Combining Isothermal Amplification Techniques: Coupled RCA-LAMP

Abstract

Loop-mediated isothermal amplification (LAMP) is a low-cost method of quasi-exponential DNA amplification which utilizes constant temperature and stem-loop structures formed during amplification to generate strong amplified signal in a short amount of time. Rolling circle amplification (RCA) is a technique for linear isothermal DNA amplification of circularized oligonucleotide templates. When combined with padlock probes, RCA can be used as a diagnostic assay with single-base specificity. Here, a process is described called RCA-LAMP, in which LAMP is coupled with RCA of padlock probes to combine the benefit of the specificity of padlock probes with the speed of the LAMP reaction. It was found that the RCA-LAMP reaction proceeded significantly faster than the comparable hyperbranched RCA (HRCA) reaction, and it could detect 100 circular probes in less than 1 h.