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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 (http://cgsc.biology.yale.edu/). 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.


Acknowledgments


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.


References 1. Breaker, R.R. 1997. DNA enzymes. Nat. Biotechnol. 15:427-431.


2. Schlosser, K. and Y. Li. 2009. Biologically inspired synthetic enzymes made from DNA. Chem. Biol. 16:311-322.


3. Silverman, S.K. 2009. Deoxyribozymes: selection design and serendipity in the development of DNA catalysts. Acc. Chem. Res. 42:1521-1531.


4. Todd, A.V., C.J. Fuery, H.L. Impey, T.L. Applegate, and M.A. Haughton. 2000. DzyNA-PCR: use of DNAzymes to detect and quantify nucleic acid sequences in a real-time fluorescent format. Clin. Chem. 46:625-630.


5. Liu, J. and Y. Lu. 2006. Fluorescent DNAzyme biosensors for metal ions based on catalytic molecular beacons. Methods Mol. Biol. 335:275-288.


6. Silverman, S.K. 2008. Catalytic DNA (deoxy- ribozymes) for synthetic applications – current abilities and future prospects. Chem. Commun. (Camb.) 14:3467-3485.


7. Carmi, N., L.A. Shultz, and R.R. Breaker. 1996. In vitro selection of self-cleaving DNAs. Chem. Biol. 3:1039-1046.


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8. Sheppard, T.L., P. Ordoukhanian, and G.F. Joyce. 2000. A DNA enzyme with N-glycosylase activity. Proc. Natl. Acad. Sci. USA 97:7802-7807.


9. Chandra, M., A. Sachdeva, and S.K. Silverman. 2009. DNA-catalyzed sequence-specific hydrolysis of DNA. Nat. Chem. Biol. 5:718-720.


10. Xiao, Y., R.J. Wehrmann, N.A. Ibrahim, and S.K. Silverman. 2012. Establishing broad generality of DNA catalysts for site-specific hydrolysis of single- stranded DNA. Nucleic Acids Res. 40:1778-1786.


11. Xiao, Y., M. Chandra, and S.K. Silverman. 2010. Functional compromises among pH tolerance, site specificity, and sequence tolerance for a DNA-hydro- lyzing deoxyribozyme. Biochemistry 49:9630- 9637.


12.Dokukin, V. and S.K. Silverman. 2012. Lanthanide ions as required cofactors for DNA catalysts. Chem. Sci. 3:1707-1714.


13.Gu, H., K. Furukawa, Z. Weinberg, D.F. Berenson, and R.R. Breaker. Small, highly-active DNAs that hydrolyze DNA. J. Am. Chem. Soc. (In press)


14.Liu, D., S.L. Daubendiek, M.A. Zillman, K. Ryan, and E.T. Kool. 1996. Rolling circle DNA synthesis: small circular oligonucleotides as efficient templates for DNA polymerases. J. Am. Chem. Soc. 118:1587- 1594.


15. Lizardi, P.M., X. Huang, Z. Zhu, P. Bray-Ward, D.C. Tomas, and D.C. Ward. 1998. Mutation detection and single-molecule counting using


isothermal rolling-circle amplification. Nat. Genet. 19:225-232.


16. Stougaard, M., S. Juul, F.F. Andersen, and B.R. Knudsen. 2011. Strategies for highly sensitive biomarker detection by rolling circle amplification of signals from nucleic acid composed sensors. Integr. Biol. (Camb). 3:982-992.


17. Asiello, P.J. and A. Baeumner. 2011. Miniaturized isothermal nucleic acid amplification, a review. Lab Chip 11:1420-1430.


18. Blondal, T., A. Torisdottir, U. Unnsteinsdottir, S. Hjorleifsdottir, A. Ævarsson, S. Ernstsson, O.H. Fridjonsson, S. Skirnisdottir, et al. 2005. Isolation and characterization of a thermostable RNA ligase 1 from a Termus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties. Nucleic Acids Res. 33:135-142.


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- er@yale.edu


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