![]() coli strain carrying a plasmid with a clone copy of DNA Polymerase I large fragment Science 273, 1392–1395.Klenow Enzyme (DNA Polymerase I Large Fragment) (1996) Crystal structure of the Aequorea victoria green fluorescent protein. (1996) FACS-optimized mutants of the green fluorescent protein (GFP). (1998) Enzymatic manipulation of the fragments obtained by cerium (IV)-induced DNA scission: Characterization of hydrolytic termini. (2000) Hydrolysis of oligonucleotides by homogeneous Ce(IV)-EDTA complex. ![]() (1996) Increased cloning efficiency by temperature-cycle ligation. (1996) Improved green fluorescent protein by molecular evolution using DNA shuffling. (1996) Incorporation of Nonnatural Amino Acids into Streptavidin through In Vitro Frame-Shift Suppression. Hohsaka, T., Ashizuka, Y., Murakami, H., and Sisido, M. (2001) Incorporation of nonnatural amino acids into proteins by using various four-base codons in an Escherichia coli in vitro translation system. Hohsaka, T., Ashizuka, Y., Taira, H., Murakami, H., and Sisido, M. (2002) Random insertion and deletion of arbitrary number of bases for codon-based random mutation of DNAs. Murakami, H., Hohsaka, T., and Sisido, M. (1998) Codon-based mutagenesis using dimer-phosphoramidites. (1998) Combination of DMT-mononucleotide and Fmoc-trinucleotide phosphoramidites in oligonucleotide synthesis affords an automatable codon-level mutagenesis method. Gaytan, P., Yanez, J., Sanchez, F., Mackie, H., and Soberon, X. (1992) A general strategy for random insertion and substitution mutagenesis: substoichiometric coupling of trinucleotide phosphoramidites. (1992) Antibody engineering by codon-based mutagenesis in a filamentous phage vector system. (1994) Codon cassette mutagenesis: a general method to insert or replace individual codons by using universal mutagenic cassettes. (1985) Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites. (1982) Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function. D., Morin, C., Itakura, K., and Richards, J. (1992) Randomization of genes by PCR mutagenesis. (ed.) (2001) In Vitro Mutagenesis Protocols, Second Edition, Humana, Totowa, NJ.Ĭadwell, R. (2001) Discovery of superior enzymes by directed molecular evolution. (2001) How enzymes adapt: lessons from directed evolution. This process is experimental and the keywords may be updated as the learning algorithm improves.Īrnold, F. These keywords were added by machine and not by the authors. These synthetic methods may be applicable only to introduce mutations within a narrow range of the target gene. To introduce codon-based mutations at various positions, the split-and-mix method ( 8) or other synthetic methods of constructing DNAs using dinucleotide or trinucleotide units have been attempted ( 9– 11). These methods are limited to a defined region of the gene and can not introduce mutations at random positions. To achieve a non-biased random replacement on the amino acid level, oligonucleotide-directed mutagenesis ( 5) and cassette mutagenesis ( 6, 7) have been carried out. For example, mutation from AUG (Met) to UGG (Trp) is unlikely to take place. However, the error-prone PCR method has an inherent drawback of biased occurrence of amino acids as the result of single base replacements in the triplet codons. ![]() ![]() Among these, error-prone PCR mutagenesis, based on inaccurate copying by DNA polymerase, is the most commonly used technique to introduce random point mutations ( 4). Several methods for introducing random mutations in vitro have been reported ( 3). Random mutagenesis combined with high-throughput screening is a versatile strategy for improving protein functions or creating artificial enzymes ( 1, 2). ![]()
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