A novel application of melting curves: utility of peak area calculation for relative methylation quantification

作者：Deligezer Ugur; Akisik Ebru Esin; Dalay Nejat^*

来源：Clinical Chemistry and Laboratory Medicine, 2007, 45(7): 867-873.

DOI：10.1515/CCLM.2007.146

• leukemia
• melting curve
• methylation
• methylation index
• peak area calculation

Background: DNA methylation markers appear to be useful in cancer detection, in evaluating the prognosis associated with disease progression, and in detecting the metastatic potential of tumors.
Methods: We present an approach for relative quantification of methylated gene sequences. The technique combines conventional PCR and LightCycler fluorescence PCR. In the conventional PCR step, bisulfite-modified molecules are selectively enriched, irrespective of their methylation status, and used as template in the second step, the LightCycler fluorescence PCR, which includes methylation-specific primers and fluorescently labeled probes. After amplification, a stepwise increase in temperature leads to the generation of melting curves reflecting fluorescence emission induced by disassociation of the probe-target complexes. Quantification is based on calculation of the peak areas for melting curves using a Gaussian fit curve. The area under this curve indicates the relative amounts of methylated sequences in each sample.
Results: By normalizing the peak areas by template concentrations, a methylation index was calculated for each sample. This approach was then used to carry out a relative quantification of p16/NK4A gene methylation in bone marrow and blood mononuclear cells from patients with positive translocation status.
Conclusions: If validated in further studies, this approach represents a highly specific technique that can be used in analyzing paired samples of cancer patients.

• 出版日期2007