Protein Engineering Protocols - Mycobacteriology research center

Degenerate Oligonucleotide Gene Shuffling 195rounds of primer hybridization and extension is enhanced by the sequence similarityof all primers in the pool; this potentially allows use of all primers in thereaction. A modification of the CODEHOP method allows efficient amplificationof overlapping segments of related genes, and subsequent overlap-extensionof adjacent segments from different genes resulting in the formation of chimericgene fragments. The careful design of the primer sequences is the most importantstep in the DOGS procedure.The modification of the CODEHOP technique entails the design of perfectlycomplementary pairs of primers. Each primer has a nondegenerate core flankedby both 5′ and 3′ degenerate ends, referred to herein as complementary degenerateend(CDE) primers. As with the CODEHOP primers, the 3′ degenerate end giveseach CDE primer their template-binding specificity, whereas the nondegenerateregion acts as a stabilizing clamp in subsequent rounds of the PCR. The 5′degenerate end is not required to contribute to the binding efficiency of the CDEprimer during PCR, however, it plays a pivotal role in allowing efficient bindingand subsequent overlap-extension of separate PCR products (gene segments)generated using, respectively, the forward or the reverse CDE primers.The nondegenerate core of individual CDE primers is generally based on thecorresponding coding sequence of one gene, designated the parental gene forshuffling. This results in the formation of chimeric fragments that retain theparental sequence at the points of segment overlap.3.1.1. Design and Use of Gene-Specific Nested End Primers1. Design and synthesize forward and reverse primer pairs suitable for PCR amplificationof each gene to be shuffled. Each primer should comprise a 17- to 20-nucleotide gene-specific 3′-end and a 17- to 20-nucleotide common 5′-end. Weincorporated restriction sites into the common ends to facilitate directional ligationof PCR products into pBSII KS–.2. Synthesize two nested primers with sequences corresponding directly to the forwardand reverse common ends of the gene-specific nested primers. The nestedprimers will be used in combination with CDE primers to amplify the first and lastsegments of each gene.3. Use the gene specific primers to amplify each gene from a suitable source of templateDNA, usually either genomic DNA or, preferably, a cloned example of the gene.3.1.2. Summary of the Features of CDE PrimersCDE primers allow efficient and specific amplification of portions (referredto herein as gene segments) of related but divergent genes. The 5′ degenerate endof CDE primers ensures that separate PCR products generated with the respectiveforward or reverse complementary CDE primers will anneal efficiently insubsequent overlap-extension PCR steps. Furthermore, multiple (one or more)pairs of CDE primers allow the generation of consecutive PCR products (genesegments) with complementary ends suitable for overlap extension and PCR,