Optimal DNA templates for rolling circle amplification revealed by in vitro selection

Chemistry. 2015 May 26;21(22):8069-74. doi: 10.1002/chem.201500994. Epub 2015 Apr 15.


Rolling circle amplification (RCA) has been widely used as an isothermal DNA amplification technique for diagnostic and bioanalytical applications. Because RCA involves repeated copying of the same circular DNA template by a DNA polymerase thousands of times, we hypothesized there exist DNA sequences that can function as optimal templates and produce more DNA amplicons within an allocated time. Herein we describe an in vitro selection effort conducted to search from a random sequence DNA pool for such templates for phi29 DNA polymerase, a frequently used polymerase for RCA. Diverse DNA molecules were isolated and they were characterized by richness in adenosine (A) and cytidine (C) nucleotides. The top ranked sequences exhibit superior RCA efficiency and the use of these templates for RCA results in significantly improved detection sensitivity. AC-rich sequences are expected to find useful applications for setting up effective RCA assays for biological sensing.

Keywords: DNA; biosensors; circular templates; in vitro selection; rolling circle amplification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aptamers, Nucleotide / chemistry
  • Bacillus Phages / enzymology
  • Base Sequence
  • Biosensing Techniques / methods*
  • DNA, Circular / chemistry*
  • DNA, Circular / metabolism
  • DNA-Directed DNA Polymerase / metabolism
  • Nucleic Acid Amplification Techniques / methods*


  • Aptamers, Nucleotide
  • DNA, Circular
  • DNA-Directed DNA Polymerase