Split -Lactamase Sensor for the Sequence-Specific Detection of DNA Methylation
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- 作者：Jason R. Porter ; Cliff I. Stains ; David J. Segal ; Indraneel Ghosh
- 刊名：Analytical Chemistry
- 出版年：2007
- 出版时间：September 1, 2007
- 年：2007
- 卷：79
- 期：17
- 页码：6702 - 6708
- 全文大小：401K
- 年卷期：v.79,no.17(September 1, 2007)
- ISSN：1520-6882
文摘
The methylation pattern of genes at CpG dinucleotide sitesis an emerging area in epigenetics. Furthermore, thehypermethylation profiles of tumor suppressor genes arelinked to specific tumor types. Thus, new molecularapproaches for the rapid determination of the methylationstatus of these genes could provide a powerful methodfor early cancer diagnosis as well as insight into mechanisms of epigenetic regulation of genetic information.Toward this end, we have recently reported the first designof a split-protein sensor for the site-specific detection ofDNA methylation. In this approach a split green fluorescent protein reporter provided a sequence-specific readout of CpG methylation. In the present work, we describea sensitive second-generation methylation detection system that utilizes the split enzymatic reporter, TEM-1
-lactamase, attached to specific DNA binding elements.This system, termed mCpG-SEER--Lac, shows a greaterthan 40-fold specificity for methylated versus nonmethylated CpG target sites. Importantly, the resulting signalenhancement afforded by the catalytic activity of split--lactamase allowed for the sensitive detection of 2.5 fmolof methylated target dsDNA in 5 min. Thus, this newsensor geometry represents a 250-fold enhancement inassay time and a 2000-fold enhancement in sensitivityover our first-generation system for the detection ofspecific sites of DNA methylation.
-lactamase, attached to specific DNA binding elements.This system, termed mCpG-SEER--Lac, shows a greaterthan 40-fold specificity for methylated versus nonmethylated CpG target sites. Importantly, the resulting signalenhancement afforded by the catalytic activity of split--lactamase allowed for the sensitive detection of 2.5 fmolof methylated target dsDNA in 5 min. Thus, this newsensor geometry represents a 250-fold enhancement inassay time and a 2000-fold enhancement in sensitivityover our first-generation system for the detection ofspecific sites of DNA methylation.