Solid-Phase Gene Synthesis for Mutant Library Construction: The Future of Directed Evolution? 2018

Aitao Li, and Zhoutong Sun, and Manfred T Reetz
Hubei Collaborative Innovation Center for Green Transformation of, Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, 368 Youyi Road, Wuchang Wuhan, 430062, China.

Directed evolution of stereo- and regioselective enzymes as catalysts in organic chemistry and biotechnology constitutes a complementary alternative to selective transition-metal catalysts and organocatalysts. Saturation mutagenesis at sites lining the binding pocket has emerged as a key method in this endeavor, but it suffers from amino acid bias, which reduces the quality of the library at the DNA level and, thus, at the protein level. Chemical solid-phase gene synthesis for library construction offers a solution to this fundamental problem, and the Sloning and Twist platforms are two possible options. This concept article analyzes these approaches and compares them to traditional PCR-based saturation mutagenesis; the superior commercial Twist technique shows almost no bias.

UI MeSH Term Description Entries
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D004247 DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D004798 Enzymes Biological molecules that possess catalytic activity. They may occur naturally or be synthetically created. Enzymes are usually proteins, however CATALYTIC RNA and CATALYTIC DNA molecules have also been identified. Biocatalyst,Enzyme,Biocatalysts
D005818 Genetic Engineering Directed modification of the gene complement of a living organism by such techniques as altering the DNA, substituting genetic material by means of a virus, transplanting whole nuclei, transplanting cell hybrids, etc. Genetic Intervention,Engineering, Genetic,Intervention, Genetic,Genetic Interventions,Interventions, Genetic
D001665 Binding Sites The parts of a macromolecule that directly participate in its specific combination with another molecule. Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D015394 Molecular Structure The location of the atoms, groups or ions relative to one another in a molecule, as well as the number, type and location of covalent bonds. Structure, Molecular,Molecular Structures,Structures, Molecular
D015723 Gene Library A large collection of DNA fragments cloned (CLONING, MOLECULAR) from a given organism, tissue, organ, or cell type. It may contain complete genomic sequences (GENOMIC LIBRARY) or complementary DNA sequences, the latter being formed from messenger RNA and lacking intron sequences. DNA Library,cDNA Library,DNA Libraries,Gene Libraries,Libraries, DNA,Libraries, Gene,Libraries, cDNA,Library, DNA,Library, Gene,Library, cDNA,cDNA Libraries
D015982 Bias Any deviation of results or inferences from the truth, or processes leading to such deviation. Bias can result from several sources: one-sided or systematic variations in measurement from the true value (systematic error); flaws in study design; deviation of inferences, interpretations, or analyses based on flawed data or data collection; etc. There is no sense of prejudice or subjectivity implied in the assessment of bias under these conditions. Aggregation Bias,Bias, Aggregation,Bias, Ecological,Bias, Statistical,Bias, Systematic,Ecological Bias,Outcome Measurement Errors,Statistical Bias,Systematic Bias,Bias, Epidemiologic,Biases,Biases, Ecological,Biases, Statistical,Ecological Biases,Ecological Fallacies,Ecological Fallacy,Epidemiologic Biases,Experimental Bias,Fallacies, Ecological,Fallacy, Ecological,Scientific Bias,Statistical Biases,Truncation Bias,Truncation Biases,Bias, Experimental,Bias, Scientific,Bias, Truncation,Biase, Epidemiologic,Biases, Epidemiologic,Biases, Truncation,Epidemiologic Biase,Error, Outcome Measurement,Errors, Outcome Measurement,Outcome Measurement Error
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D016296 Mutagenesis Process of generating a genetic MUTATION. It may occur spontaneously or be induced by MUTAGENS. Mutageneses

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