ArticleSynthetic biology (Oxford, England)2019
Optimization of the experimental parameters of the ligase cycling reaction.
Article in Synthetic biology (Oxford, England), 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 2 papers.
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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Who cites it
2 citing papers in PubMed, 7 citations in OpenAlex.
- HyperXpress: Rapid Single Vessel DNA Assembly and Protein Production in Microliterscale.Frontiers in bioengineering and biotechnology · 2022Article
- Recent Advances in Strategies for the Cloning of Natural Product Biosynthetic Gene Clusters.Frontiers in bioengineering and biotechnology · 2021Review
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The ligase cycling reaction (LCR) is a scarless and efficient method to assemble plasmids from fragments of DNA. This assembly method is based on the hybridization of DNA fragments with complementary oligonucleotides, so-called bridging oligos (BOs), and an experimental procedure of thermal denaturation, annealing and ligation. In this study, we explore the effect of molecular crosstalk of BOs and various experimental parameters on the LCR by utilizing a fluorescence-based screening system. The results indicate an impact of the melting temperatures of BOs on the overall success of the LCR assembly. Secondary structure inhibitors, such as dimethyl sulfoxide and betaine, are shown to negatively impact the number of correctly assembled plasmids. Adjustments of the annealing, ligation and BO-melting temperature further improved the LCR. The optimized LCR was confirmed by validation experiments. Based on these findings, a step-by-step protocol is offered within this study to ensure a routine for high efficient LCR assemblies.
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