Hybridization Properties of Support-Bound Oligonucleotides:  The Effect of the Site of Immobilization on the Stability and Selectivity of Duplex Formation

Abstract

Four 12-mer oligodeoxyribonucleotide sequences were immobilized to uniformly sized (50 microm) polymer particles through C5-tethered thymine and N(4)-tethered cytosine bases at four different sites in each sequence. The effect of the site of immobilization on the efficiency and selectivity of hybridization of the particle-bound probes was quantified by a sandwich-type assay based on a time-resolved fluorometric measurement of an oligonucleotide probe labeled with a photoluminescent europium(III) chelate directly from the surface of a single particle. Immobilization through a base in the central part of the sequence was observed to destablize the duplex more markedly than tethering through a terminal base. The effect of a one-base mismatch on the duplex stability increased with the increasing distance from the site of immobilization.

A generic approach for the design of whole-genome oligoarrays, validated for genomotyping, deletion mapping and gene expression analysis on Staphylococcus aureus

“…A drawback of these approaches is to apply thermodynamics laws on probe/target interaction as determined in solution [37], but ignoring effects related to oligonucleotide immobilization on a microarray surface [15,38]. …”

Selective Circular Oligonucleotide Probes Improve Detection of Point Mutations in DNA

“…A desired proportion (8 ± 28 mmol g À1 ) of the amino groups was acylated with [(4,4'-dimethoxytrityl)oxy]acetic acid, and the remaining groups were capped by repeated acetylations [21]. Two oligonucleotide probes, 3'-TTT AAGCTGTCGCTGCACC-5' and 3'-TTT CGACAGCTTCGCGAGC-5', were assembled on these supports via a normal phosphoramidite strategy, as described in [6] in detail.…”

“…The mixtures were shaken in sealed tubes with a rotamix (15 rpm) at 258 overnight. The particles were then rapidly washed with buffer, and 10 of the particles were subjected to a time-resolved measurement of the fluorescence emission directly from the particle, one after another [6]. The standard deviation ranged from 5 to 20%.…”

“…For example, quantitative measurements with microparticles on a solid phase have shown that reliable detection of point mutations and deletions is still problematic, as the selectivity of hybridization is markedly changed when going from one sequence to another [2] [4]. In addition, the distance between mismatch and site of immobilization within a probe [5] [6], the loading of oligonucleotide probes onto the particle, and, to a lesser extent, the homogeneity of probes all affect selectivity [4] [7] [8]. Accordingly, detection of singlebase alterations within certain sequences may still be difficult and, even when possible, depends on time-consuming optimization of the probe length.…”

SNP genotyping by DNA photoligation: application to SNP detection of genes from food crops

“…Most methods of DNA detection involve hybridization by an oligodeoxynucleotide (ODN) probe to its complementary single-strand nucleic acid target, leading to signal generation [6,7]. In as far as the detection relies on hybridization events [8][9][10][11], however, such ODN probes have inherent limitations in their selectivity. In our previous studies, we developed an SNP-typing method with high selectivity and sensitivity by introducing a form of photochemical ligation with 5-carboxyvinyl-2 -deoxyuridine ( CV U) into an existing allele-specific hybridization [12][13][14][15][16][17].…”