A novel application of melting curves: utility of peak area calculation for relative methylation quantification
A novel application of melting curves: utility of peak area calculation for relative methylation quantification
复制标题
DOI：
10.1515/cclm.2007.146
复制
发表时间：
2007-01-01
影响因子：
6.8
通讯作者：
Dalay, Nejat
中科院分区：
文献类型：
--
作者：
Deligezer, Ugur;Akisik, Ebru Esin;Dalay, Nejat
背景资料：DNA甲基化标志物似乎是有用的癌症检测，在评估与疾病进展相关的预后，并在检测肿瘤的转移潜能。方法：我们提出了一种方法，甲基化基因序列的相对定量。该技术结合了常规PCR和LightCycler荧光PCR。在常规PCR步骤中，选择性富集亚硫酸氢盐修饰的分子，而不管其甲基化状态如何，并在第二步LightCycler荧光PCR中用作模板，该步骤包括甲基化特异性引物和荧光标记探针。扩增后，温度的逐步升高导致产生反映探针-靶复合物解离诱导的荧光发射的解链曲线。定量基于使用高斯拟合曲线计算熔解曲线的峰面积。该曲线下面积表示每个样品中甲基化序列的相对量。结果：通过用模板浓度标准化峰面积，计算每个样品的甲基化指数。然后，这种方法被用来进行相对定量的p16/NK 4A基因甲基化的患者的骨髓和血液单核细胞阳性translocationstatus.Conclusions：如果在进一步的研究中验证，这种方法代表了一种高度特异性的技术，可用于分析配对样本的癌症患者。
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.