Microarrays

Traditionally, arrays have been categorized into different formats: conventional (cDNA and oligonucleotide), elec-trokinetic, fiberoptic, microfluidic, and more recently, applications in the nanotechnology field. The most popular format today is the conventional arrays, which will be discussed in detail here (Fig. 1). Within this paper, ''probe'' will refer to the macromolecule printed on the array surface and ''target'' will refer to the macromole cule derived from the sample and hybridized to the array. The other array formats have just recently been commercialized or are in development.

The conventional array format involves either printing macromolecules onto a surface or synthesizing the macromolecules in situ one building block at a time. The first technique, usually referred to as ''cDNA microarrays,'' is the printing of DNA onto a substrate. The initial papers describing this technique were derived from collaboration between the laboratories of Ron Davis and Pat Brown at Stanford University.[4] The second technique, usually referred to as ''oligonucleotide arrays,'' builds the macromolecular sequence in situ or at a specific location on the surface. Early applications used photolithography. A mask is put over the array surface to allow chemical reactions to take place only at specified sites. The reactions involve photolabile groups activated by light to free hydroxyl groups. The hydroxyl groups were then covalently bound with phosphoramidite-activated deoxynucleosides. The bound molecule is capped and oxidized. After rinsing, a new mask covers the array surface and a new set of reactions occur. With each round of reactions, an activated nucleoside is added and the oligonucleotide (''oligo'') is synthesized to approximately 25 nucleotide oligomers or ''25-mers.'' This method was originated by Stephen Fodor and David Lockhart at Affymetrix.[5] Two newer in situ methods have been developed: one which uses a digital micro-mirror to create a virtual mask for the photolithographic manufacturing process of microarrays[6] and another allows parallel synthesizing of several different types of molecules.[7] The first technology was initially developed at Texas Instruments and is being commercialized by NimbleGen and the second is being commercialized by Xeotron.

If an investigator wishes to set up his/her own arraying facility, cDNA microarrays are the easiest methodology to start with. (Here the authors are referring to printing microarrays of cDNAs or oligos by methods other than photolithography.) However, before starting, several choices of materials and processes are required.

The first choice is to decide which substrate the array will be printed on. Current choices are listed in Table 1

Fig. 1 The schematic depicts the hybridization reaction for cDNA or oligonucleotide microarrays. Briefly, the probes are oligonucleotides or expressed sequence tags (ESTs, actual form of cDNA used) printed onto a glass slide. The target molecule is the cDNA (fluorescently labeled with either Cy3 or Cy5) present in the hybridization solution. During the reaction, the fluo-rescently labeled cDNA in solution will hybridize to the probe on the glass surface with a homologous sequence.

and, traditionally, the most common substrates used are glass slides and nylon filters. Because of their larger surface area (several square centimeters), nylon filter arrays are sometimes referred to as ''macroarrays.'' Other surfaces listed in Table 1 are being used by formats just recently commercialized or in development, such as microfluidic arrays. Today, most conventional arrays use glass; however, it is extremely important that glass surfaces are cleaned prior to printing. Some preferred methods include ultrasonication[23] or washing with acid[10'12] or alkali.[24]

Table 1 Microarray substrate choices

Substrates

References

Beads

[8]

Compact discs

[9]

Fused silica slides

[10]

Gel pads (fixed to glass)

[11]

Glass slides

[4,5,12]

Nylon filters

[13,14]

Polypropylene film

[15]

Polystyrene microwell plates

[16]

Silicon wafer

[17-20]

Waveguides, planar

[21,22]

Table 2 Derivatizing agents for microarray surfaces

Modifications

Derivative

References

Agarose film

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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