either by means of indicator-free assays [5], or using electroactive indicators [6], the electrochemical signal depending on the interaction of a DNA probe immobilized on the sensing surface with its complementary target sequences in solution [7,8].…”

3D-nanostructured Au electrodes for the event-specific detection of MON810 transgenic maize

“…The obtained extracts Consequently, the relative position of the recognition site in the amplicon is of paramount importance to ensure a proper performance. Overhangs as small as 21-nt were found deleterious for the magnitude of the electrochemical signal when using long amplicons (over 200 bp), but not for smaller ones [31]. Recently, an equivalent behavior was also observed with amplicons smaller than 150 bp [32].…”