Isothermal DNA Amplification Systems

The second non-PCR-mediated isothermal amplification technology is based on the rolling circle replication (RCR) strategy used by small plasmids, viroids, and a variety of bacteriophage (Doermann, 1973; Diener, 1991; del Solar et al., 1998). The minimalist view presented in Fig. 12.2 illustrates the salient points of this technology: (i) extension of a primer by DNA polymerase around a circular or closed

Figure 12.2. Rolling circle replification: variations on a theme. (A-D) Rolling circle amplification uses the strand displacement activity of an exonuclease-deficient DNA polymerase to synthesize concatemeric DNA molecules. (E) An example of a "padlock" probe that can be used for allelic and mutation discrimination. A linear ssDNA containing either a perfect or imperfect complement at the ligation site is added to the enzyme-target mixture. Ligation and subsequent amplification will only occur if the target hybridization and ligation is successful. This will only occur when a perfectly complementary ssDNA probe sequence is used.

Figure 12.2. Rolling circle replification: variations on a theme. (A-D) Rolling circle amplification uses the strand displacement activity of an exonuclease-deficient DNA polymerase to synthesize concatemeric DNA molecules. (E) An example of a "padlock" probe that can be used for allelic and mutation discrimination. A linear ssDNA containing either a perfect or imperfect complement at the ligation site is added to the enzyme-target mixture. Ligation and subsequent amplification will only occur if the target hybridization and ligation is successful. This will only occur when a perfectly complementary ssDNA probe sequence is used.

template, (ii) strand displacement upon reaching a double-stranded area, and (iii) accumulation of concatameric molecules upon continued synthesis of the circular strand (Figs. 12.2A-12.2D). These concatamers can then serve as detection targets. The simplicity and robustness of this system has spawned the development of strategies for the detection of nucleic acids as well as proteins, as will be discussed below. Rolling circle amplification (RCA) and strand displacement amplification (SDA) are the major two designs used with RCR technologies. The ability of DNA polymerase to carry out strand displacement lies at the heart of these technologies.

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