Figure 1

Recombination between PCR fragment and genome leading to marker replacement. A) Standard SSG mutagenesis (i) and gsSSG mutagenesis (ii). Diagrams show recombination between PCR fragment (top line) and chromosome (bottom line with centromere represented as open circle). Asterisk indicates the targeted mutation, and the "S" in (ii) indicates position of the SceI site, which is introduced on one of the primers. B) Relationship between the distance between marker insertion site and closest end of PCR fragment and recombinant fraction. Recombinant fraction, defined as the fraction of FOA^R isolates that derive from marker replacement is shown for standard SSG mutagenesis transformants with the nearest end at varying distances between the marker insertion site and the nearest fragment end. Only transformations where ≥ 5 FOA^R isolates were analyzed are included in this graph. C) Logistic regression analysis of the same data for SSG mutagenesis as in B) except that all data was included regardless of the number of isolates analyzed for a given transformation. The solid line represents the fitted logistic model, and the dashed line represents 95% confidence bands around the model.