Alternating Recombination Method for Assembling DNA Fragments

A process for recombination assembly of a series of cloned DNA fragments into a large ordered arrangement. Potential uses of this technology include: . Analysis of function of large interrupted coding regions . Construction of gene sets involved in complex metabolic processes . Chromosome engineering . Extensive genetic reprogramming of microbes for optimal production processes (metabolic engineering) . Large scale "editing" of known genomes based on engineering optimization considerations . Exploration of global structural effects in the function of genomes With the structural analysis of DNA proceeding at a rapid pace based on the advances in DNA sequencing techniques and encouraged by potential applications of information from completely sequenced genomes of important organisms, a new horizon is the synthesis of large DNA structures. In the case of microbial genomes, there has been discussion of preparing a minimal genome. However, general methods for constructing large precisely designed DNA segments have not been developed. Presently, commercially available oligonucleotide synthesis can routinely produce molecules on the order of a hundred nucleotides, and through PCR amplification of known segments of a genome, defined fragments of up to 40 kilobase pairs can be prepared. Through cleavage with specific restriction enzymes and joining by ligation, designed DNA molecules (e.g., large vectors) have been made. However, this method becomes complicated as larger fragments with more restriction sites are used and each molecule to be made must have its unique route of synthesis depending on its particular arrangement of restriction endonuclease sites. Further complicating this process is the presence of endogenous DNA methylation enzymes that may prevent cleavage at restriction endonuclease sites. Finally, ordered and oriented concatenation of DNA sequences is possible with these methods, but can be painstaking and relies on paired restriction sites and planned or fortuitous placement of sites in both donor and recipient molecules. The method developed at Rice University avoids the complications of endonuclease-based construction through an �alternating recombination� system that allows for ordered, oriented insertion of fragments into a construct. This general synthetic approach allows for the formation of designed unique DNA molecules of a size of hundreds of kilobase pairs. In general, the technique uses site-specific recombinases to insert a vector containing a fragment of interest into a specific location on DNA in the cell, and excisionases to remove unnecessary vector sequences. Using alternating excisionases, an unlimited number of fragments can be aligned adjacent in the chromosome, BAC or YAC DNA. When fragment assembly is complete, a final excisionase can be used to excise and circularize the assembled fragment.

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