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of RNA markers. Samples of the ss100 DNA ladder can be obtained for evaluation or appli- cation from the Coli Genetics Stock Center at Yale University ( In summary, we have developed a simple and

effective method to produce ssDNAs of defined sequence and length from engineered circular DNA templates. Tis approach permits the efficient synthesis of DNAs that can be much longer and carry less chemical damage than those prepared by existing solid-phase DNA synthesis methods. In the current study, we demonstrate the use of such ssDNA products as markers for gel electrophoresis applications. Markers of this type could be useful when conducting experiments on natural ssDNAs (e.g., bacterio- phage genomes) or on cDNA products made from natural RNAs. Additional applications involving complete digestion with a deoxy- ribozyme should permit the production of uniform-length sequence specific ssDNAs for other uses. Moreover, one could envision the incorporation of deoxyribozymes or DNA aptamers with other functions that would yield multifunctional DNA constructs produced by RCA.


We thank the Breaker laboratory for helpful discussions. Tis work was supported by grants from DARPA and the NIH (GM022778). Research in the Breaker laboratory is also supported by the Howard Hughes Medical Institute.

Competing interests

Te authors have filed for intellectual property protection on aspects of this work.

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Received: 7 November 2012; accepted: 5 February 2013.

Address correspondence to Ronald R Breaker, Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA. Email: ronald.break-

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