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. 2010 Jan 4;11(1):71-4.
doi: 10.1002/cbic.200900630.

Enzymatic incorporation of multiple dyes for increased sensitivity in QD-FRET sensing for DNA methylation detection

Vasudev J Bailey  1 , Brian P Keeley, Yi Zhang, Yi-Ping Ho, Hariharan Easwaran, Malcolm V Brock, Kristen L Pelosky, Hetty E Carraway, Stephen B Baylin, James G Herman, Tza-Huei Wang
Affiliations

Enzymatic incorporation of multiple dyes for increased sensitivity in QD-FRET sensing for DNA methylation detection

Vasudev J Bailey et al. Chembiochem. .
No abstract available

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Figures

Figure 1
Figure 1
A) Schematic of enzymatic incorporation of Cy5-dCTP (formula image) and detection through QD-FRET. B) Normalized FRET efficiency was computed from SMD traces for Cy5-incorporated p16INK4a product generated for different numbers of enzymatic replications. C) Photon counts for multilabeled product generated from genomic bisulfite-treated IVD DNA (positive control). D) Photon counts for water (negative) control. The measurements illustrated are a representative 10 out of thousands using an integration time of 0.01 ms. As indicated by comparing the two panels, the number of photon counts seen in the presence of Cy5-incorporated product is visibly higher than that in the water control.
Figure 2
Figure 2
FRET efficiencies for singly (formula image) and multiply (■) labeled p16INK4a methylated product calculated and normalized against the maximal FRET efficiency observed with a large excess of DNA/QD.
Figure 3
Figure 3
Analysis of p16INK4a methylation of patient serum. A) Comparison of normalized FRET efficiency for singly (formula image) and multiply (■) labeled methylated product. IVD=in vitro-methylated DNA. Values equal or lower than normal lymphocytes (NL) were scored as 0. B) Representative trace demonstrating a marked increase in Cy5 signal at 670 nm for multiply labeled product when compared to singly labeled product. C) Results summary of PCR. Green boxes represent unmethylated product and red boxes represent methylated product.
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