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To date, sequencing the genome has been keeping mankind busy. Now is the time to decipher this sequence information and to relate it to the mechanisms of the various biological processes. To achieve this, one of the efficient approaches is to build up oligonucleotide arrays that address various sequences for high throughput applications, in areas ranging from medicine to environmental analysis.
Our new approach in this field is based on an in situ photosynthesis platform that uses digital photolithography to activate photogenerated reactions in a microfluidic chip to produce large amounts of DNA’s or peptides using inexpensive, conventional DNA/peptide building blocks. This technology enables us to gain control over each individual reaction at each reactive site; to eliminate the usage of photomask-guided photolithography thus, reducing the cost considerably; yet maintaining flexibility in design, and feasibility for massively parallel in situ synthesis in our three-dimensional chambers on microfluidic chips .
Schematic illustration of the structure (left) and the operation of a microfluidic array device (right).
Oligonucleotide Design for DNA Microarrays: A Thermodynamics Approach
Our technology of DNA synthesis on chips combined with the availability of an increasing number of sequenced genomes, have prompted us to implement a software to design specific oligonucleotides for microarrays.
Ideally, such oligonucleotides should be totally specific to their respective targets to avoid any cross-hybridization and should not form stable secondary structures that may interfere with the labeled probes during hybridization.
We have developed OligoArray (Rouillard et al, 2002), a program to design specific oligonucleotides at genome scale. The latest version, OligoArray 2.0 (Rouillard et al. submitted), uses a thermodynamic approach to predict secondary structures and calculate the specificity of a probe on the chips to a unique target in a mixture of labeled targets.
Pangenomic Database of Oligonucleotides
We have used our OligoArray 2.0 software to design pangenomic sets of oligonucleotides devoted to gene expression analysis for most of the organisms with a sequenced genome.
As of today, we have designed 774,642 oligonucleotides representing 277,976 transcripts from 80 organisms (14 archaea, 59 bacteria and 7 eucaryotes including Homo sapiens and some genetic models). We have successfully designed at least one oligonucleotide for more than 99% of all transcripts from all organisms processed and there is at least one specific oligonucleotide for 94% of these transcripts.
All these oligonucleotide sets are stored into OligoArrayDb, a public database that allows not only to retrieve a complete set of oligonucleotides, but also to build subsets according to user defined keywords (Rouillard et al, submitted).
Our fabrication of three-dimensional microchambers is simply a combination of photolithographical techniques, surface and bulk micromachining, applied on silicon based substrate, with the glass bonded at the top; forming a closed system.
We are able to fabricate hundreds to thousands of nanochambers in an area the size of a dime; each of them being an isolated site for chemical synthesis.
These microfluidic chips provide several attractive benefits for use as biological microarrays:
- Flexiblity in design
- Small inner volume; less than 10 µl per chip
- Controllable temperature and flow conditions
using a chip processing instrument
- Easy to handle because molecules are inside the chip; not on the chip
Our microfluidic chip with nano chambers.
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Last Updated: December 2004
© 2004 Gulari's Group. All rights reserved.