TY - JOUR
T1 - Split β-lactamase sensor for the sequence-specific detection of DNA methylation
AU - Porter, Jason R.
AU - Stains, Cliff I.
AU - Segal, David J.
AU - Ghosh, Indraneel
PY - 2007/9/1
Y1 - 2007/9/1
N2 - The methylation pattern of genes at CpG dinucleotide sites is an emerging area in epigenetics. Furthermore, the hypermethylation profiles of tumor suppressor genes are linked to specific tumor types. Thus, new molecular approaches for the rapid determination of the methylation status of these genes could provide a powerful method for early cancer diagnosis as well as insight into mechanisms of epigenetic regulation of genetic information. Toward this end, we have recently reported the first design of a split-protein sensor for the site-specific detection of DNA methylation. In this approach a split green fluorescent protein reporter provided a sequence-specific readout of CpG methylation. In the present work, we describe a sensitive second-generation methylation detection system that utilizes the split enzymatic reporter, TEM-1 β-lactamase, attached to specific DNA binding elements. This system, termed mCpG-SEER-β-Lac, shows a greater than 40-fold specificity for methylated versus nonmethylated CpG target sites. Importantly, the resulting signal enhancement afforded by the catalytic activity of split-β-lactamase allowed for the sensitive detection of 2.5 fmol of methylated target dsDNA in 5 min. Thus, this new sensor geometry represents a 250-fold enhancement in assay time and a 2000-fold enhancement in sensitivity over our first-generation system for the detection of specific sites of DNA methylation.
AB - The methylation pattern of genes at CpG dinucleotide sites is an emerging area in epigenetics. Furthermore, the hypermethylation profiles of tumor suppressor genes are linked to specific tumor types. Thus, new molecular approaches for the rapid determination of the methylation status of these genes could provide a powerful method for early cancer diagnosis as well as insight into mechanisms of epigenetic regulation of genetic information. Toward this end, we have recently reported the first design of a split-protein sensor for the site-specific detection of DNA methylation. In this approach a split green fluorescent protein reporter provided a sequence-specific readout of CpG methylation. In the present work, we describe a sensitive second-generation methylation detection system that utilizes the split enzymatic reporter, TEM-1 β-lactamase, attached to specific DNA binding elements. This system, termed mCpG-SEER-β-Lac, shows a greater than 40-fold specificity for methylated versus nonmethylated CpG target sites. Importantly, the resulting signal enhancement afforded by the catalytic activity of split-β-lactamase allowed for the sensitive detection of 2.5 fmol of methylated target dsDNA in 5 min. Thus, this new sensor geometry represents a 250-fold enhancement in assay time and a 2000-fold enhancement in sensitivity over our first-generation system for the detection of specific sites of DNA methylation.
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U2 - 10.1021/ac071163+
DO - 10.1021/ac071163+
M3 - Article
C2 - 17685552
AN - SCOPUS:34548580356
SN - 0003-2700
VL - 79
SP - 6702
EP - 6708
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 17
ER -