Screening Gene Family-Enriched cDNA Sublibraries with an Unamplified cDNA Probe: Focusing on Moderately to Abundantly Expressed Clones

The examination of gene expression and the search for novel and/or tissue-specific genes has been facilitated by the presence of conserved domains among the members of gene families (e.g., ref. 1 ). As discussed in the previous chapters of this section, such a conserved domain can be exploited by designing degenerate primer pairs that allow for the polymerase chain reaction (PCR)-based amplification of this domain. Although the PCR product may appear as a single band after typical agarose gel electrophoresis, it is likely to be composed of multiple species of cDNA fragments. Cloning of these fragments (i.e., construction of a sublibrary of cDNA fragments) is necessary for the analysis of individual cDNA fragments. Initial screens (in addition to blue-white selection) can be employed to obviate numerous individual analyses of many clones and allow the investigator to focus on a fraction of clones that display certain characteristics.