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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different alleles are designed in distinct lengths to allow separation of amplified DNA
fragments by gel electrophoresis or melting curve analysis (Figure 2.1b).
Real-time PCR is developed to amplify and simultaneously quantify target DNA
molecules. One approach is to include a double-stranded DNA-binding dye in the
PCR reaction. The binding of the dye to double-stranded DNA causes fluorescence
that can be measured, therefore generating a real-time quantification of the amount
of amplified DNA molecules in the reaction (Ponchel et al. 2003; Zipper et al. 2004).
An alternative strategy is the use of fluorophore-conjugated probes that anneal to
different alleles, as exemplified by the TaqMan ® assay (ThermoFisher Scientific;
www.lifetechnologies.com) (Figure 2.1c). In this assay, allele-specific oligonucleotide
(or oligo) probes are labeled with different fluorophores (e.g., 6-carboxyfluorescein
or tetrachlorofluorescein) at the 5′-ends and with a quencher molecule (e.g., tetramethylrhodamine)
at the 3′-ends. The quencher molecules quench the fluorescence of
the fluorophores by fluorescence resonance energy transfer, which is most effective
when the fluorophore and the quencher are in close proximity in the same probe. The
allele-specific probes are included in the PCR reaction with a specific set of primers
that amplify the region complementary to the probes. In the annealing phase,
the probes and primers hybridize to their target. In the extension phase, the 5′–3′
exonuclease activity of the DNA polymerase (Holland et al. 1991) degrades the perfectly
matched, annealed probes. The degraded probes are released into the solution
as single nucleotide, separating the fluorophore from the quencher, which results
in an increase in fluorescence. In contrast, mismatched probes are not annealed to
their targets and will not be degraded by the DNA polymerase, resulting in fluorescence
still quenched. The difference in fluorescence can be monitored in a quantitative
PCR thermal cycler, in which fluorescence released from degraded nucleotides
of perfectly matched probes indicate how much the target SNP is amplified. The
TaqMan assay can be multiplexed by combining the detection of up to seven SNPs
in one reaction. However, since each SNP requires a distinct probe, the TaqMan
assay is limited by how close the SNPs locate from each other on the DNA template.
Generally, TaqMan is limited to applications that involve a small number of SNPs
since optimal probes and reaction conditions must be designed for each SNP.
More recently, digital PCR has been developed and routinely used for clonal
amplification of samples in next-generation sequencing (NGS). The digital PCR procedure
was originally aimed to precisely quantify the input template rather than the
final PCR product. The very first clinical application of digital PCR was to measure
the absolute lowest number of leukemic cells in a leukemia patient with a goal to
monitor residue disease and detect recurrence in patients as early as possible (Sykes
et al. 1992). Evolution of this technology has allowed for a broad use in studying
variations in gene sequences—such as CNVs and point mutations. The key difference
between digital PCR and traditional PCR lies in the methods of treating the
DNA templates. Digital PCR separates each single starting DNA molecule into
distinct partitioned reactors and carries out one single reaction within each partition
individually (Kalinina et al. 1997). The localization of individual DNA molecules
in separate partitions provides an estimation of the starting molecule number
by assuming that the population follows the Poisson distribution. In other words,
each partition is assumed to contain either zero or one starting template for PCR.