Homology-Based Cloning Methods

Abstract

Cloning strategies based on homology provide an attractive methodology for identifying novel members of a multigene family. The techniques are straightforward, with well-established protocols, and high-quality reagents are readily available. Most steps are relatively forgiving, and there is a short learning curve. The value of this should not be underestimated when comparing it with expression-based strategies, such as oocyte injections or transient cell transfections, in which success depends on meticulous replication of very exacting conditions. From a technical standpoint, homology-based approaches are the fastest and simplest to initiate and master. Of course, implicit in this is the assumption that novel family members will share significant homology to the known gene, which may or may not be true. Also, because the level of homology may be modest, low-stringency conditions are generally required, which results in a significant background of “false positives” in the assay. Indeed, a major challenge in the development of a homology-based approach lies in devising strategies that seek to maximize the efficiency of the process. In the course of our efforts we utilized four independent strategies, all of which led to the identification of novel genes. Each approach brought with it a set of advantages and disadvantages. Notably, no single strategy would have been sufficient to isolate all receptors.