Nucleic Acids Research Advance Access published online on March 15, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn107
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Labeling of unique sequences in double-stranded DNA at sites of vicinal nicks generated by nicking endonucleases
Center for Advanced Biotechnology and Department of Biomedical Engineering, Boston University, MA 02215, USA
*To whom correspondence should be addressed. Tel: +1 617 353 8498; Fax: +1 617 353 8501; Email: mfk{at}bu.edu Correspondence may also be addressed to Heiko Kuhn. Tel: +1 617 353 8492; Fax: +1 617 353 8501; Email: hkuhn{at}bu.edu
Received January 14, 2008. Revised February 18, 2008. Accepted February 26, 2008.
We describe a new approach for labeling of unique sequences within dsDNA under nondenaturing conditions. The method is based on the site-specific formation of vicinal nicks, which are created by nicking endonucleases (NEases) at specified DNA sites on the same strand within dsDNA. The oligomeric segment flanked by both nicks is then substituted, in a strand displacement reaction, by an oligonucleotide probe that becomes covalently attached to the target site upon subsequent ligation. Monitoring probe hybridization and ligation reactions by electrophoretic mobility retardation assay, we show that selected target sites can be quantitatively labeled with excellent sequence specificity. In these experiments, predominantly probes carrying a target-independent 3' terminal sequence were employed. At target labeling, thus a branched DNA structure known as 3'-flap DNA is obtained. The single-stranded terminus in 3'-flap DNA is then utilized to prime the replication of an externally supplied ssDNA circle in a rolling circle amplification (RCA) reaction. In model experiments with samples comprised of genomic 
-DNA and human herpes virus 6 type B (HHV-6B) DNA, we have used our labeling method in combination with surface RCA as reporter system to achieve both high sequence specificity of dsDNA targeting and high sensitivity of detection. The method can find applications in sensitive and specific detection of viral duplex DNA.
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