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Fig. 3. ( continued from opposite page ) of mRNA may also be studied after reverse
transcription. The sample with the greater amount of DNA (or cDNA) will show an
earlier increase in fluorescence. The second derivative maxima of the curves (vertical
dotted lines) are determined as fractional cycle numbers. The relative copy number
between samples is the PCR efficiency (eff) raised to the difference between frac-
tional cycle numbers (
C). The calculation assumes that the PCR efficiency is the
same between samples. The PCR efficiency is usually between 1.7 and 2.0. As a first
approximation, an efficiency of 2 is often assumed. This analysis assumes that the
starting amount of material (DNA or cDNA) in each sample is the same. (B) Another
option is to use a test target normalized to a reference target. The amount of starting
material in each sample is normalized to a reference (Ref) or housekeeping gene. Both
experimental and control samples are amplified for both the test and reference targets.
Any difference in the amount of starting material is normalized by the results of the
reference target amplification. This method assumes that the reference target is invari-
ant between samples and that the PCR efficiency for each target does not vary between
samples. As a first approximation, an efficiency of 2 is often assumed for both targets
and has become known as the
∆∆
C method.
Fig. 4. A single-nucleotide polymorphism (SNP) is demonstrated in a 544-bp frag-
ment by melting analysis. Shown are high-resolution melting curves of polymerase
chain reaction amplicons from the HTR2A gene locus carrying an SNP. Results are
shown for six individuals, two different individuals for each of the three genotypes:
wild type homozygote (TT), mutant homozygote (CC), and heterozygote (TC). The
inset is a magnified portion of the data showing that all three genotypes can be dis-
criminated. (Adapted from ref. 8 , with the permission of AACC Press.)
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