Mechanical Microspotting, Polylysine, DNA printing, Eukaryotic system, Genome, Aldehydes, Modern gridding robots

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Mechanical microspotting.
Most convenient microspotting substrates are 1 x 3 inch microscope slides, with their surfaces chemically modified. A variety of surface coating substances including polylysine and reactive aldehydes are used for this purpose. Modern gridding robots can easily print up to 100 chips in a single session. Availability of high-precision print heads, each containing 32 pins allows preparation of 100 micro arrays containing> 10,000 features in an area of 3.6 cm² in 12 hours. Thus a set of four microarrays should allow the study of expression of 40,000 genes. Such high density DNA printing, together with suitable fluorescence detection equipment will allow efficient and inexpensive genome analysis of any eukaryotic system.

The following steps are involved in microspotting: (i) cDNA samples are prepared generally through amplification of cloned cDNA fragments using PCR; (ii) a 1 x 3 inch microslide with chemically modified surface is used for microspotting; (Hi) biochemical sample (e.g. cDNA) is loaded into a spotting pin by capillary action and a small volume is transferred to the chemically modified solid surface at a specified position; this is achieved by a physical

Three different approaches for microarrays production technologies

(a) Photolithography

Photolithography

b) Mechanical Microspotting

Mechanical Microspotting

c) Ink Jetting

Ink Jetting

contact between the pin and the solid surface; (iv) after the first spotting cycle, the pin is washed and second sample is similarly loaded and deposited at an adjacent position. These steps are speeded up by robotic control systems and multiplexed print-heads.

Microspotting is relatively simpler and cheaper. However, a disadvantage is that each sample needs to be synthesized purified and stored prior to microarrays fabrication. Although, it is believed that microspotting can never produce the densities achievable through photolithography, impovements in microspotting may allow production of chips containing 1.00,000 features in an area of 6.5cm² In view of this and the low cost, microspotting may become the microarray technology of future.

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Ink-jetting. This technology makes use of piezoelectric and other forms of propulsion to transfer biochemical substances from miniature nozzles to solid surfaces. Piezoelectric effect makes use of the property of certain materials such as ceramics, which expand when exposed to an electric potential. Therefore, piezojets attached to suitable control systems ate capable of delivering 100 picoliter droplets at a rate of 10,000 droplets per second. Several companies including lncyte Pharmaceuticals and Protogene Laboratories have proprietary ink-jetting technologies. High-density cDNA microarrays for gene expression analysis have been prepared for several organisms including humans using ink-jeting.

The above two technologies described for cDNA microarrays can be used for any set of molecules of interest including genomic DNAs, antibodies, lipids, carbohydrates, etc. Bacterial artificial chromosomes (BACs) can also be used for these micro arrays, which may cover the entire human genome. These may not be suitable for single nucleotide resolution (e.g. SNPs = single nucleotide polymorphisms), but can resolve individual genes.

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