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Tc 80oC
0.2%
1%
Supplemental Data Figure 1. Detection of
critical temperature (Tc) for COLD-PCR /
HRM assays.
5%
25%
Tc 79.5oC
0.2%
1%
5%
25%
Tc 79oC
0.2%
1%
5%
25%
Using uMelt software, we predicted that the wildtype DNA melting point for the amplified fragment
would be around 80.5oC. Therefore, we
experimentally tested three temperatures, 80.0 oC,
79.5 oC and 79.0 oC, to find out a Tc providing
highest enrichment for KRAS mutations detection.
We used serial dilutions of DNA extracted from
KRAS mutant A549 cell line (harbours p.G12S,
c.34G>A mutation) in DNA from KRAS wild-type
TT1 cell line. Using the dilutions of 25%, 5%, 1%,
and 0.2% and automatic software calling of the
variants, we identified the best Tc to be 79.0 oC for
fast COLD-PCR and 79.5 oC for full COLD-PCR.
Arrows indicate the curves corresponding to the
dilution percentages of the mutant DNA.
A.
0.024%
0.098%
0.391%
1.563%
6.25%
25%
100%
B.
0.410%
1.024%
2.56%
6.4%
16%
40%
100%
Supplemental Data Figure 2. Analytic
sensitivity of COLD-PCR / HRM assays.
Analytic sensitivity of (A) fast and (B) full
COLD-PCR/HRM assays to detect mutations
in codons 12/13 of KRAS gene was
established by the analysis of serial
dilutions of DNA extracted from KRAS
mutant A549 cell line (harbours p.G12S,
c.34G>A mutation) in DNA from KRAS wildtype TT1 cell line. Similar results were
obtained for serial dilutions of H358 cell
line (heterozygote on G12C, c.34G>T
mutation; data not shown). Arrows indicate
the curves corresponding to the dilution
percentages of the mutant DNA.