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PCR Applications Guide
Rare Mutation and Sequence Detection
RMD Experiment Considerations
The first consideration for low-level detection is the amount of DNA available. If 1 mutant
in 100,000 wild-type sequences, or 0.001%, is to be detected, then statistically at least
300,000 haploid genomes must be screened. For human DNA this is 1 µg of DNA.
The challenge for RMD assay development is that it must discriminate between two
highly similar sequences, one of which is significantly more abundant than the other.
An example of an RMD assay is detection of a single nucleotide polymorphism (SNP) in
a cancer biopsy. An RMD assay comprises a single set of primers plus two competitive
probes (each probe with a different fluorophore), one detecting the wild-type allele,
and one detecting the variant allele.
Testing an RMD Assay
RMD assays should be tested for specificity by first running a temperature gradient at a
relatively high wild-type DNA concentration and then by running a concentration gradient
of wild-type DNA with a spike-in of mutant DNA to assess specificity.
First, run a temperature gradient using both a row/column of restriction digested,
100% wild-type DNA at a concentration between 2,000–5,000 copies/µl previously
restriction digested and a row/column of 2,000–5,000 copies/µl wild-type DNA including
50–200 copies/µl mutant DNA. Using the 1-D amplitude plot, select the T
m
with no false
positives, good separation between positive and negative populations, and minimal probe
cross-reactivity (Figure 5.3). Also, be sure to simultaneously check the concentration plots
to make sure the concentration is the expected result at the temperature selected.
In Figure 5.3, wells A–H are an annealing/extension gradient from 65–55°C, respectively.
Panel A is a 1-D amplitude plot showing the FAM-labeled mutant assay results from a
temperature gradient on the SNP PI3Kca_E545K. Wells A03–H03 contain 100% wild-
type DNA, and A04–H04 contain 50% wild-type/mutant DNA. Well D was selected at an
annealing/extension temperature of 61.4°C. At lower annealing/extension temperatures,
false positives appear. Panel B is a 1-D amplitude plot of FAM-labeled mutant assay results
of PI3Kca_H1047R. Wells A06–H06 contain 100% wild-type DNA, and A07–H07 contain
50% wild-type/mutant DNA. Well B was selected at an annealing/extension temperature of
64.5°C. At lower annealing/extension temperatures, the positive clusters for the VIC channel
have increasing probe cross-reactivity with the nonspecific target, causing the wild-type
droplets to have some level of FAM fluorescence in the FAM channel.