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Optimization of Golden Gate assembly through application of ligation sequence-dependent fidelity and bias profiling

biorxiv. 2018-05; 
Potapov Vladimir, Jennifer L. Ong, Rebecca B. Kucera, Bradley W. Langhorst, Katharina Bilotti, John M. Pryor, Eric J. Cantor, Barry Canton, Thomas F. Knight, Thomas C. Evans Jr., Gregory J. S. Lohman
Products/Services Used Details Operation
GenParts™ DNA Fragments … protocols Synthetic oligonucleotides were obtained from Integrated DNA Technologies as lyophilized solid (Coralville, IA) Fragments for Golden Gate assembly assays were obtained from GenScript (Piscataway, NJ), as precloned … Get A Quote

摘要

Modern synthetic biology depends on the manufacture of large DNA constructs from libraries of genes, regulatory elements or other genetic parts. Type IIS restriction enzyme-dependent DNA assembly methods (e.g., Golden Gate) enable rapid one-pot, ordered, multi-fragment DNA assembly, facilitating the generation of high-complexity constructs. The order of assembly of genetic parts is determined by the ligation of flanking Watson-Crick base-paired overhangs. The ligation of mismatched overhangs leads to erroneous assembly, and the need to avoid such pairings has typically been accomplished by using small sets of empirically vetted junction pairs, limiting the number of parts that can be joined in a single reaction... More

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