build oligodeoxyribonucleotides on glass surfaces. An entirely new carbonate-based protecting group chem-
istry enables deprotection and oxidation in a single step, reducing time and cost for microarray synthesis.^23
5.5.4.4 Microarray Analysis. How does one analyse the information encoded in thousands of indi-
vidual gene sequences on a small glass or silicon chip? The process is based on hybridisation probing, a
technique that uses fluorescently labelled nucleic acid molecules as ‘mobile probes’ to identify complemen-
tary DNA sequences using base pair recognition. The DNA probes to be hybridised to the array are labelled
by incorporating fluorescently tagged nucleotides (such as Cy3-dUTP, Figure 5.14b) during oligo-primed
reverse transcription of mRNA. Alternatively, they can be chemically tagged by 5-end labelling (Figure
5.14c). Different green and red fluorophores are used to label cDNAs from control (reference) and exper-
imental (test) RNAs. The labelled cDNAs are then mixed together prior to hybridisation to the array so that
relative amounts of a particular gene transcript in the two samples are determined by measuring the signal
intensities detected for both green and red fluorophores. Because the arrays are constructed on a rigid surface
(glass), they can be inverted and mounted in a temperature-controlled hybridisation chamber. When the fluor-
escent mobile probe, DNA, cDNA or mRNA, locates a complementary sequence on the chip, it will lock
onto that immobilised target, and the probe is identified by fluorescence microscopy. The fluorescent tag on
the probe is excited by a laser and the digital image of the array is captured. These data are then stored in a
computer for analysis. Thus, for example, cDNA from a normal cell and a diseased cell can be separately
labelled with green and red fluorescent markers to enable comparative analysis. The location and intensity of
both colours shows whether the gene, or a mutant, is present in either the control and/or sample DNA (Figure
5.15). It can also provide an estimate of the expression level of the gene(s) in the sample and control DNA.
5.5.4.5 Types of Microarray. There are three basic types of samples used to construct DNA
microarrays, two are genomic and the other is ‘transcriptomic’, for measuring mRNA levels. They differ
in the kind of immobilised DNA used to generate the array and, ultimately, the kind of information that is
derived from the chip. The target DNA used will also determine the type of control and sample DNA that
is used in the hybridisation solution.
5.5.4.5.1 Changes in Gene Expression Levels. Determining the level, or volume, at which a par-
ticular gene is expressed is called microarray expression analysis, and the arrays used in this kind of
analysis are called ‘expression chips’. The immobilised DNA is cDNA derived from the mRNA of known
Nucleic Acids in Biotechnology 187
Figure 5.15 Microarray analysis to compare the hybridisation of expressed genes in a control cDNA sample (left) and
in a mutant (or diseased) cDNA sample (right) to an immobilised reference gene set. Red dots show where
the gene is expressed only in the control. Grey dots (green channel) show where the gene is expressed only
in the mutant. White dots (normally green plus redyellow) show here the gene is expressed in both con-
trol and mutant sample. Absence of a dot indicates that the gene is not expressed in either DNA sample