Page 1341 - Clinical Immunology_ Principles and Practice ( PDFDrive )

1302 ParT ElEvEN Diagnostic Immunology

(Illumina Inc., San Diego, CA). 22,23 These processes are very resulting hybridization intensities can be used to derive the
similar, in principle, to the fabrication of microelectronics devices, genotypes. These assays are fixed in the sense that the investigator
and so have given these arrays the nickname “gene chips.” uses the information from a standardized array but cannot add
Microarrays have been used in very-large-scale studies to SNPs for particular purposes. An alternative approach, incor-
genotype common polymorphisms. Robust chemistries that can porated in the Infinium assays (Illumina Inc., San Diego, CA),
analyze standard sets of up to 5 000 000 SNPs in a single assay involves the detection of allele-specific primer extension or single
are now routine (see Fig. 96.3). Nonamplified genomic DNA is base extension products on a self-assembling high-density
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too complex (i.e., the concentration of any particular sequence microbead array. In the Infinium assays, genomic DNA is first
is too low) to be analyzed directly. High-throughput genotyping subjected to whole genome amplification (WGA), and those
assays require an array-based readout and scalable, multiplex products are hybridized to an array of locus-specific 50-mer
assay chemistry. This is extremely challenging because of the capture probes. Either primer extension or ligation reactions on
complexity of the human genome (i.e., any particular unique the array surface are used to accomplish allele discrimination.
sequence in the mixture has a low molar concentration and Signal amplification with methods familiar to users of enzyme-
because of the need for exquisite specificity at the single base linked immunosorbent assay (ELISA) is used to enhance the
level). An important general principle for achieving single-base sensitivity. A distinct advantage of this class of assays is that the
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specificity is the detection of physically coincident events. This “assay conversion” (percent SNPs that actually allow inference
is exactly the route taken by the polymerase chain reaction (PCR), of the genotype in the final assay mixture) is very high and thus
in which specific annealing of both primers is required for the allows for flexibility in designing tests customized to particular
reaction to take place (Fig. 96.4). problems.
Whole genome sampling and amplification (WGSA) imple-
mented in the Affymetrix SNP arrays uses parallel amplification GENE EXPRESSION
of short DNA segments (200–1100 base pairs [bp]) that are then
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labeled and hybridized to oligonucleotide probe arrays. The Global gene expression analysis has been enabled by microarray
oligonucleotides are specific for each allele of SNPs, and the technology giving rise to so called “transcriptomics.” For RNA
Primer A Primer B

1. Denature
1 st Cycle 2. Anneal
3. Extend

2 Cycle
nd

1. Denature
2. Anneal
3. Extend

.
.
th
n Cycle .
n
2 n
FIG 96.4 Polymerase Chain Reaction. Primers that hybridize to the target sequence are used
to initiate multiple cycles of synthesis, melting, annealing, and synthesis. In practice, the potential
geometrical increase in DNA tapers off as reaction components become limiting.