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The combinatorial synthesis has brought about novel opportunities for efficiently creating reactions of a scaffold chemical moiety with a number of different compounds to yield a library of various compounds with different substituent groups, i.e. nucleotides, amino acids.
By in situ synthesis, an array of oligonucleotides is synthesized using conventional DMT-protected phosphoramidite chemistry. This is different from conventional synthesis by the use of a photogenerated acid (PGA) rather than an acid in DMT deprotection step to control the parallel synthesis.
Deprotection is initiated by directing light at selected three-dimensional chambers in microfluidic chips. Upon activation, acid forms in seconds, removing DMT group.
An incoming phosphoramidite nucleoside monomer is then coupled to the growing oligomer chain.
The synthesis cycle is repeated for each additional monomer until an array of thousands of oligonucleotides is formed.
The main advantage in this technique is to be able to produce long chains of oligonucleotides, i.e. 150mer, with a considerably high stepwise efficiency (>98.5 %).
The key to this parallel synthesis is the generation of a photogenerated acid to remove the acid-labile protecting group. The removal of the protecting group is spatially controlled. Based on this fact, many conventional chemical reactions can be adapted to form diverse combinatorial molecular libraries; RNA sequences, peptide analogs and a variety of organic molecules.
A digital microarray platform consisting of a microarray reactor, a micromirror array image projector, a controller computer and an automated synthesizer.
Parallel synthesis using acid-labile protecting groups. The DNA chain is deprotected in a spatially controlled manner using photogenerated acid (PGA) (b and d), followed by coupling and oxidation reactions (c and e). This cycle is repeated until the desired lengths and sequences are obtained (f).
Miniaturized spatially addressable microchips of oligonucleotides and peptides are powerful tools for high-throughput medical, biomedical, environmental research, and the advancement of genomics and proteomics.
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Oligonucleotide arrays single base mismatch detection
We have validated our oligonucleotide sequence fidelity by testing for the ability to discriminate single base mismatches by the means of hybridization using fluorescein-labeled target sequences.
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Gene expression analysis
Moving from single mismatch detection, we have started to work on genomic studies for different organisms; investigating gene expression. Some on-going projects:
- Gene screening to detect microbial activity in water
- Gene expression tracking for beast cancer
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Oligopeptide arrays metal binding peptides
Different peptide analogues have shown to have affinities for different metal ions.
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Epitope Screening using A p53 Antibody, PAb240
The peptidomimetic squences on the microchip shows specific antibody binding and provide insights into molecular details for specificity of epitope binding.
FRE images of o-DNA chip hybridization with fluorescein-labeled target sequence.
Microfluidic array used in differential gene expression of brain and skeletal muscle samples. This chip contains 253 cancer genes in 15 replicates throughout the chip. Green shows over expressed genes in brain and red shows over expressed genes in muscle.
The right image demonstrates well-differentiation as well as uniform spot intensities.
Microarray of tetrapeptides for Pb(II) and As(III) binding: (Left) Pattern of localized parallel synthesis of labeled tetrapeptide Glu-Cys-Glu-Glu (white), Glu-Glu-Glu-Glu (strip), Cys-Cys-Cys-Cys (gray) and Gly-Gly-Gly-Gly (black).
(Middle) Fluorescence image of labeled tetrapeptides when Pb(II) sensing is introduced.
(Right) Fluorescence image of the same array when As(III) is introduced.
The fluorescence intensity in the image is the increased intensity as a result of metal-peptide binding.
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Last Updated: December 2004
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