title: 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.
authors.affiliation: Department of Chemistry, University of Florence, via della Lastruccia 3, 50019 Sesto F.no, Florence, Italy.
authors.forename: Maria Lisa
authors.initials: ML
authors.lastname: Del Giallo
authors.forename: Fausto
authors.initials: F
authors.lastname: Lucarelli
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Cosulich
authors.forename: Erika
authors.initials: E
authors.lastname: Pistarino
authors.forename: Barbara
authors.initials: B
authors.lastname: Santamaria
authors.forename: Giovanna
authors.initials: G
authors.lastname: Marrazza
authors.forename: Marco
authors.initials: M
authors.lastname: Mascini
mesh_terms: D001483:Base Sequence; D005784:Gene Amplification / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity