Cloning-Free PCR-Based Allele Replacement Methods

Use of adaptamer A^mut and a mutamer to create a de novo mutation. Adaptamer A^mut contains the sequence tag described for adaptamer A (see text) followed by an altered nucleotide(s) (indicated by the blue dot on adaptamerA^mut ) and an additional 20 nts of sequence adjacent to the desired change. In combination with adaptamer B, PCR is used to generate mutated fragment 1. As described in Fig. 2, fragment 1 is fused to fragment 3 generating fusion R. To create fusion L, fragments 1 and 2 are mixed with K. lactis int 3′ and a mutamer. The mutamer consists of an additional 17 nts of sequence upstream of the desired change followed by the desired change itself and 14 nts downstream. The asterisk (*) depicts the introduced mutation. Fusions L and R recombine as described in Fig. 3.