of a test and a standard probe provides an immediate signal of the presence or absence of a particular
sequence. Ultimately, such studies promise to expand the size of existing gene families, reveal new patterns
of coordinated gene expression across gene families, and uncover entirely new categories of genes.
5.5.4.1 Technical Foundations. Two technologies are central to the production and use of DNA
microarrays. The first is the fabrication of tens to hundreds of thousands of polynucleotides at high spatial
resolution in precise locations on a 2-D surface. The second involves the measurement of molecular
hybridisation events on the array using laser fluorescence scanning. By use of one of three different
methodologies, DNA is synthesised, spotted or printed onto the support, which is usually a glass micro-
scope slide, but can also be a silicon chip or a nylon membrane. The DNA sequences in a microarray are
attached to the support in a fixed way, so that the location of each spot in the 2-D grid identifies a particu-
lar sequence. The spots themselves are either oligodeoxynucleotides, DNA or cDNA.
5.5.4.2 Use of DNA Microarrays. The five steps for carrying out a microarray experiment are
DNA chippreparation using the chosen target DNAs,
making a hybridisation solution containing a mixture of fluorescently labelled cDNAs,
incubating the hybridisation mixture of fluorescently labelled cDNAs with the DNA chip,
detecting bound cDNA using laser technology and data storage in a computer, and
data analysis using computational methods.5.5.4.3 Microarray Preparation. The first chip technology came in 1984 from the work of Stephen
Fodor in the California-based company, Affymetrix, and is based on photolithography. A synthetic linker
with a photochemically removable protecting group is bonded to a flat glass substrate. Light is then directed
through a photolithographic mask to specific areas on the surface to produce localised photodeprotection
(Figure 5.13). The first of a series of DNA phosphoramidite monomers (Section 4.1.2), also having a
5 -(-methyl-2-nitropiperonyloxycarbonyl), photochemically labile protecting group^20 (Figure 5.14a) is
Nucleic Acids in Biotechnology 185
Figure 5.13 Light directed oligonucleotide synthesis. Derivatised solid support has hydroxyl groups protected with
a photolabile group. Light is directed through a mask to effect selective deprotection. The first dT-
phosphoramidite with 5-photolabile protection is introduced. A new mask enables deprotection of a
second set of spots on the array which are then linked to the second nucleotide, dA. Repetition of this
procedure for next dC and finally dG completes the cycle for the first nucleotides in the oligomer array.
The cycle is then repeated with new masks to install the second nucleotides in the array, and so on