Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Sequence-specific Labeling of Duplex DNA Using Nicking Enzymes and Oligonucleotide Probes
Labeling of specific target sites on genomic, double-stranded DNA (dsDNA) in combination with ultra-sensitive detection technologies may result in valuable diagnostic assays for pathogen detection and identification. In particular, such methods may offer rapid time-to-results and decreased probability for error as amplification steps are avoided. We propose a method, in which dsDNA labeling is accomplished through strand exchange with oligonucleotide probes at sites of vicinal nicks. Such sites are generated by treatment of genomic DNA with nicking endonucleases. Following probe hybridization, probes are covalently linked to the target DNA by ligation (Fig. 1). So far, we have successfully labeled sites that contained two nicks on the same DNA strand at distances between 13 nt and 24 nt (1). On DNA fragments we have shown that target sites with significant homology could be labeled with very high sequence specificity. As a result, our approach offered the possibility to directly label and detect unique target sites in genomic DNA.
Figure 1: Sequence-specific labeling at sites of vicinal nicks in dsDNA. In the initial step, site-specific nicks are introduced into dsDNA. Regions between vicinal nicks serve in our procedure as target sites for probe oligonucleotide binding. Through subsequent ligation, a hybridized probe, which may carry a fluorescence label (blue pentagon), becomes covalently linked to the dsDNA at the selected target sites. In our preliminary data, we have shown that probes with different design, resulting in either linkage at one terminus (structure I) or at both termini (structure II), performed equally well in site-specific labeling reactions.
Previously, we used a signal amplification step for the final detection (1). However, the general approach carries the potential for single-molecule detection. We have therefore begun to explore this possibility by performing genomic DNA labeling reactions with fluorophore-tagged probes with subsequent analysis of labeled DNA on US Genomic?s single-molecule detection platform (2). This technology allows accurate determination of labeling locations through measurement of fluorescent signals in individual, stretched DNA molecules. Data will be presented that show remarkable agreement between calculated and measured label positions in genomic DNA.
References and Footnotes
1Centrer of Advanced Biotechnology