One strategy for DNA detection utilizes the target DNA strand as the template in a ligation reaction. Through hybridization, the template strand brings two small complementary DNA probes in close proximity, so that ligation between the two strands occurs in the presence of enzyme. Generally, the resulting product duplex is very stable, so the product will not dissociate from the template under isothermal conditions. To make the DNA template catalytic we replaced one of its nucleotides with different destabilizing modifications. Consequently, the ligated product duplexes were less stable and dissociated under isothermal conditions. We next introduced a cross-catalytic approach where a target strand catalyzed the formation of this destabilizing template, which in turn templated more target. In this way we observed amplification of target DNA under isothermal conditions. Achieving isothermal amplification and detection of DNA is a promising step towards the development of point-of-care disease diagnostics.

Tuning stability of DNA to achieve turnover in template-catalyzed enzymatic ligation reactions

243rd National Meeting (2012)

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