Use of Polymerase Chain Reaction (PCR) to Detect Homologous Recombination in Transfected Cell Lines

Abstract

When DNA is introduced into eukaryotic cells, it can be integrated into the genome by homologous or illegitimate recombination 1,2. Despite great efforts to gain insight into the molecular mechanisms, our understanding of the recombination process is still in its infancy. In the absence of a molecular model, predictions concerning the frequency of homologous recombination compared to illegitimate recombination cannot be precisely made. In mammalian cells, illegitimate recombination is the most predominant event (for review, seeref. (3). Thus, if the integration of DNA via homologous recombination into mammalian cells is the goal of the experiment, a single homologous recombination event has to be detected among many illegitimate recombination events. Described here is a method of detecting homologous recombination events in a small subpopulation of cells by using the polymerase chain reaction (PCR), a primer-directed enzymatic amplification of specific DNA sequences. This method can be used to determine the homologous recombination frequency and to facilitate the cloning of homologously recombined cells 1,2. Homologously recombined alleles are identified by their amplification products, which are generated by the PCR reaction (see Fig. 1). The specificity of the reaction is dependen t on the two primers. One primer (primer 2 in Fig. 1) primes DNA synthesis specifically at the nonhomologous sequences of the exogenous DNA. The other primer (primer 1 in Fig. 1) is specific for the target locus, but outside of the exogenous DNA. Thus, both priming sites are physically linked in a predictable manner only after homologous recombination. Fig. 1. Detection of homologous recombination events by PCR. Homologous recombination between a target locus and exogenous DNA yields a new recombinant molecule in which a small portion of the target locus is replaced by a heterologous sequence of the exogenous DNA. This recombination event links two priming sites for the PCR. Primer 1 primes DNA synthesis from the target sequence, outside of the region homologous to the exogenous DNA. Primer 2 is specific for the heterologous sequence residing in the exogenous DNA. These priming sites are not linked (in the same manner) after the integration of the exogenous DNA by illegitimate recombination. Yet, DNAs of a heterogeneous length are made from the target and the illegitimately recombined sequence during every PCR cycle. Those molecules that terminate in the region of homology could anneal, thus creating a recombinant molecule.