Feng, Xiaodong_ Xie, Hong-Guang - Applying pharmacogenomics in therapeutics-CRC Press (2016)

Principles of Pharmacogenetic Biotechnology and Testing in Clinical Practice
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A allele
G allele
CAGATCGG
CAGGTCGG
Target
nucleic acids
Genotype
A/A
A/G
G/G
No/weak
signal
Signal
Probes
Signal Signal Signal
No/weak
signal
GTCCAGCC
CAGATCGG
GTCTAGCC
CAGGTCGG
GTCCAGCC
CAGATCGG
GTCTAGCC
CAGGTCGG
GTCCAGCC
GTCTAGCC
Array 1 Array 2 Array 3
FIGURE 2.2 (See color insert.) Simplified illustration of an SNP microarray. This simplified
array has only two probes, each corresponding to a different SNP allele (A or G). The
affinities of A or G targets to allele-specific probes are different, which result in differential
hybridization strength. Targets are often labeled with fluorophores or chemiluminescence.
After nonbound targets are washed off the microarray, the scanning of fluorescence signal
emitted by bound targets on the probe spots can reveal SNP information of the targets. In fact,
thousands to millions of probes can be printed on designated spots of each microarray, with
each probe representing a unique sequence variation.
Microarray fabrication differs in the types and number of probes, the technology
for attaching probes to the test point, and the solid surface used for probe fixation.
The number of probes can range from hundreds to millions, depending on the
experi mental designs and scientific questions to be addressed. A widely used fabrication
method is to spot oligos on the solid surface of the array using robotic arm–
controlled pins or needles, which capture presynthesized DNA probes by dipping
into solutions of the probes. The array can be customized for specialized research
purposes. The use of such miniaturized microarrays for gene expression profiling
was first reported by Schena et al. (1995), and the first analysis of a complete eukaryotic
genome was published by Lashkari et al. (1997).
An alternative approach to manufacturing a microarray is to directly synthesize
probes on solid surfaces. Probes as long as 60 bases (e.g., Agilent design) or as short
as 25 bases (e.g., Affymetrix design) can be generated in this manner. Longer probes
are more specific to individual target genes, whereas shorter ones may be spotted in
higher density across the array and are economic for manufacturing. The biotechnology
company Roche NimbleGen Systems (www.nimblegen.com) also developed
a new method called maskless array synthesis, which combines sequence flexibility
with the large-scale probes (Nuwaysir et al. 2002). In standard microarrays, the probes