DNA Assembly Technologies Based on Homologous Recombination

The origins of the recombinant DNA technology can be traced back to the discovery of restriction enzymes and the generation of the first recombinant DNA molecule over 40 years ago (1–3). Since then, and with the emergence of PCR, scientists have generated a number of elegant cloning systems that enable the manipulation of DNA fragments in various ways (for recent reviews see Refs 4–7). Some remarkable examples include the poly-merase cycling assembly of oligonucleotides (8), thermostable ligation (9), topoisomerase-based cloning Due to the value that these technologies have, many of them have turned into commercial products. In the postgenomic era and with the advent of the emerging synthetic biology field, which uses complex combinations of genetic elements to design circuits with new properties, the manipulation and analyses of large set of genes becomes a crucial necessity. In this chapter we offer a review of DNA assembly strategies based on homologous recombination, which present important advantages over other methods. Homologous recombination is the exchange of genetic information between two similar or identical molecules of DNA. This exchange occurs in a precise, specific, and faithful manner, and thus presents an excellent tool for genetic engineering for seamless gene fusion. The mechanism requires the presence of homologous regions, DNA sequence stretches shared by the recombining fragments. The term " homologous recombination in vitro " is commonly referred to the joining of two or more DNA fragments that share end-terminal homology. The reaction is driven by purified enzymes involved in the double-strand DNA break-repair mechanisms such as DNA polymerases, DNA ligases, exonucleases, and single-strand DNA binding proteins. In vitro recombina-tion protocols take advantage of improved PCR techniques to introduce sequence identity at the ends of adjacent DNA fragments that otherwise do not share significant homology. A common element in the in vitro recombi-nation methods is the generation of complementary single-strand overhangs in the DNA fragments to be joined. Different techniques employ different approaches to generating complementary overhangs. A number of in vitro methods use digestion with exonucleases to generate long complementary overhangs, thereby allowing efficient base pairing between adjacent fragments. For example " ligation-independent cloning " (LIC), uses PCR primers that are designed in such a way that the first 12 bases at the 5 0-end must lack one particular nucleotide. The (3 0 ! 5 0) exonuclease activity of T4 DNA polymerase is used in combination with the corresponding dNTP to specifically remove …