Protein mutations are often introduced at the DNA level in the corresponding gene, which takes advantage of the central dogma of biology (DNA is transcribed to mRNA; mRNA is translated to proteins. The introduction of mutations in target genes has become trivial with the advances in synthetic biology. Major classes of library construction include (1) random mutagenesis, (2) recombination, (3) semi-rational design, and (4) scanning mutagenesis. Together, these classes of library construction are termed “combinatorial design” and require little to no prior knowledge of the protein, albeit more knowledge can help design libraries more efficiently. The counter technique is termed “rational design” and requires detailed information about the
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Based on knowledge about hot spots in the protein (e.g. binding pockets, catalytically active sites, dimerization domains), specific residues are selected for mutagenesis due to their importance in the hot spot. Residues are substituted randomly using techniques such as overlap- extension PCR (oe-PCR) and QquickC change mutagenesis PCR (QC-PCR). The former is popular for introducing multiple mutations in the gene. Degenerate oligonucleotides are used as primers in PCR to amplify fragments of the gene. Each amplified fragment contains a region of homology with the appropriate adjacent gene fragment. Fragments are mixed, similarly to DNA shuffling, in the absence of primer and allowed to extend until the full length gene has been amplified. Though this technique has proven successful, it can be tedious with multiple fragments and introduce bias during each round of PCR amplification. Recently with the advances in DNA synthesis, an entire gene can be synthesized with the proper degenerate sites. The preferred method in academia remains oe-PCR because of the cost of degenerate gene synthesis, but the prices of DNA synthesis is constantly declining and may lead to more attractive pricing when compared with the time and labor involved with in-house library
Based on knowledge about hot spots in the protein (e.g. binding pockets, catalytically active sites, dimerization domains), specific residues are selected for mutagenesis due to their importance in the hot spot. Residues are substituted randomly using techniques such as overlap- extension PCR (oe-PCR) and QquickC change mutagenesis PCR (QC-PCR). The former is popular for introducing multiple mutations in the gene. Degenerate oligonucleotides are used as primers in PCR to amplify fragments of the gene. Each amplified fragment contains a region of homology with the appropriate adjacent gene fragment. Fragments are mixed, similarly to DNA shuffling, in the absence of primer and allowed to extend until the full length gene has been amplified. Though this technique has proven successful, it can be tedious with multiple fragments and introduce bias during each round of PCR amplification. Recently with the advances in DNA synthesis, an entire gene can be synthesized with the proper degenerate sites. The preferred method in academia remains oe-PCR because of the cost of degenerate gene synthesis, but the prices of DNA synthesis is constantly declining and may lead to more attractive pricing when compared with the time and labor involved with in-house library