Bounce PCR v2

Bounce PCR: optimisation free PCR for synthetic biology cloning applications. Synthetic biology projects require the cloning of multiple DNA components, and increasingly this is done though in-vitro gene synthesis. This remains an expensive alternative to traditional cloning by PCR. However, PCR often requires time-consuming optimisation. Bounce PCR is a largely optimisation-free PCR method suitable for the rapid cloning of multiple targets. Bounce PCR is a modified version of Touchdown PCR that takes advantage of primer-extension sequences -commonly used in molecular cloning – to successfully amplify most DNA fragments without time consuming optimisation. Touchdown (and related methods) employ a sequential lowering of the annealing temperature over successive cycles to minimise non-specific primer-binding during the early amplification cycles, but maximise amplification efficiency in later cycles when the target amplicon is more abundant (Don et al. 1991; Rowther et al. 2012). However, the lower annealing temperature in later cycles may still allow for non-specific amplification. Bounce PCR takes advantage of the increased primer melting temperature caused by the addition to the template of primer-extension sequences (for example: restriction enzyme recognition sites, Gateway recombination sites, or vector overlaps for Gibson cloning) to maintain the specificity in later cycles- without sacrificing efficiency- by increasing the annealing temperature again after an initial round of touchdown. Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K., & Mattick, J. S. (1991). 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic acids research, 19(14), 4008. Rowther, F. B., Kardooni, H., & Warr, T. (2012). TOUCH-UP gradient amplification method. Journal of biomolecular techniques : JBT, 23(1), 1-3.