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While Kluyveromyces marxianus is a promising yeast strain for biotechnological applications, genetic engineering of this strain is still challenging, especially when multiple genes are to be transformed. Sequential gene integration, which takes advantage of repetitive insertion/excision of the URA3 gene as a marker, has been the best option until now, because the URA3-deletion mutant is the only precondition...
In the present study, we analyzed the kinetics of the PCR-suppression effect (PS-effect) and firstly demonstrate that lower annealing temperature enhances PS-effect. Furthermore, we controlled the average size of complex PCR products over a wide range, and simultaneously amplified targets from 0.25 to 10kb by regulating the degree of suppression in a single-primer PCR. In addition, we describe an...
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