Investigation of the multiple anchors approach in oligonucleotide microarray preparation using linear and stem-loop structured probes (original) (raw)
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Analysis of DNA-microarrays produced by inverse in situ oligonucleotide synthesis
Journal of Biotechnology, 2002
5%-Phosphoramidites protected by 2-nitrophenylethyl (NPE) and 2-(4-nitrophenyl)ethoxy carbonyl (NPEOC) functions were employed for in situ synthesis of oligonucleotides in 5% 3% direction on flat glass surfaces. By this inverse synthesis format, the oligonucleotides are attached to the solid support via their 5%-ends while the free 3%-hydroxyl groups are available as substrates for enzymatic reactions such as elongation by polymerases, thereby adding another feature to the portfolio of chip-based applications. Having a fluorescence dye present at the first base during synthesis, the quality of the oligonucleotides was analysed quantitatively by capillary electrophoresis after release from the solid support. With about 95% yield per condensation, it was found to be equivalent to synthesis results achieved on CPG support. The chip-bound oligonucleotides could be extended enzymatically upon hybridisation of a DNA-template. Surprisingly, however, only 63% of the oligonucleotides were elongated in polymerase reactions, while oligonucleotides that were released from the support behaved normally in standard PCR amplifications. This rate of 63% nevertheless compares favourably with an extension rate of only 50%, which was achieved under identical conditions, if pre-fabricated oligonucleotides of identical sequence had been spotted to the glass support.
DNA microarrays with stem-loop DNA probes: preparation and applications
Nucleic acids research, 2001
We have developed DNA microarrays containing stem-loop DNA probes with short single-stranded overhangs immobilized on a Packard HydroGel chip, a 3-dimensional porous gel substrate. Microarrays were fabricated by immobilizing self-complementary single-stranded oligonucleotides, which adopt a partially duplex structure upon denaturing and re-annealing. Hybridization of single-stranded DNA targets to such arrays is enhanced by contiguous stacking interactions with stem-loop probes and is highly sequence specific. Subsequent enzymatic ligation of the targets to the probes followed by stringent washing further enhances the mismatched base discrimination. We demonstrate here that these microarrays provide excellent specificity with signal-to-background ratios of from 10- to 300-fold. In a comparative study, we demonstrated that HydroGel arrays display 10-30 times higher hybridization signals than some solid surface DNA microarrays. Using Sanger sequencing reactions, we have also developed...
Reproducible and inexpensive probe preparation for oligonucleotide arrays
Nucleic Acids Research, 2001
We present a new protocol for the preparation of nucleic acids for microarray hybridization. DNA is fragmented quantitatively and reproducibly by using a hydroxyl radical-based reaction, which is initiated by hydrogen peroxide, iron(II)-EDTA and ascorbic acid. Following fragmentation, the nucleic acid fragments are densely biotinylated using a biotinylated psoralen analog plus UVA light and hybridized on microarrays. This non-enzymatic protocol circumvents several practical difficulties associated with DNA preparation for microarrays: the lack of reproducible fragmentation patterns associated with enzymatic methods; the large amount of labeled nucleic acids required by some array designs, which is often combined with a limited amount of starting material; and the high cost associated with currently used biotinylation methods. The method is applicable to any form of nucleic acid, but is particularly useful when applying double-stranded DNA on oligonucleotide arrays. Validation of this protocol is demonstrated by hybridizing PCR products with oligonucleotidecoated microspheres and PCR amplified cDNA with Affymetrix Cancer GeneChip microarrays.
Langmuir, 2003
A versatile chemistry utilizing the homobifunctional cross-linker 1,4-phenylene diisothiocyanate (PDC) to attach both amine-and thiol-terminated oligonucleotides to aminosilane-coated slides was examined in a microarray format. Three common aminosilanes, 3-aminopropyltriethoxysilane (APS), N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, and m,p-(aminoethyl-aminomethyl) phenethyltrimethoxysilane, were coated onto glass slides and silicon wafers and characterized using contact angle goniometry, ellipsometry, and X-ray photoelectron spectroscopy. Evaluation of the aminosilane-modified surfaces using contact angle measurements, UV-vis spectroscopy, and covalent attachment of a Cy5-conjugated N-hydroxysuccinimide ester reporter molecule suggested that derivatization of the surface with APS + PDC resulted in the best overall coverage. Microarrays printed using APS + PDC chemistry to immobilize both amine-and thiolterminated oligonucleotides resulted in rapid attachment, uniform spot morphology, and minimal background fluorescence. Both amine-and thiol-terminated oligonucleotides showed comparable attachment, although greater attachment and hybridization efficiencies were observed with amine-functionalized molecules at saturating printing densities. The data highlight the influence of surface chemistry on both immobilization and hybridization and, by extrapolation, on microarray data analysis.
Analytical Chemistry, 2010
and Southern blots, although details of this process and their effects on DNA functionality are not well understood. By using Cy5-labeled model oligonucleotides for UV immobilization and Cy3-labeled complementary sequences for hybridization, we measured independently the number of immobilized and hybridized oligonucleotides on the microarray surface. By using a two-color fluorescence LED setup and a novel method to compile the data, a full analysis has been made of the effects of oligonucleotide composition (length and sequence) on both immobilization and hybridization. Short homo-oligomer sequences (tails) of uracils, thymines, and, to a limited extent, guanines attached to a hybridization sequence improve immobilization. We propose a possible mechanism explaining the grafting of these nucleotides to amine-functionalized substrates, and we found evidence that the DNA backbone is possibly involved in the immobilization process. Hybridization, on the other hand, greatly improves as a function of tail length regardless of tail composition. On the basis of statistical arguments, the probes increasingly bind via their tail, with the hybridization sequence becoming more accessible to its complement. We conclude that all tails, sequence independent, improve hybridization signals, which is caused by either improved immobilization (especially thymine and uracil) or improved hybridization (most pronounced with guanine tails).
Hybridization to surface-bound oligonucleotide probes: Influence of point defects
Microarray-based genotyping is based on the high discrimination capability of oligonucleotide probes. For detection of Single Nucleotide Polymorphisms (SNPs) single-base discrimination is required. We investigate how various point-mutations, comprising single base mismatches (MMs), insertions and deletions, affect hybridization of DNA-DNA oligonucleotide duplexes. Employing light-directed in situ synthesis we fabricate DNA microarrays with comprehensive sets of cognate point-mutated probes, allowing us to systematically investigate the influence of defect type, position and nearest neighbor effects. Defect position has been identified as the dominating influential factor. This positional effect which is almost identical for the different point-mutation types, is biased from the local sequence environment. The impact of the MM type is largely determined by the type of base pair (either AT or CG) affected by the mismatch. We observe that single base insertions next to like-bases resul...
[Oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips]
Bioorganicheskaia khimiia
A possibility of using oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips was studied. The oligonucleotide conjugates contain a hairpin ligand (MGB) composed of two tripyrrolcarboxamide residues with an aminocaproic acid residue as a linker and bound to the oligonucleotide duplex AT tract in a site-specific manner. We used as (5'-3') probes GACAAGAp, GACAAAAp, GACAAGA-MGB, and GACAAAA-MGB. The oligonucleotides labeled with Cy3 cyanine dye, Cy3-ACTAATTTTGTC and Cy3-ACTAATCTTGTC, were used as targets. The maximal MGB effect on the fluorescence level of microarray chip spots, which caused its fourfold increase as compared with the initial unmodified duplex, was observed for the duplex containing only AT pairs in the ligand binding site. The presence of A-C and G-T mutations in the binding site (imperfect duplexes) or a C-G pair (perfect duplex) affects the change in fluorescence level to a considerably lesser degree.
Immobilization of oligodeoxyribonucleotides with multiple anchors to microchips
Nucleic Acids Research, 2001
Facile modification of oligodeoxyribonucleotides is required for efficient immobilization to a pre-activated glass surface. This report presents an oligodeoxyribonucleotide which contains a hairpin stem-loop structure with multiple phosphorothioate moieties in the loop. These moieties are used to anchor the oligo to glass slides that are pre-activated with bromoacetamidopropylsilane. The efficiency of the attachment reaction was improved by increasing the number of phosphorothioates in the loop, as shown in the remarkable enhancement of template hybridization and single base extension through catalysis by DNA polymerase. The loop and stem presumably serve as lateral spacers between neighboring oligodeoxyribonucleotides and as a linker arm between the glass surface and the single-stranded sequence of interest. The oligodeoxyribonucleotides of this hairpin stem-loop architecture with multiple phosphorothioate moieties have broad application in DNA chip-based gene analysis.