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

38 Applying Pharmacogenomics in Therapeutics
5’
3’
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DNA template
Primer
dNTP
DNA polymerase
A allele
G allele
Denaturation
(a)
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A
T
A
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3’
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C 5’
G
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C
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(b)
G
TaqMan probe
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T 5’
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T
Annealing
Elongation
Cycling
Genotype
A/A A/G G/G
Sample A Partitioning Amplification
Sample B
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3’
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(d)
FIGURE 2.1 (See color insert.) Illustration of polymerase chain reaction (PCR)–related
methods. (a) A PCR cycle generally includes denaturation, annealing, and elongation steps.
(b) A representative design for a tetra-primer set, which integrates allele-specific nucleotides
into the 3′-ends of primers. (c) A modified graph showing the mechanism of SNP genotyping
by TaqMan assay (Life Technologies) using allele-specific fluorophore-conjugated probes.
The perfectly matched TaqMan probe that anneals to the template will be degraded by DNA
polymerase and release the fluorescence (yellow), while the fluorophore (blue) of the mismatched
TaqMan probe that does not anneal will remain quenched. (d) Strategy of digital
PCR to quantitatively estimate the starting DNA templates. Sample A has fewer starting templates,
which are shown in fewer positive digital PCR reactions. For sample B, more starting
templates are shown in more positive digital PCR reactions.
to target different allele-specific SNPs (You et al. 2008) (Figure 2.1b). These primer
pairs are designed such that one single mismatch at the 3′-end will make the primers
nonfunctional and terminate the amplification. As a result, the tetra-primer can
amplify only the specific allele present in the PCR reaction but not the alternative
allele with a different SNP. To distinguish the SNPs, PCR regions spanning the two