The DNA Microarray:

        Approaching Personalized Medicine

What is a DNA-Microarray?    Fabrication    Gene Expression Profiling

Probe selection: cDNA vs. Oligonucleotide

cDNA - cDNA is short for complementary DNA which is made from reverse transcription of mRNA.  mRNA is a good indicator of gene expression, however the reverse transcription is required because DNA is more chemically stable then RNA.  The primary benefit to using cDNA probes is that the entire gene does not have to be fully characterized to be included in the assay.  With the fully expressed gene, this arrays have a very high specificity, often allowing a target to be identified with just one probe location.  Because the process is less controlled than oligonucleotide synthesis there is the potential for less uniformity of the physiochemical characteristics of the array.

Oligonucleotide - Synthesized oligonucleotides are smaller than cDNA probes; approximately 25-70 bases in length.  Their shorter length makes them more accessible to target hybridization.  The short length can also be used to detect differences in highly similar 'gene families.'  The primary downside to oligo probes is their potential for cross-hybridization do to there short length.  This is compensated for by probing for the same target on several  locations of the array.  This will reduce the total number of targets tested by the same factor.

Substrate

Glass, typically in the form of a microscope slide, is this most common substrate for the microarray.  Other substrate materials have been silicon, gold, and assorted polymers.  Depending on the chemical treatment of the substrate, probes can be attached covalently or non-covalently, e.g. through hydrophobic interaction or electrical attraction, to the substrate.

In Situ Synthesis

With In Situ synthesis, the probes are constructed base by base directly onto the substrate.  The primary way to do this is through photolithography.  In this process, light is directed to specific area with a lithographic mask.  The exposed area becomes 'activated' by the removal of a photo labile group.  This area will then covalently bond to the next nucleotide it is exposed to.  This process is repeated as the oligo is built up.  This process can be used to make highly dense arrays.  The primary disadvantage is that a less than 100% yield per cycle limits the probe length that can be synthesized with high fidelity.  Use of this technique is also limited because of the complexity and cost of this process. 

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Contact and Non-Contact spotting techniques

For the majority of microarrays, probes are synthesized in advance then 'spotted' on the substrate.  This is the  original technique and is still commonly used today is pinprinting.  A solid pin is dipped into well containing the reagent and removed.  The resulting droplet that forms on the end of the pin is then transferred to the substrate via direct contact between the pin and the substrate.  This is a simple and cheap process which can even be built relatively easy in lieu of purchase a commercially built spotting machine.  The downside to this process is the pin must be washed after every liquid transfer, slowing down the process.

An alternative process has been developed which involves no contact between the substrate and the printing device. Typically referred to as reagent jetting, this technology is similar to the technology behind an inkjet printer.  Delivery of the reagent comes from the rapid displacement of a small amount of the reagent inside the fluid channel of the dispenser.  This technology allows for the rapid delivery of very small amounts of the solution to the substrate.  While much faster than pinprinting, this technique is also significantly more complex. 

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