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Nucleic Acids Research, 2002, Vol. 30, No. 2 574-580
© 2002 Oxford University Press

Rolling-circle amplification under topological constraints

Heiko Kuhn, Vadim V. Demidov* and Maxim D. Frank-Kamenetskii

Center for Advanced Biotechnology, Department of Biomedical Engineering, Boston University, 36 Cummington Street, Boston, MA 02215, USA

We have performed rolling-circle amplification (RCA) reactions on three DNA templates that differ distinctly in their topology: an unlinked DNA circle, a linked DNA circle within a pseudorotaxane-type structure and a linked DNA circle within a catenane. In the linked templates, the single-stranded circle (dubbed earring probe) is threaded, with the aid of two peptide nucleic acid openers, between the two strands of double-stranded DNA (dsDNA). We have found that the RCA efficiency of amplification was essentially unaffected when the linked templates were employed. By showing that the DNA catenane remains intact after RCA reactions, we prove that certain DNA polymerases can carry out the replicative synthesis under topological constraints allowing detection of several hundred copies of a dsDNA marker without DNA denaturation. Our finding may have practical implications in the area of DNA diagnostics.

* To whom correspondence should be addressed. Tel: +1 617 353 8490; Fax: +1 617 353 8501; Email: vvd{at}bu.edu The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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