[show abstract][hide abstract] ABSTRACT: We have developed an efficient method for the simultaneous introduction of up to three mutations in a plasmid DNA via homologous recombination. The strategy is compatible with a variety of mutations, including degenerate codons in plasmids of different sizes. In contrast to other methodologies, this approach employs the same set of reagents for both single- and multi-site mutagenesis assays, minimizes the required protocol steps, and exhibits remarkably high mutagenesis efficiencies.
[show abstract][hide abstract] ABSTRACT: Recombinant DNA technologies have had a fundamental impact on drug discovery. The continuous emergence of unique gene assembly techniques resulted in the generation of a variety of therapeutic reagents such as vaccines, cancer treatment molecules and regenerative medicine precursors. With the advent of synthetic biology there is a growing need for precise and concerted assembly of multiple DNA fragments of various sizes, including chromosomes. In this article, we summarize the highlights of the recombinant DNA technology since its inception in the early 1970s, emphasizing on the most recent advances, and underscoring their principles, advantages and shortcomings. Current and prior cloning trends are discussed in the context of sequence requirements and scars left behind. Our opinion is that despite the remarkable progress that has enabled the generation and manipulation of very large DNA sequences, a better understanding of the cell's natural circuits is needed in order to fully exploit the current state-of-the-art gene assembly technologies.
Expert Opinion on Drug Discovery 03/2012; 7(5):371-4. · 2.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: In recent years there has been a growing interest in the precise and concerted assembly of multiple DNA fragments of diverse sizes, including chromosomes, and the fine tuning of gene expression levels and protein activity. Commercial DNA assembly solutions have not been conceived to support the cloning of very large or very small genetic elements or a combination of both. Here we summarize a series of protocols that allow the seamless, simultaneous, flexible, and highly efficient assembly of DNA elements of a wide range of sizes (up to hundred thousand base pairs). The protocols harness the power of homologous recombination and are performed either in vitro or within the living cells. The DNA fragments may or may not share homology at their ends. An efficient site-directed mutagenesis protocol enhanced by homologous recombination is also described.
Methods in molecular biology (Clifton, N.J.) 01/2012; 834:93-109.
[show abstract][hide abstract] 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.
Methods in enzymology 01/2011; 498:327-48. · 1.90 Impact Factor