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{"work":{"id":42307705,"created_at":"2020-03-24T09:24:02.167-07:00","from_world_paper_id":null,"updated_at":"2024-11-14T17:26:23.969-08:00","_data":{"grobid_abstract":"Genetic diversity creation is a core technology in directed evolution where a high quality mutant library is crucial to its success. Owing to its importance, the technology in genetic diversity creation has seen rapid development over the years and its application has diversified into other fields of scientific research. The advances in molecular cloning and mutagenesis since 2008 were reviewed. Specifically, new cloning techniques were classified based on their principles of complementary overhangs, homologous sequences, overlapping PCR and megaprimers and the advantages, drawbacks and performances of these methods were highlighted. New mutagenesis methods developed for random mutagenesis, focused mutagenesis and DNA recombination were surveyed. The technical requirements of these methods and the mutational spectra were compared and discussed with references to commonly used techniques. The trends of mutant library preparation were summarised. Challenges in genetic diversity creation were discussed with emphases on creating \"smart\" libraries, controlling the mutagenesis spectrum and specific challenges in each group of mutagenesis methods. An outline of the wider applications of genetic diversity creation includes genome engineering, viral evolution, metagenomics and a study of protein functions. 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if (!viewCountBody) { throw new Error('Failed to find work views element'); } viewCountBody.textContent = `${commaizedViewCount} views`; } catch (error) { // Remove the whole views element if there was some issue parsing. document.getElementById('work-metadata-view-count')?.parentNode?.remove(); throw new Error(`Failed to parse view count: ${viewCount}`, error); } }; // If the DOM is still loading, wait for it to be ready before updating the view count. if (document.readyState === "loading") { document.addEventListener('DOMContentLoaded', () => { updateViewCount(viewCount); }); // Otherwise, just update it immediately. } else { updateViewCount(viewCount); } })();</script></div><p class="ds-work-card--work-abstract ds-work-card--detail ds2-5-body-md">Genetic diversity creation is a core technology in directed evolution where a high quality mutant library is crucial to its success. Owing to its importance, the technology in genetic diversity creation has seen rapid development over the years and its application has diversified into other fields of scientific research. The advances in molecular cloning and mutagenesis since 2008 were reviewed. Specifically, new cloning techniques were classified based on their principles of complementary overhangs, homologous sequences, overlapping PCR and megaprimers and the advantages, drawbacks and performances of these methods were highlighted. New mutagenesis methods developed for random mutagenesis, focused mutagenesis and DNA recombination were surveyed. The technical requirements of these methods and the mutational spectra were compared and discussed with references to commonly used techniques. The trends of mutant library preparation were summarised. Challenges in genetic diversity creation were discussed with emphases on creating "smart" libraries, controlling the mutagenesis spectrum and specific challenges in each group of mutagenesis methods. An outline of the wider applications of genetic diversity creation includes genome engineering, viral evolution, metagenomics and a study of protein functions. The review ends with an outlook for genetic diversity creation and the prospective developments that can have future impact in this field.</p><div class="ds-work-card--button-container"><button class="ds2-5-button js-swp-download-button" data-signup-modal="{"location":"continue-reading-button--work-card","attachmentId":62459830,"attachmentType":"pdf","workUrl":"https://www.academia.edu/42307705/Polishing_the_craft_of_genetic_diversity_creation_in_directed_evolution"}">See full PDF</button><button class="ds2-5-button ds2-5-button--secondary js-swp-download-button" data-signup-modal="{"location":"download-pdf-button--work-card","attachmentId":62459830,"attachmentType":"pdf","workUrl":"https://www.academia.edu/42307705/Polishing_the_craft_of_genetic_diversity_creation_in_directed_evolution"}"><span class="material-symbols-outlined" style="font-size: 20px" translate="no">download</span>Download PDF</button></div><div class="ds-signup-banner-trigger-container"><div class="ds-signup-banner-trigger ds-signup-banner-trigger-control"></div></div><div class="ds-signup-banner ds-signup-banner-control"><div id="ds-signup-banner-close-button"><button class="ds2-5-button ds2-5-button--secondary ds2-5-button--inverse"><span class="material-symbols-outlined" style="font-size: 20px" translate="no">close</span></button></div><div class="ds-signup-banner-ctas" data-impression-entity-id="42307705" data-impression-entity-type="2" data-impression-source="signup-banner"><img src="//a.academia-assets.com/images/academia-logo-capital-white.svg" /><h4 class="ds2-5-heading-serif-sm">Sign up for access to the world's latest research</h4><button class="ds2-5-button ds2-5-button--inverse ds2-5-button--full-width js-swp-download-button" data-signup-modal="{"location":"signup-banner"}">Sign up for free<span class="material-symbols-outlined" style="font-size: 20px" translate="no">arrow_forward</span></button></div><div class="ds-signup-banner-divider"></div><div class="ds-signup-banner-reasons"><div class="ds-signup-banner-reasons-item"><span class="material-symbols-outlined" style="font-size: 24px" translate="no">check</span><span>Get notified about relevant papers</span></div><div class="ds-signup-banner-reasons-item"><span class="material-symbols-outlined" style="font-size: 24px" translate="no">check</span><span>Save papers to use in your research</span></div><div class="ds-signup-banner-reasons-item"><span class="material-symbols-outlined" style="font-size: 24px" translate="no">check</span><span>Join the discussion with peers</span></div><div class="ds-signup-banner-reasons-item"><span class="material-symbols-outlined" style="font-size: 24px" translate="no">check</span><span>Track your impact</span></div></div></div><script>(() => { // Set up signup banner show/hide behavior: // 1. 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At the current state of random mutagenesis technologies mutation frequencies have often been adjusted to values that cause a limited number of amino acid changes (often one to four amino acid changes per protein). For harvesting the power of directed evolution algorithms it is therefore important that generated mutant libraries are rich in diversity and enriched in active population. Insufficient knowledge about protein traits, mutational robustness of protein folds and technological limitations in diversity generating methods are main challenges for managing the complexity of protein sequence space. This review covers computational and experimental advances for high quality mutant library generation that have been achieved in the past two years.</p><div class="ds-related-work--ctas"><button class="ds2-5-text-link ds2-5-text-link--inline js-swp-download-button" data-signup-modal="{"location":"wsj-grid-card-download-pdf-modal","work_title":"Advances in generating functional diversity for directed protein evolution","attachmentId":62459819,"attachmentType":"pdf","work_url":"https://www.academia.edu/42307699/Advances_in_generating_functional_diversity_for_directed_protein_evolution","alternativeTracking":true}"><span class="material-symbols-outlined" style="font-size: 18px" translate="no">download</span><span class="ds2-5-text-link__content">Download free PDF</span></button><a class="ds2-5-text-link ds2-5-text-link--inline js-wsj-grid-card-view-pdf" href="https://www.academia.edu/42307699/Advances_in_generating_functional_diversity_for_directed_protein_evolution"><span class="ds2-5-text-link__content">View PDF</span><span class="material-symbols-outlined" style="font-size: 18px" translate="no">chevron_right</span></a></div></div><div class="ds-related-work--container js-wsj-grid-card" data-collection-position="1" data-entity-id="8357672" data-sort-order="default"><a class="ds-related-work--title js-wsj-grid-card-title ds2-5-body-md ds2-5-body-link" href="https://www.academia.edu/8357672/Improved_PCR_method_for_the_creation_of_saturation_mutagenesis_libraries_in_directed_evolution_application_to_difficult_to_amplify_templates">Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates</a><div class="ds-related-work--metadata"><a class="js-wsj-grid-card-author ds2-5-body-sm ds2-5-body-link" data-author-id="16778362" href="https://independent.academia.edu/JeromePeyralans">Jerome Peyralans</a></div><p class="ds-related-work--metadata ds2-5-body-xs">Applied Microbiology and Biotechnology, 2008</p><p class="ds-related-work--abstract ds2-5-body-sm">Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene’s QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.</p><div class="ds-related-work--ctas"><button class="ds2-5-text-link ds2-5-text-link--inline js-swp-download-button" data-signup-modal="{"location":"wsj-grid-card-download-pdf-modal","work_title":"Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates","attachmentId":48124612,"attachmentType":"pdf","work_url":"https://www.academia.edu/8357672/Improved_PCR_method_for_the_creation_of_saturation_mutagenesis_libraries_in_directed_evolution_application_to_difficult_to_amplify_templates","alternativeTracking":true}"><span class="material-symbols-outlined" style="font-size: 18px" translate="no">download</span><span class="ds2-5-text-link__content">Download free PDF</span></button><a class="ds2-5-text-link ds2-5-text-link--inline js-wsj-grid-card-view-pdf" href="https://www.academia.edu/8357672/Improved_PCR_method_for_the_creation_of_saturation_mutagenesis_libraries_in_directed_evolution_application_to_difficult_to_amplify_templates"><span class="ds2-5-text-link__content">View PDF</span><span class="material-symbols-outlined" style="font-size: 18px" translate="no">chevron_right</span></a></div></div><div class="ds-related-work--container js-wsj-grid-card" data-collection-position="2" data-entity-id="12473887" data-sort-order="default"><a class="ds-related-work--title js-wsj-grid-card-title ds2-5-body-md ds2-5-body-link" href="https://www.academia.edu/12473887/Random_mutagenesis_libraries_optimization_and_simplification_by_PCR">Random mutagenesis libraries: optimization and simplification by PCR</a><div class="ds-related-work--metadata"><a class="js-wsj-grid-card-author ds2-5-body-sm ds2-5-body-link" data-author-id="31311594" href="https://independent.academia.edu/jZucmanRossi">jessica Zucman-Rossi</a></div><p class="ds-related-work--metadata ds2-5-body-xs">BioTechniques, 1999</p><div class="ds-related-work--ctas"><button class="ds2-5-text-link ds2-5-text-link--inline js-swp-download-button" data-signup-modal="{"location":"wsj-grid-card-download-pdf-modal","work_title":"Random mutagenesis libraries: optimization and simplification by PCR","attachmentId":46156382,"attachmentType":"pdf","work_url":"https://www.academia.edu/12473887/Random_mutagenesis_libraries_optimization_and_simplification_by_PCR","alternativeTracking":true}"><span class="material-symbols-outlined" style="font-size: 18px" translate="no">download</span><span class="ds2-5-text-link__content">Download free PDF</span></button><a class="ds2-5-text-link ds2-5-text-link--inline js-wsj-grid-card-view-pdf" href="https://www.academia.edu/12473887/Random_mutagenesis_libraries_optimization_and_simplification_by_PCR"><span class="ds2-5-text-link__content">View PDF</span><span class="material-symbols-outlined" style="font-size: 18px" translate="no">chevron_right</span></a></div></div><div class="ds-related-work--container js-wsj-grid-card" data-collection-position="3" data-entity-id="24798419" data-sort-order="default"><a class="ds-related-work--title js-wsj-grid-card-title ds2-5-body-md ds2-5-body-link" href="https://www.academia.edu/24798419/A_Statistical_Analysis_of_Random_Mutagenesis_Methods_Used_for_Directed_Protein_Evolution">A Statistical Analysis of Random Mutagenesis Methods Used for Directed Protein Evolution</a><div class="ds-related-work--metadata"><a class="js-wsj-grid-card-author ds2-5-body-sm ds2-5-body-link" data-author-id="36408593" href="https://ulincoln.academia.edu/DaniloRoccatano">Danilo Roccatano</a></div><p class="ds-related-work--metadata ds2-5-body-xs">Journal of Molecular Biology, 2006</p><div class="ds-related-work--ctas"><button class="ds2-5-text-link ds2-5-text-link--inline js-swp-download-button" data-signup-modal="{"location":"wsj-grid-card-download-pdf-modal","work_title":"A Statistical Analysis of Random Mutagenesis Methods Used for Directed Protein Evolution","attachmentId":45126674,"attachmentType":"pdf","work_url":"https://www.academia.edu/24798419/A_Statistical_Analysis_of_Random_Mutagenesis_Methods_Used_for_Directed_Protein_Evolution","alternativeTracking":true}"><span class="material-symbols-outlined" style="font-size: 18px" translate="no">download</span><span class="ds2-5-text-link__content">Download free PDF</span></button><a class="ds2-5-text-link ds2-5-text-link--inline js-wsj-grid-card-view-pdf" href="https://www.academia.edu/24798419/A_Statistical_Analysis_of_Random_Mutagenesis_Methods_Used_for_Directed_Protein_Evolution"><span class="ds2-5-text-link__content">View PDF</span><span class="material-symbols-outlined" style="font-size: 18px" translate="no">chevron_right</span></a></div></div><div class="ds-related-work--container js-wsj-grid-card" data-collection-position="4" data-entity-id="48700516" data-sort-order="default"><a class="ds-related-work--title js-wsj-grid-card-title ds2-5-body-md ds2-5-body-link" href="https://www.academia.edu/48700516/Transposon_Based_Approaches_for_Generating_Novel_Molecular_Diversity_During_Directed_Evolution">Transposon-Based Approaches for Generating Novel Molecular Diversity During Directed Evolution</a><div class="ds-related-work--metadata"><a class="js-wsj-grid-card-author ds2-5-body-sm ds2-5-body-link" data-author-id="57344666" href="https://independent.academia.edu/AmyBaldwin2">Amy Baldwin</a></div><p class="ds-related-work--metadata ds2-5-body-xs">Methods in Molecular Biology, 2014</p><p class="ds-related-work--abstract ds2-5-body-sm">This chapter introduces a set of transposon-based methods that were developed to sample trinucleotide deletion, trinucleotide replacement and domain insertion. Each approach has a common initial step that utilises an engineered version of the Mu transposon called MuDel. The inherent low sequence specificity of MuDel results in its random insertion into target DNA during in vitro transposition. Removal of the transposon using a type IIS restriction endonuclease generates blunt end random breaks at a frequency of one per target gene and the concomitant loss of 3 bp. Self-ligation or insertion of another DNA cassette results in the sampling of trinucleotide deletion or trinucleotide substitution/domain insertion, respectively.</p><div class="ds-related-work--ctas"><button class="ds2-5-text-link ds2-5-text-link--inline js-swp-download-button" data-signup-modal="{"location":"wsj-grid-card-download-pdf-modal","work_title":"Transposon-Based Approaches for Generating Novel Molecular Diversity During Directed Evolution","attachmentId":67177860,"attachmentType":"pdf","work_url":"https://www.academia.edu/48700516/Transposon_Based_Approaches_for_Generating_Novel_Molecular_Diversity_During_Directed_Evolution","alternativeTracking":true}"><span class="material-symbols-outlined" style="font-size: 18px" translate="no">download</span><span class="ds2-5-text-link__content">Download free PDF</span></button><a class="ds2-5-text-link ds2-5-text-link--inline js-wsj-grid-card-view-pdf" href="https://www.academia.edu/48700516/Transposon_Based_Approaches_for_Generating_Novel_Molecular_Diversity_During_Directed_Evolution"><span class="ds2-5-text-link__content">View PDF</span><span class="material-symbols-outlined" style="font-size: 18px" translate="no">chevron_right</span></a></div></div><div class="ds-related-work--container js-wsj-grid-card" data-collection-position="5" data-entity-id="19004931" data-sort-order="default"><a class="ds-related-work--title js-wsj-grid-card-title ds2-5-body-md ds2-5-body-link" href="https://www.academia.edu/19004931/Random_priming_in_vitro_recombination_An_effective_tool_for_directed_evolution">Random-priming in vitro recombination: An effective tool for directed evolution</a><div class="ds-related-work--metadata"><a class="js-wsj-grid-card-author ds2-5-body-sm ds2-5-body-link" data-author-id="39156946" href="https://independent.academia.edu/FrancesArnold">Frances Arnold</a></div><p class="ds-related-work--metadata ds2-5-body-xs">Nucleic Acids Research, 1998</p><p class="ds-related-work--abstract ds2-5-body-sm">A simple and efficient method for in vitro mutagenesis and recombination of polynucleotide sequences is reported. The method involves priming template polynucleotide(s) with random-sequence primers and extending to generate a pool of short DNA fragments which contain a controllable level of point mutations. The fragments are reassembled during cycles of denaturation, annealing and further enzyme-catalyzed DNA polymerization to produce a library of full-length sequences. Screening or selecting the expressed gene products leads to new variants with improved functions, as demonstrated by the recombination of genes encoding different thermostable subtilisins in order to obtain enzymes more stable than either parent.</p><div class="ds-related-work--ctas"><button class="ds2-5-text-link ds2-5-text-link--inline js-swp-download-button" data-signup-modal="{"location":"wsj-grid-card-download-pdf-modal","work_title":"Random-priming in vitro recombination: An effective tool for directed evolution","attachmentId":42119475,"attachmentType":"pdf","work_url":"https://www.academia.edu/19004931/Random_priming_in_vitro_recombination_An_effective_tool_for_directed_evolution","alternativeTracking":true}"><span class="material-symbols-outlined" style="font-size: 18px" translate="no">download</span><span class="ds2-5-text-link__content">Download free PDF</span></button><a class="ds2-5-text-link ds2-5-text-link--inline js-wsj-grid-card-view-pdf" href="https://www.academia.edu/19004931/Random_priming_in_vitro_recombination_An_effective_tool_for_directed_evolution"><span class="ds2-5-text-link__content">View PDF</span><span class="material-symbols-outlined" style="font-size: 18px" translate="no">chevron_right</span></a></div></div><div class="ds-related-work--container js-wsj-grid-card" data-collection-position="6" data-entity-id="24798403" data-sort-order="default"><a class="ds-related-work--title js-wsj-grid-card-title ds2-5-body-md ds2-5-body-link" href="https://www.academia.edu/24798403/Steering_directed_protein_evolution_strategies_to_manage_combinatorial_complexity_of_mutant_libraries">Steering directed protein evolution: strategies to manage combinatorial complexity of mutant libraries</a><div class="ds-related-work--metadata"><a class="js-wsj-grid-card-author ds2-5-body-sm ds2-5-body-link" data-author-id="36408593" href="https://ulincoln.academia.edu/DaniloRoccatano">Danilo Roccatano</a></div><p class="ds-related-work--metadata ds2-5-body-xs">Environmental Microbiology, 2007</p><p class="ds-related-work--abstract ds2-5-body-sm">How to explore protein sequence space efficiently and how to generate high-quality mutant libraries that allow to identify improved variants with current screening technologies are key questions for any directed protein evolution experiment. High-quality mutant libraries can be generated through improved random mutagenesis methodologies and by restricting diversity generation through computational methods to residues which have high success probabilities. Advances in mutant library design and computational tools to focus diversity generation are summarized in this minireview and discussed from an experimentalist point of view in the context of directed protein evolution.</p><div class="ds-related-work--ctas"><button class="ds2-5-text-link ds2-5-text-link--inline js-swp-download-button" data-signup-modal="{"location":"wsj-grid-card-download-pdf-modal","work_title":"Steering directed protein evolution: strategies to manage combinatorial complexity of mutant libraries","attachmentId":45126666,"attachmentType":"pdf","work_url":"https://www.academia.edu/24798403/Steering_directed_protein_evolution_strategies_to_manage_combinatorial_complexity_of_mutant_libraries","alternativeTracking":true}"><span class="material-symbols-outlined" style="font-size: 18px" translate="no">download</span><span class="ds2-5-text-link__content">Download free PDF</span></button><a class="ds2-5-text-link ds2-5-text-link--inline js-wsj-grid-card-view-pdf" href="https://www.academia.edu/24798403/Steering_directed_protein_evolution_strategies_to_manage_combinatorial_complexity_of_mutant_libraries"><span class="ds2-5-text-link__content">View PDF</span><span class="material-symbols-outlined" style="font-size: 18px" translate="no">chevron_right</span></a></div></div><div class="ds-related-work--container js-wsj-grid-card" data-collection-position="7" data-entity-id="12327548" data-sort-order="default"><a class="ds-related-work--title js-wsj-grid-card-title ds2-5-body-md ds2-5-body-link" href="https://www.academia.edu/12327548/A_one_pot_simple_methodology_for_cassette_randomisation_and_recombination_for_focused_directed_evolution">A one-pot, simple methodology for cassette randomisation and recombination for focused directed evolution</a><div class="ds-related-work--metadata"><a class="js-wsj-grid-card-author ds2-5-body-sm ds2-5-body-link" data-author-id="30984679" href="https://independent.academia.edu/JuanHermoso">Juan Hermoso</a></div><p class="ds-related-work--metadata ds2-5-body-xs">2008</p><p class="ds-related-work--abstract ds2-5-body-sm">Protein engineering is currently performed either by rational design, focusing in most cases on only a few positions modified by site-directed mutagenesis, or by directed molecular evolution, in which the entire protein-encoding gene is subjected to random mutagenesis followed by screening or selection of desired phenotypes. A novel alternative is focused directed evolution, in which only fragments of a protein are randomised while the overall scaffold of a protein remains unchanged. For this purpose, we developed a PCR technique using long, spiked oligonucleotides, which allow randomising of one or several cassettes in any given position of a gene. This method allows over 95% incorporation of mutations independently of their position within the gene, yielding sufficient product to generate large libraries, and the possibility of simultaneously randomising more than one locus at a time, thus originating recombination. The high efficiency of this method was verified by creating focused mutant libraries of Pseudomonas fluorescens esterase I (PFEI), screening for altered substrate selectivity and validating against libraries created by error-prone PCR. This led to the identification of two mutants within the OSCARR library with a 10-fold higher catalytic efficiency towards p-nitrophenyl dodecanoate. These PFEI variants were also modelled in order to explain the observed effects.</p><div class="ds-related-work--ctas"><button class="ds2-5-text-link ds2-5-text-link--inline js-swp-download-button" data-signup-modal="{"location":"wsj-grid-card-download-pdf-modal","work_title":"A one-pot, simple methodology for cassette randomisation and recombination for focused directed evolution","attachmentId":46240339,"attachmentType":"pdf","work_url":"https://www.academia.edu/12327548/A_one_pot_simple_methodology_for_cassette_randomisation_and_recombination_for_focused_directed_evolution","alternativeTracking":true}"><span class="material-symbols-outlined" style="font-size: 18px" translate="no">download</span><span class="ds2-5-text-link__content">Download free PDF</span></button><a class="ds2-5-text-link ds2-5-text-link--inline js-wsj-grid-card-view-pdf" href="https://www.academia.edu/12327548/A_one_pot_simple_methodology_for_cassette_randomisation_and_recombination_for_focused_directed_evolution"><span class="ds2-5-text-link__content">View PDF</span><span class="material-symbols-outlined" style="font-size: 18px" translate="no">chevron_right</span></a></div></div><div class="ds-related-work--container js-wsj-grid-card" data-collection-position="8" data-entity-id="21239965" data-sort-order="default"><a class="ds-related-work--title js-wsj-grid-card-title ds2-5-body-md ds2-5-body-link" href="https://www.academia.edu/21239965/Rapid_generation_of_random_mutant_libraries">Rapid generation of random mutant libraries</a><div class="ds-related-work--metadata"><a class="js-wsj-grid-card-author ds2-5-body-sm ds2-5-body-link" data-author-id="13503719" href="https://tamu.academia.edu/MichaelBenedik">Michael Benedik</a></div><p class="ds-related-work--metadata ds2-5-body-xs">Bioengineered Bugs, 2010</p><p class="ds-related-work--abstract ds2-5-body-sm">A simple and efficient method utilizing in vivo recombination to create recombinant libraries incorporating the products of PCR amplification is described. This will be especially useful for generating large pools of randomly mutagenized clones after error-prone PCR mutagenesis. Here we investigate various parameters to optimize this approach and we demonstrate that as little as 1 pmole of PCR fragment can generate a library with greater than 10 4 clones in a single transformation without ligation.</p><div class="ds-related-work--ctas"><button class="ds2-5-text-link ds2-5-text-link--inline js-swp-download-button" data-signup-modal="{"location":"wsj-grid-card-download-pdf-modal","work_title":"Rapid generation of random mutant libraries","attachmentId":41777604,"attachmentType":"pdf","work_url":"https://www.academia.edu/21239965/Rapid_generation_of_random_mutant_libraries","alternativeTracking":true}"><span class="material-symbols-outlined" style="font-size: 18px" translate="no">download</span><span class="ds2-5-text-link__content">Download free PDF</span></button><a class="ds2-5-text-link ds2-5-text-link--inline js-wsj-grid-card-view-pdf" href="https://www.academia.edu/21239965/Rapid_generation_of_random_mutant_libraries"><span class="ds2-5-text-link__content">View PDF</span><span class="material-symbols-outlined" style="font-size: 18px" translate="no">chevron_right</span></a></div></div><div class="ds-related-work--container js-wsj-grid-card" data-collection-position="9" data-entity-id="20222442" data-sort-order="default"><a class="ds-related-work--title js-wsj-grid-card-title ds2-5-body-md ds2-5-body-link" href="https://www.academia.edu/20222442/dRTP_and_dPTP_a_complementary_nucleotide_couple_for_the_Sequence_Saturation_Mutagenesis_SeSaM_method">dRTP and dPTP a complementary nucleotide couple for the Sequence Saturation Mutagenesis (SeSaM) method</a><div class="ds-related-work--metadata"><a class="js-wsj-grid-card-author ds2-5-body-sm ds2-5-body-link" data-author-id="39827752" href="https://wichita.academia.edu/RajniVerma">Rajni Verma</a><span>, </span><a class="js-wsj-grid-card-author ds2-5-body-sm ds2-5-body-link" data-author-id="36408593" href="https://ulincoln.academia.edu/DaniloRoccatano">Danilo Roccatano</a></div><p class="ds-related-work--abstract ds2-5-body-sm">Methods to generate random mutant libraries in directed evolution are limited in functional diversity generation. The Sequence Saturation Mutagenesis (SeSaM) method was reported as a four step random mutagenesis method overcoming the limitations of epPCR based mutagenesis methods. SeSaM targets in contrast to epPCR each nucleotide "equally" avoiding mutagenic hot spots, achieving subsequent mutations in a codon (up to 37.1%), and allowing to adjust mutational biases through employed universal bases. In this manuscript, we report an advanced SeSaM method in which a protocol was developed and optimized for implementing the R (ribavirin) base in a SeSaM experiment. The R-based protocol was subsequently combined with the original P-base SeSaM protocol. Combining P-and R-base allows in SeSaM experiments to generate transversions at all four nucleotides of a given sequence with an unmatched chemical diversity. Following the later strategy, we developed a combined P-(at A & G positions) and R-base (at T & C positions) protocol, nearly doubled in comparison to the SeSaM-P [27] the number of mutations that are unobtainable by epPCR and removed the requirement of a single stranded template in the SeSaM method.</p><div class="ds-related-work--ctas"><button class="ds2-5-text-link ds2-5-text-link--inline js-swp-download-button" data-signup-modal="{"location":"wsj-grid-card-download-pdf-modal","work_title":"dRTP and dPTP a complementary nucleotide couple for the Sequence Saturation Mutagenesis (SeSaM) method","attachmentId":41077733,"attachmentType":"pdf","work_url":"https://www.academia.edu/20222442/dRTP_and_dPTP_a_complementary_nucleotide_couple_for_the_Sequence_Saturation_Mutagenesis_SeSaM_method","alternativeTracking":true}"><span class="material-symbols-outlined" style="font-size: 18px" translate="no">download</span><span class="ds2-5-text-link__content">Download free PDF</span></button><a class="ds2-5-text-link ds2-5-text-link--inline js-wsj-grid-card-view-pdf" href="https://www.academia.edu/20222442/dRTP_and_dPTP_a_complementary_nucleotide_couple_for_the_Sequence_Saturation_Mutagenesis_SeSaM_method"><span class="ds2-5-text-link__content">View PDF</span><span class="material-symbols-outlined" style="font-size: 18px" translate="no">chevron_right</span></a></div></div></div></div><div class="ds-sticky-ctas--wrapper js-loswp-sticky-ctas hidden"><div class="ds-sticky-ctas--grid-container"><div class="ds-sticky-ctas--container"><button class="ds2-5-button js-swp-download-button" data-signup-modal="{"location":"continue-reading-button--sticky-ctas","attachmentId":62459830,"attachmentType":"pdf","workUrl":null}">See full PDF</button><button class="ds2-5-button ds2-5-button--secondary js-swp-download-button" data-signup-modal="{"location":"download-pdf-button--sticky-ctas","attachmentId":62459830,"attachmentType":"pdf","workUrl":null}"><span class="material-symbols-outlined" style="font-size: 20px" translate="no">download</span>Download PDF</button></div></div></div><div class="ds-below-fold--grid-container"><div class="ds-work--container js-loswp-embedded-document"><div class="attachment_preview" data-attachment="Attachment_62459830" style="display: none"><div class="js-scribd-document-container"><div class="scribd--document-loading js-scribd-document-loader" style="display: block;"><img alt="Loading..." src="//a.academia-assets.com/images/loaders/paper-load.gif" /><p>Loading Preview</p></div></div><div style="text-align: center;"><div class="scribd--no-preview-alert js-preview-unavailable"><p>Sorry, preview is currently unavailable. 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wide coding sequence","attachmentId":45871409,"attachmentType":"pdf","work_url":"https://www.academia.edu/25539631/A_fast_and_simple_method_for_simultaneous_mixed_site_specific_mutagenesis_of_a_wide_coding_sequence","alternativeTracking":true}"><span class="material-symbols-outlined" style="font-size: 18px" translate="no">download</span><span class="ds2-5-text-link__content">Download free PDF</span></button><a class="ds2-5-text-link ds2-5-text-link--inline js-related-work-grid-card-view-pdf" href="https://www.academia.edu/25539631/A_fast_and_simple_method_for_simultaneous_mixed_site_specific_mutagenesis_of_a_wide_coding_sequence"><span class="ds2-5-text-link__content">View PDF</span><span class="material-symbols-outlined" style="font-size: 18px" translate="no">chevron_right</span></a></div></div><div class="ds-related-work--container js-related-work-sidebar-card" data-collection-position="8" data-entity-id="20996693" data-sort-order="default"><a class="ds-related-work--title 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data-collection-position="16" data-entity-id="112560573" data-sort-order="default"><a class="ds-related-work--title js-related-work-grid-card-title ds2-5-body-md ds2-5-body-link" href="https://www.academia.edu/112560573/A_novel_random_mutagenesis_approach_using_human_mutagenic_DNA_polymerases_to_generate_enzyme_variant_libraries">A novel random mutagenesis approach using human mutagenic DNA polymerases to generate enzyme variant libraries</a><div class="ds-related-work--metadata"><a class="js-related-work-grid-card-author ds2-5-body-sm ds2-5-body-link" data-author-id="32743728" href="https://insa-toulouse.academia.edu/PMonsan">P. 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