PCR-Based Strategies to Clone Unknown DNA Regions from Known Foreign Integrants: An Overview

Many foreign DNAs, such as some virus DNAs and almost all transposable elements (transposons), are capable of integrating host genomes, and the effects of integration can be pleiotropic. To investigate the mechanism and biological effect of foreign DNA insertions, characterization of the integration site, called integrant-host junction (IHJ), in the host genome becomes important. Traditional genomic library construction and screening for the cloning and analysis of IHJ are time-consuming, labor-intensive, and tedious. Therefore, a variety of efficient and reliable polymerase chain reaction (PCR)-based techniques have been developed. Application of the PCR to yield enough amounts of DNA for cloning and analysis is highly recommended especially for those specimens that are in a minute amount. Because the amplification process of PCR requires a pair of primers that can anneal to known sites at two end of the target DNA template, it seems that PCR is not applicable to IHJ searching because only one side of the fragment sequence in the integrant is known. A number of PCR-based techniques, however, have been developed to amplify the unknown cellular DNA flanking sequence from the foreign DNA. In this chapter, we introduce the PCRbased methodologies for the rapid acquisition of unknown DNA sequences. Based on the underlying principles, we classified these techniques into five categories: 1) PCR after intramolecular circularization; 2) interspersed repetitive sequence PCR (IRS-PCR); 3) ligation-anchored PCR (LA-PCR); 4) arbitrarily primed PCR (AP-PCR); and 5) reverse transcription PCR (RT-PCR).