Cloning-free PCR-based allele replacement methods

Abstract

Efficient homologous recombination permits the directed introduction of specific mutations into the yeast genome. Here we describe a cloning-free, PCR-based allele replacement method that simplifies allele transfer between yeast strains. The desired allele from one strain is amplified by PCR, along with a selectable/counterselectable marker. After transformation, the resident allele in the target strain is replaced by creating a duplication of the new allele. Selection for direct repeat recombinants results in a single copy of the new allele in the target strain. Specifically, the desired allele is amplified by PCR with a pair of adaptamers, which are chimeric oligonucleotides that are used to amplify the allele and differentially tag its 5' and 3' ends. These tags allow the directed fusion to two different, but overlapping, regions of an appropriately tagged selectable/counterselectable marker after a second round of PCR amplification. Following cotransformation of the two fusion fragments into yeast, homologous recombination efficiently generates a duplication of the amplified allele flanking the intact selectable marker in the genome. After counterselection, only the desired allele is retained as a result of direct repeat recombination. A simple modification of this method allows the creation of de novo mutations in the genome.

Figure 1

Use of adaptamers to fuse two fragments. The matched adaptamers A and a contain complementary sequence tags at their 5′ ends, as described in the text (indicated as A and a on the PCR products). The 3′ ends of each adaptamer are homologous to two different DNA sequences, respectively. Adaptamer A, in conjunction with adaptamer B, differentially tags one fragment at each end. The primer is designed to permit the PCR amplification of the other fragment, as shown. After amplification, the fragments are mixed and excess primer and adaptamer B are added for an additional PCR step. The complementary sequence tags in adaptamers A and a direct the fusion of the two fragments leading to a chimeric product.

Figure 2

Generating fusion fragments for allele replacement. The gene of interest with an altered site [indicated by an asterisk (*)] is amplified by PCR using adaptamers A and B (fragment 1). Similarly, two overlapping K. lactis URA3 fragments are generated separately by PCR with the K. lactis URA3 adaptamers and two internal K. lactis URA3 primers (fragments 2 and 3). Fragments 2 and 3 do not encode full-length URA3 and thus are represented as K. lactis and lactis URA3, respectively. The ends tagged by adaptamers A, B, a, and b are labeled on the initial PCR products. As described in Fig. 1, fragments 1 and 2 are mixed with the K. lactis int 3′ primer and adaptamer A for an additional PCR step to generate a new fusion product (fusion L). In a separate PCR, fragments 1 and 3 are mixed with the K. lactis int 5′ primer and adaptamer B to generate a second chimeric fragment (fusion R). Both fusions L and R contain the altered site.

Figure 3

Integration of fusion fragments and subsequent pop-out event for allele replacement. Fusions L and R (Fig. 2) are cotransformed into the appropriate yeast strain. Recombination between the two fusion fragments generates a functional, intact K. lactis URA3 gene. Recombination between each fragment and the homologous chromosomal locus results in a duplication of the gene of interest where both copies contain the altered site (*). During the integration, the tags are deleted from the ends of the fragments. The left copy of the duplication lies adjacent to the endogenous promoter (purple box labeled Pro). After the subsequent pop-out event, the altered site is always preserved in the genome.

Figure 4

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 adaptamer A^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.

Figure 5

Fragmentation of large sequences for allele transfer. (A) When the mutation is close to the 3′ end of a large gene (>2.5 kb), a new adaptamer A^int is needed to amplify the 3′ portion of the ORF. After amplification using adaptamer A^int and adaptamer B, the fragment is fused to K. lactis URA3 fragments and cotransformed into yeast as described in Figs. 2 and 3. Integration results in a full-length ORF only in the left copy following recombination that fuses the promoter (purple box labeled Pro) and the endogenous, nonamplified region of the ORF (shaded box) with the duplicated 3′-amplified fragment (open box). The fragment on the right is truncated upstream of the sequence homologous to adaptamer A^int. (B) When the mutation is close to the 5′ end in long essential genes, allele transfer requires two new adaptamers. Adaptamer A^pro and adaptamer B^int are used to amplify the 5′ portion of the ORF including the promoter. After fusion to K. lactis URA3 and cotransformation (Figs. 2 and 3), integration results in the generation of the full-length ORF with its promoter in the right repeat. The left copy contains a 3′ truncation downstream of the sequence homologous to adaptamer B^int.