Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection.
Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection.
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实时甲基化特异性PCR（SMART-MSP）之后的敏感熔解分析：高通量和无探针定量DNA甲基化检测。
DOI：
10.1093/nar/gkn113
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发表时间：
2008-04
影响因子：
14.9
通讯作者：
Dobrovic, Alexander
中科院分区：
文献类型：
--
作者：
Kristensen, Lasse S.;Mikeska, Thomas;Krypuy, Michael;Dobrovic, Alexander
在癌症中复发的DNA甲基化变化作为早期检测和监测癌症的潜在生物标志物引起了极大的兴趣。在这种情况下，如果甲基化检测是纯定性的，则会错过重要信息。我们描述了一种新的无探针定量甲基化特异性PCR（MSP）检测，采用高分辨率熔解（HRM）分析的扩增子的评价。根据扩增子设计，可以从HRM分析获得不同类型的信息。这些信息中的大部分不能通过电泳分析获得。特别是，由于亚硫酸氢盐转化不完全或错误启动而导致的假阳性识别是可能的。异源甲基化也可以与同质甲基化区分开。作为原理的证明，我们已经开发了针对CDH 1、DAPK 1、CDKN 2A（p16 INK 4a）和RARB基因的启动子区域的测定。我们表明，高度准确的定量是可能的范围从100%至0.1%甲基化模板时，25 ng的亚硫酸氢盐修饰的DNA被用作PCR模板。我们将这种定量甲基化检测的新方法命名为真实的时间后灵敏解链分析（SMART）-MSP。
DNA methylation changes that are recurrent in cancer have generated great interest as potential biomarkers for the early detection and monitoring of cancer. In such situations, essential information is missed if the methylation detection is purely qualitative. We describe a new probe-free quantitative methylation-specific PCR (MSP) assay that incorporates evaluation of the amplicon by high-resolution melting (HRM) analysis. Depending on amplicon design, different types of information can be obtained from the HRM analysis. Much of this information cannot be obtained by electrophoretic analysis. In particular, identification of false positives due to incomplete bisulphite conversion or false priming is possible. Heterogeneous methylation can also be distinguished from homogeneous methylation. As proof of principle, we have developed assays for the promoter regions of the CDH1, DAPK1, CDKN2A (p16INK4a) and RARB genes. We show that highly accurate quantification is possible in the range from 100% to 0.1% methylated template when 25 ng of bisulphite-modified DNA is used as a template for PCR. We have named this new approach to quantitative methylation detection, Sensitive Melting Analysis after Real Time (SMART)-MSP.
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影响因子：
20.3
作者：
Cameron, EE;Baylin, SB;Herman, JG
通讯作者：
Herman, JG
DOI：
10.1073/pnas.93.18.9821
发表时间：
1996-09-03
影响因子：
11.1
作者：
Herman, JG;Graff, JR;Baylin, SB
通讯作者：
Baylin, SB
影响因子：
14.9
作者：
Giglio, S;Monis, PT;Saint, CP
通讯作者：
Saint, CP
影响因子：
4.1
作者：
Mikeska, Thomas;Bock, Christoph;Waha, Andreas
通讯作者：
Waha, Andreas
影响因子：
4.5
作者：
Hong YS;Roh MS;Kim NY;Lee HJ;Kim HK;Lee KE;Kwak JY;Kim JY
通讯作者：
Kim JY