Melting profiles of bisulfite-converted DNA from clinical samples following <i>fast</i>-COLD-MS-PCR.

<p>The effect of lowering the denaturation temperature in PCR is depicted. <b>Panel A.</b> Glioma sample no. 3 was subjected to different critical denaturation temperature-T_c during <i>fast</i>-COLD-MS-PCR. The modulation of the preferential amplification of the un-methylated DNA fraction is shown. <b>Panel B. </b><i>fast</i>-COLD-MS-PCR performed at a T_c of 84°C demonstrates that the amplification of the methylated DNA fraction is completely inhibited.</p

Detection of un-methylated DNA within different abundances of methylated DNA background by conventional or <i>fast</i>-COLD-MS-PCR.

<p><b>Panel A.</b> Post-PCR melting profile of the 255 bp bisulfite-converted-specific amplicon after conventional PCR or COLD-PCR. Serial dilutions of un-methylated (U) to methylated (M) genomic DNA are depicted (top half). Higher abundances of un-methylated DNA can be discriminated from methylated DNA by the melt peak, whereas lower abundances are only detectable if <i>fast</i>-COLD-MS-PCR replaces conventional PCR. <b>Panel B.</b> Sanger sequencing results of the 0.05% un-methylated (U): methylated (M) DNA sample as amplified by conventional and <i>fast</i>-COLD-MS-PCR are shown (bottom half). Chromatograms are aligned and compared using SeqDoc, and the CpG methylation positions are revealed in the middle panel.</p

Melting profiles of bisulfite-converted DNA from clinical samples following conventional PCR.

<p>Post-PCR melting profiles of the 255 bp bisulfite-converted <i>MGMT</i> gene amplicon after conventional PCR. Examples of fully un-methylated DNA samples isolated from infant blood (<b>Panel A</b>) and glioma samples (<b>Panel B</b>) are depicted. 100% methylated (M) and 100% un-methylated (U) DNA controls are used as reference standards, demonstrating a ∼5°C melting temperature difference among the two. <b>Panel C.</b> A glioma sample with mixed methylation/unmethylation pattern is shown.</p