A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA):: analysis of the retinoblastoma locus -: art. no. e125
A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA):: analysis of the retinoblastoma locus -: art. no. e125
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DOI：
10.1093/nar/gnh122
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Lohmann, DR
中科院分区：
文献类型：
--
作者：
Zeschnigk, M;Böhringer, S;Lohmann, DR
已发现改变的甲基化模式在发育障碍、癌症和衰老中起作用。DNA甲基化的变化越来越多地被用作疾病的分子标记。因此，需要可靠且易于使用的技术来检测和测量研究和常规诊断中的DNA甲基化。我们已经建立了一种新的甲基化等位基因的定量分析（QAMA），这基本上是一个重大的改进，在以前的方法的基础上实时PCR（MethyLight）。该方法基于亚硫酸氢盐处理的DNA的实时PCR。通过使用基于小沟结合剂（MGB）技术的TaqMan探针，获得了优于传统MethyLight的显著优势。其改进的序列特异性有利于在单管中同时扩增的甲基化和未甲基化等位基因的相对定量。这种改进允许精确测量甲基化与未甲基化等位基因的比率，并减少了测定间变异的潜在来源。因此，需要较少的对照试验。我们已经使用这种新的技术方法来鉴定位于视网膜母细胞瘤（RB1）基因启动子区域的CpG岛的超甲基化，并发现QAMA有助于可靠和快速测量甲基化等位基因的相对数量，并改善诊断甲基化分析的处理。此外，简化的反应设置和单管测定固有的稳健性促进了高通量甲基化分析。由于MGB技术固有的高序列特异性被广泛用于区分单核苷酸多态性，因此QAMA潜在地可用于区分单个CpG二核苷酸的甲基化状态。
Altered methylation patterns have been found to play a role in developmental disorders, cancer and aging. Increasingly, changes in DNA methylation are used as molecular markers of disease. Therefore, there is a need for reliable and easy to use techniques to detect and measure DNA methylation in research and routine diagnostics. We have established a novel quantitative analysis of methylated alleles (QAMA) which is essentially a major improvement over a previous method based on real-time PCR (MethyLight). This method is based on real-time PCR on bisulfite-treated DNA. A significant advantage over conventional MethyLight is gained by the use of TaqMan probes based on minor groove binder (MGB) technology. Their improved sequence specificity facilitates relative quantification of methylated and unmethylated alleles that are simultaneously amplified in single tube. This improvement allows precise measurement of the ratio of methylated versus unmethylated alleles and cuts down potential sources of inter-assay variation. Therefore, fewer control assays are required. We have used this novel technical approach to identify hypermethylation of the CpG island located in the promoter region of the retinoblastoma (RB1) gene and found that QAMA facilitates reliable and fast measurement of the relative quantity of methylated alleles and improves handling of diagnostic methylation analysis. Moreover, the simplified reaction setup and robustness inherent to the single tube assay facilitates high-throughput methylation analysis. Because the high sequence specificity inherent to the MGB technology is widely used to discriminate single nucleotide polymorphisms, QAMA potentially can be used to discriminate the methylation status of single CpG dinucleotides.