Coupled rolling circle amplification loop-mediated amplification for rapid detection of short DNA sequences

BioTechniques, Vol. 45, No. 3, September 2008, pp. 275–280

Coupled rolling circle amplification loop-mediated amplification for rapid detection of short DNA sequences
Jennifer Y. Marciniak 1, Andrew C. Kummel 2, Sadik C. Esener 3, Michael J. Heller 1, 3, Bradley T. Messmer 41, Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
2, Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA
3, Department of Nanoengineering, University of California San Diego, La Jolla, CA, USA
4, Rebecca and John Moores Cancer Center, University of California San Diego, La Jolla, CA, USA

Circularizable oligonucleotide probes can detect short DNA sequences with single-base resolution at the site of ligation and can be amplified by rolling circle amplification (RCA) using strand displacing polymerases. A secondary amplification scheme was developed that uses the loop-mediated amplification reaction concurrent with RCA to achieve rapid signal development from the starting circular molecules. This isothermal reaction was found to be significantly faster than the comparable hyperbranching amplification method and could detect 100 circular copies in less than 1 h.