Abstract
With the completion of myriad genome sequencing projects, genetic bioengineering has expanded into many applications including the integrated analysis of complex pathways, the construction of new biological parts and the redesign of existing, natural biological systems. All these areas require the precise and concerted assembly of multiple DNA fragments of various sizes, including chromosomes, and the fine-tuning of gene expression levels and protein activity. Current commercial cloning products are not robust enough to support the assembly of very large or very small genetic elements or a combination of both. In addition, current strategies are not flexible enough to allow further modifications to the original design without having to undergo complicated cloning strategies. Here, we present a set of protocols that allow the seamless, simultaneous, flexible, and highly efficient assembly of genetic material, designed for a wide size dynamic range (10s to 100,000s base pairs). The assembly can be performed either in vitro or within the living cells and the DNA fragments may or may not share homology at their ends. A novel site-directed mutagenesis approach enhanced by in vitro recombineering is also presented.
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