A simple and rapid method for the analysis of genetic polymorphisms has been developed using allele-specific oligonucleotide arrays bound to glass supports. Allele-specific oligonucleotides are covalently immobilized on glass slides in arrays of 3 mm spots. Genomic DNA is amplified by PCR using one fluorescently tagged primer oligonucleotide and one biotinylated primer oligonucleotide. The two complementary DNA strands are separated, the fluorescently tagged strand is hybridized to the support-bound oligonucleotide array, and the hybridization pattern is detected by fluorescence scanning. Multiple polymorphisms present in the PCR product may be detected in parallel. The effect of spacer length, surface density and hybridization conditions were evaluated, as was the relative efficacy of hybridization with single or double-stranded PCR products. The utility of the method was demonstrated in the parallel analysis of 5 point mutations from exon 4 of the human tyrosinase gene.
A highly selective and robust catalyst based on Pt nanoclusters (NCs) confined inside the cavities of an amino-functionalized Zr-terephthalate metal-organic framework (MOF), UiO-66-NH 2 was developed. The Pt NCs are monodisperse and confined in the cavities of UiO-66-NH 2 even at 10.7 wt % Pt loading. This confinement was further confirmed by comparing the catalytic performance of Pt NCs confined inside and supported on the external surface of the MOF in the hydrogenation of ethylene, 1-hexene, and 1,3-cyclooctadiene. The benefit of confining Pt NCs inside UiO-66-NH 2 was also demonstrated by evaluating their performance in the chemoselective hydrogenation of cinnamaldehyde. We found that both high selectivity to cinnamyl alcohol and high conversion of cinnamaldehyde can be achieved using the MOFconfined Pt nanocluster catalyst, while we could not achieve high cinnamyl alcohol selectivity on Pt NCs supported on the external surface of the MOF. The catalyst can be recycled ten times without any loss in its activity and selectivity. To confirm the stability of the recycled catalysts, we conducted kinetic studies for the first 20 h of reaction during four recycle runs on the catalyst. Both the conversion and selectivity are almost overlapping for the four runs, which indicates the catalyst is very stable under the employed reaction conditions.
A new method for constructing oligonucleotide arrays on gold surfaces has been developed, and these arrays have been used in DNA hybridization experiments with in situ surface plasmon resonance (SPR) imaging detection. The detection technique was able to differentiate between single- and double-stranded DNA regions on the gold surface. The hybridization of both oligonucleotides and PCR-amplified DNA fragments was detectable, with the latter exhibiting slower hybridization kinetics. Temperature control of the in situ SPR cell was used to discriminate between perfectly matched duplexes and single-base-mismatched duplexes. The SPR detection technique requires no label on the DNA, but fluorescently labeled targets were also tested and detected by fluorescence imaging as an independent verification of the hybridization behavior of these DNA arrays. The in situ SPR imaging method for detection of DNA hybridization is expected to complement other existing methods for study of DNA interactions and might find future uses in mutation screening assays and DNA resequencing.
A strategy for DNA computing on surfaces using linked sets of 'DNA words' that are short oligonucleotides (16mers) is proposed. The 16mer words have the format 5'-FFFFvvvvvvvvFFFF-3' in which 4-8 bits of data are stored in 8 variable ('v') base locations, and the remaining fixed ('F') base locations are used as a word label. Using a template and map strategy, a set of 108 8mers each of which possesses at least a 4 base mismatch with the complements to all the other members of the set (4bm complements) are identified for use as a variable base sequence set. In addition, sets of 4 and 12 word labels of the form ABCD....DCBA that are respectively 8bm and 6bm complements with each other are identified. The 16mers are chosen to have a G/C content of 50% in order to make the thermodynamic stability of the perfectly matched hybridized DNA duplexes similar; a simple pairwise additive method is used to estimate the perfect match and mismatch hybridization thermodynamics. A series of preliminary experiments are presented that use small arrays of 16mers attached to chemically modified gold surfaces and fluorescently labeled complements to study the hybridization adsorption and enzymatic manipulation of the oligonucleotides.
Addressable DNA arrays are created by photopatterning self-assembled monolayers to form hydrophilic and hydrophobic regions on a gold surface. The hydrophilic regions aid to contain small volumes of different DNA solutions placed on them using an automated pin-tool loading strategy. The method allows for efficient attachment, manipulation, and hybridization of pure DNA strands on the surface.
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