MS-FLAG, a novel real-time signal generation method for methylation-specific PCR
MS-FLAG, a novel real-time signal generation method for methylation-specific PCR
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DOI：
10.1373/clinchem.2007.094011
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发表时间：
2007-12-01
影响因子：
9.3
通讯作者：
Makrigiorgos, G. Mike
中科院分区：
文献类型：
--
作者：
Bonanno, Cinzia;Shehi, Erlet;Makrigiorgos, G. Mike
背景：异常启动子甲基化是肿瘤抑制基因沉默的主要机制。超甲基化的检测被用作早期癌症诊断的分子标志物，作为预后指标，或定义异常甲基化逆转的治疗靶点。我们报告了一种新的实时PCR检测基因启动子methylation.Methods的信号产生技术：FLAG（荧光扩增子产生）是一种均匀的信号产生技术的基础上异常热稳定的核酸内切酶PspGI。FLAG在PCR期间通过PspGI介导的双链PCR产物的5'末端处淬灭的荧光团的切割提供实时信号产生。将应用于亚硫酸氢盐处理的DNA的甲基化特异性PCR（MSP）调整为实时格式（甲基化特异性FLAG; MS-FLAG），用于定量CDKN 2A（p16）、GATA 5和RASSF 1的启动子中的甲基化。我们在已知甲基化状态的质粒和基因组DNA上验证了MS-FLAG，并将其应用于检测有限数量的临床样本中的甲基化。我们还对这些样品进行了亚硫酸氢盐测序。结果：通过MS-FLAG获得的实时PCR结果与通过常规的基于凝胶的MSP获得的结果一致。该新技术在对照样品上显示出高特异性、灵敏度（2-3个质粒拷贝）和选择性（0.01%的甲基化DNA）。它能够正确预测21个肺腺癌样品中所有3个基因启动子的甲基化状态，如通过亚硫酸氢盐测序所证实的。我们还开发了GATA 5和RASSF 1启动子的多重MS-FLAG检测方法。结论：MS-FLAG提供了一种新的、定量的、高通量的检测基因启动子甲基化的方法，是基于琼脂糖凝胶的MSP筛查甲基化的方便替代方法。除了甲基化之外，基于FLAG的实时信号生成可能在DNA诊断中具有广泛的应用。(C)2007年美国临床化学协会。
Background: Aberrant promoter methylation is a major mechanism for silencing tumor suppressor genes in cancer. Detection of hypermethylation is used as a molecular marker for early cancer diagnosis, as a prognostic index, or to define therapeutic targets for reversion of aberrant methylation. We report on a novel signal generation technology for real-time PCR to detect gene promoter methylation.Methods: FLAG (fluorescent amplicon generation) is a homogeneous signal generation technology based on the exceptionally thermostable endonuclease PspGI. FLAG provides real-time signal generation during PCR by PspGI-mediated cleavage of quenched fluorophories at the 5' end of double-stranded PCR products. Methylation-specific PCR (MSP) applied on bisulfite-treated DNA was adapted to a real-time format (methylation-specific FLAG; MS-FLAG) for quantifying methylation in the promoter of CDKN2A (p16), GATA5, and RASSF1. We validated MS-FLAG on plasmids and genomic DNA with known methylation status and applied it to detection of methylation in a limited number of clinical samples. We also conducted bisulfite sequencing on these samples.Results: Real-time PCR results obtained via MS-FLAG agreed with results obtained via conventional, gel-based MSP. The new technology showed high specificity, sensitivity (2-3 plasmid copies), and selectivity (0.01% of methylated DNA) on control samples. It enabled correct prediction of the methylation status of all 3 gene promoters in 21 lung adenocarcinoma samples, as confirmed by bisulfite sequencing. We also developed a multiplex MS-FLAG assay for GATA5 and RASSF1 promoters.Conclusion: MS-FLAG provides a new, quantitative, high-throughput method for detecting gene promoter methylation and is a convenient alternative to agarose gel-based MSP for screening methylation. In addition to methylation, FLAG-based real-time signal generation may have broad applications in DNA diagnostics. (C) 2007 American Association for Clinical Chemistry.