Construction of the Arrays

To make the arrays, the sample DNA must be fixed onto glass microscope slides.

Microarray slides

DNA microarray slides are the slides that have the DNA sequences printed onto them robotically, in the specific grid pattern.

The surface-bound molecules are termed 'probes' because they are the molecules that probe, or interrogate the composition of the fluorescently labelled DNA population.

Substrates

Microarrays are unlike the traditional hybridisation assays in the substrate they use. Previous techniques used flexible membranes, for example nylon and nitrocellulose, with radioactivity and autoradiography. Microarray technology uses solid substrates like glass, with fluorescent labelling and detection.
The solid substrates used in microarray technology have a number of advantages for this technique, because they are non-porous. These include:
  • deposition of minute amounts of material is possible in a precisely defined location. This is not possible with porous substrates, because of diffusion of the materials;

  • prevention of the absorption of material into the substrate, because the substrate is non-porous;

  • a uniform attachment surface is provided by a solid substrate, and this improves the quality of the array elements
A Comparison of Filter and Microarrays Assays

Criterion

Surface
Substrate
Conformation
Format
Compatible with

  • miniaturisation
  • photolithography
  • piezoelectric
  • microspotting
  • automated production
  • fluorescence
  • confocal scanning
  • sample multiplexing
    Sample concentration
    Hybridisation kinetics
    Reagent volumes
    Data acquisition

    • Filter

    Porous
    Non-uniform
    Flexible
    Semi-parallel








    Low
    Slow
    Large
    Slow

    Microarray

    Non-porous
    Uniform
    Solid
    Parallel








    High
    Fast
    Small
    Fast

    DNA Microarrays: A Practical Approach

    Methods of Construction

    See Equipment for details of Arrayers.

    The construction of the microarrays involves laying the DNA onto the slide quickly, with a high degree of accuracy and reproducibility. There are a number of methods for producing the microarray slides, and these are mainly done using robotic systems.They each have their own advantages and disadvantages.

    The 3 main methods for doing this are:

    1. Spotting of DNA fragments directly onto the slide,
    2. Arraying of prefabricated oligonucleotides, and
    3. In-situ synthesis of oligonucleotides, done on the chip.

    Microspotting Techniques

    The use of prefabricated oligonucleotides greatly simplifies the construction of microarrays. Conventional methods can be used to produce the sequences, and these can then be printed directly onto the microscope slide, which is first overlaid with a coating that is positively charged.

    The cDNA or oligonucleotide material can be deposited straight from a reagent tray, where it is suspended in a denaturing solution, onto the glass surface by a printhead containing microspotting pins or micropipettes.

    A recent development in this technique has been accomplished by Genetic Microsystems:

      They have developed a novel 'pin and ring' device. The ring holds a droplet of solution collected from a reservoir. The pin then punches a smaller droplet onto the substrate. This means that sample spotting can be done much faster, as the printhead does not have to return to the microtitre dish as often.
    Other robotic systems for spotting arrays are offered by BioDiscovery, BioRobotics, Cartesian Technologies.

    Another more sophisticated variant of this technology is that recently developed by Nanogen.

    This "grabber" relies on the presence of a charge - either positive or negative - on most biological molecules. The technique uses this property to enable the active movement of the charged molecules on the microchip.

    • The microchip is swamped with a solution of a DNA probe, and a spot, row or column is activated electrically.
    • This introduces a positive electrical charge onto the surface, and the negatively charged DNA probe concentrates on this.
    • The DNA is then chemically bonded in position, the chip is washed, and further probe solutions can be added.

      This means an array of specifically bound DNA probes can be made on the microchip - site by site, row by row.

    The use of controlled electric fields in this way makes the process of hybridisation considerably faster.
    There is also the added advantage of being able to reverse the polarity of certain sites. This forces any DNA molecules that are unbound, or nonspecifically bound, back into solution and away from the probes. This improves the quality, as it ensures that bound pairs of DNA are truly complementary. This in turn means that the technique increases application in the detection of

    • single point mutations
    • single base pair mismatches
    • other genetic mutations

    This will affect a number of research and diagnostic areas.

    A reduction in time and labour is another benefit of Nanogen's technology: a lower concentration of target DNA molecules is required in this technique, because the electric field causes the DNA molecules to be concentrated over the test site.

    Nanogen's technology has considerable advantages, and its importance and application will no doubt increase in the future.

    Piezoelectric Printing

    Minute volumes of reagents are delivered to defined locations on the slide similar to 'ink-jet' printing methods.

    The printhead moves across the array, and at each spot electrical stimulation causes the DNA bases, cDNAs or other molecules to be delivered onto the surface via tiny jets. This is a non-contact process.

    Photolithography

    This makes use of semiconductor technologies. Light is used in association with a series of photomasks.

    The light from a mercury lamp activates modified photospecific versions of the four DNA bases. The photolithographic masks ensure the DNA synthesis is stimulated in defined positions - the masks predetermine which of the nucleotides are activated.
    This 'in situ' fabrication technique was developed by Affymetrix, and is used to produce their GeneChips.

    These 'oligonucleotide arrays' are described in Oligonucleotide Arrays: their production, uses and advantages.
    The disadvantages of photolithography for the production of microarrays are also discussed here.

    There is an alternative method for the in situ fabrication of oligonucleotides to the one using these photomasks.

    Singh-Gasson et al., have developed a Maskless Array Synthesiser (MAS).

      This method replaces the chrome masks usually used with virtual masks, generated on a computer. This image is then relayed to a digital micromirror array.
      The micromirrors are individually addressable, and can be used to make any set image or pattern.
      The series of steps can be repeated many times using different virtual masks, so growing the required oligonucleotides in the correct patterns.

    The main disadvantages of the usual photolithography method are:

    1. the amount of time taken to produce the masks, and
    2. the expense of their production.

    The MAS technique overcomes both of these problems:

    1. with adequate computing resources, a new array based on a new sequence can be synthesised in less than 24 hours;
    2. the cost of arrays produced this way is less too, because there are no costly masks to produce. The chemistry involved here is the main expense, and that is limited to around $60.

    The limiting factor of this technique is in fact the lack of sufficient resolution of commercial scanners. An improvement in this technology will improve the potential of this new technique.


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