Rolling-circle amplification under topological constraints.
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.
Kuhn
Heiko
H
Center for Advanced Biotechnology, Department of Biomedical Engineering, Boston University, 36 Cummington Street, Boston, MA 02215, USA.
Demidov
Vadim V
VV
Frank-Kamenetskii
Maxim D
MD
D001483:Base Sequence; D004270:DNA, Circular / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008957:Models, Genetic; D008958:Models, Molecular; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004259:DNA-Directed DNA Polymerase