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Technology

Directed Evolution

Overview

Next-generation sequencing (NGS) workflows have several steps with each step potentially allowing for the introduction of errors that could significantly impact the quality and reliability of sequencing results. Reagents and methods used to generate NGS data should therefore be of the highest quality, and need to be tested and validated for robust precision and accuracy. Although several reagent options are available for various steps of the NGS workflow, the difference among them exists primarily due to the composition of buffers or the concentration of enzymes. The majority of DNA- and RNA-modifying enzymes used in common NGS reagents are “wild-type”, isolated from nature without modification, and these enzymes were never intended as tools for molecular biology. Directed evolution technology selects enzymes used in NGS reagents through a simulated natural selection process in the lab for high performance efficiency.

How does directed evolution work?

The process starts with a gene coding for a “wild-type”, or unmodified, enzyme of interest. Random variation is introduced into the gene through a process of mutagenesis, generating a library of millions of genes, each coding for a unique enzyme variant. A functional selection pressure is then applied to the library where only the genes that coded for the highest performing enzymes “survive”. This process of random mutation and selection is repeated until the desired enzyme function evolves.

How is directed evolution different?

Since introducing random mutations into a gene is mostly harmful to the function of the encoded enzyme, unlocking the power of directed evolution requires the ability to screen very large numbers of enzyme variants. Our core technology combines a high-throughput emulsion format where aqueous droplets suspended in oil, which serve as microreactors, are combined with high-throughput selection assays to enable the functional screening of hundreds of millions of enzyme variants in parallel.

What are the benefits of evolved enzymes versus wild-type enzymes?

Directed evolution enables us to engineer improvements to enzyme function, such as thermostability, specific activity, processivity, or resistance to inhibitors, which require global changes to protein structure. These functions are not amenable to rational design, a method of protein engineering that requires detailed information about enzyme structure and function. These evolved enzymes are different from other “engineered” enzymes because they are optimized for functions that cannot be addressed using rational design.

directed evolution steps
Applications of enzymes selected through directed evolution
  • NGS library preparation
  • Amplicon-based target enrichment
  • Library amplification
  • Quality control
  • PCR
  • qPCR
Explore PCR and qPCR products
Product overview

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    References
     
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    4. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17: 405–424. doi:10.1038/gim.2015.30
    5. Song W, Gardner SA, Hovhannisyan H, Natalizio A, Weymouth KS, Chen W, et al. Exploring the landscape of pathogenic genetic variation in the ExAC population database: insights of relevance to variant classification. Genet Med. 2016;18: 850–854. doi:10.1038/gim.2015.180
    6. Stenson PD, Mort M, Ball E V, Howells K, Phillips AD, Thomas NS, et al. The Human Gene Mutation Database: 2008 update. Genome Med. 2009;1: 13. doi:10.1186/gm13

     

    For Research Use Only. Not for use in diagnostic procedures.