In addition, the construction of synthetic DNA remains expensive, labor intensive and impractical for gigabase genomes such as those of animals and plants 11. However, despite significant advances in DNA synthesis over the last two decades 10, current methods are still restricted by size limits, synthesis fidelity and long lead times. ![]() At the genome scale, de novo synthesis and genome assembly can be used to explore synthetic genome designs 5, 6, 7, 8, 9. At the gene and pathway level, synthetic or engineered sequences can be applied to a design-build-test-learn (DBTL) framework to optimize for a desired function 3, 4. Thus, the MEGAA platform enables generation of multi-site sequence variants quickly, cheaply, and in a scalable manner for diverse applications in biotechnology.Ĭonstruction and manipulation of kilobase-sized DNA building blocks is fundamental to synthetic biology and synthetic genomics 1, 2. Furthermore, 125 defined combinatorial adeno-associated virus-2 cap gene variants were easily built using the system, which exhibited viral packaging enhancements of up to 10-fold compared with wild type. Using this system, we demonstrated the construction of 31 natural SARS-CoV2 spike gene variants and 10 recoded Escherichia coli genome fragments, with each 4 kb region containing up to 150 mutations. We devised a robust and iterative protocol for an open-source laboratory automation robot that enables desktop production and long-read sequencing validation of variants. ![]() ![]() With this method, many mutations can be generated at a time to a DNA template at more than 90% efficiency per target in a predictable manner. Here, we present a new approach, mutagenesis by template-guided amplicon assembly (MEGAA), for the rapid construction of kilobase-sized DNA variants. Efficient methods for the generation of specific mutations enable the study of functional variations in natural populations and lead to advances in genetic engineering applications.
0 Comments
Leave a Reply. |