PCR-Mediated Recombination: A General Method Applied to Construct Chimeric Infectious Molecular Clones

Molecular cloning has proven to be a powerful tool in biology, and chimeric clones are useful in a variety of fields including microbial pathogenesis and the development of vaccines. Chimeras can be created from DNA by using conventional cloning techniques, specifically restriction cleavage and DNA ligation. Such techniques, however, have limitations; most commonly, limitations result from the lack of restriction sites to provide points of entry for inserts in the desired regions or the multiplicity of restriction sites in other regions of the DNA. Because recombinant DNA molecules may be created during polymerase chain reaction (PCR) when two or more different DNA sequences are brought together (1 ,2 ), PCR-mediated recombination has been exploited to join DNA fragments of a few hundred bases (3 -8 ). There are two drawbacks to these methods. First, they often involve multiple steps, and second, sequence errors frequently are introduced by certain thermostable polymerases during the PCR reaction (9 ,10 ).