Abstract Method for Assembling PCR Fragments of DNA

A process for assembling a series of DNA fragments generated by PCR into an ordered circular arrangement for replication and genetic work in cells. The method uses the 3' overhangs created by excision repair enzymes to direct the sequential ligation of PCR fragments on a solid scaffold. The resulting assembled DNA is removed with a site-specific recombinase.

Potential uses for this technology include: . Assembly of large DNA fragments . Automated assembly of DNA fragments . Construct development which is independent of restriction enzymes . Synthesis of ready-to-transform plasmid DNA . Combinatorial library development Current methods of manipulating DNA fragments are each limited by size. Plasmid sized fragments of up to 10 kb can be easily assembled, but specifically ordered fragments much larger are difficult to assemble by current techniques and require careful handling to avoid breakage. Other types of cloning vehicles allow larger fragments to be cloned and manipulated, but even these have their limits (lambda ~15-20 kb; cosmids ~ 35-40 kb, BAC ~100 kb and YACs ~1000 kb). Therefore, there is a need in the art for the assembly and manipulation of very large DNA fragments. PCR fragments can be assembled into larger arrangements for useful purposes. This is usually done with the creation of restriction sites in the primer sequences. When the amplified DNA is cut with a particular restriction enzyme, a short overhang is generated that can be used to assemble two PCR fragments with complementary overhang sequences. However, PCR fragments often have like ends, so that orientation of the resulting fragment is not defined. Further, many restriction enzymes also cleave within a large PCR fragment and cannot be used in this way. When different restriction enzyme sites are used in each of the two amplification primers, the likelihood that one of restriction enzymes will cut within the PCR fragment is doubled. A method that did not depend on restriction enzymes would be of general application and advantageous. The invention developed at Rice University uses a modified nucleotide at a specific position in the primer that is removed with DNA excision repair enzyme and AP endonuclease or AP lyase. The AP endonuclease or lyase activity may be part of the excision repair enzyme. Sequential ligation is facilitated if the first fragment attached to modified streptavidin-coated magnetic beads, or otherwise immobilized. Several such fragments can be assembled on the solid substrate to give an in-phase, functional gene. Inclusion of a site-specific recombinase site in the first and last PCR fragments of the assembled DNA allows the joined fragments to be removed from the solid assembly system and circularized for transformation. The method developed at Rice University has several useful features that overcome limitations of current methods. Several fragments can be joined in a defined order allowing the PCR generated DNA fragments to be assembled into an ordered arrangement and formed into a replicating plasmid without use of restriction enzymes. The final product is in a form for transformation into cells and the use of immobilized DNA facilitates the steps and allows for scaling with automated devices

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