PCR-based procedures to isolate insertion sites of DNA elements

During the past several years, retroviral insertional mutagenesis has been fruitfully applied to search for genes/pathways involved in tumorigenesis. Techniques used to identify proviral insertion sites are critical for fulfilling these projects. Although a variety of approaches have been described, an improvement over existing methods is required to recover every possible insertion site for cancer gene discovery, so-called saturation analysis. Here, we have described the development of two ligation-mediated PCR variants, SplinkTA-PCR (STA-PCR) and SplinkBlunt-PCR, for efficient isolation of insertion sites in retrovirus-induced leukemia. Our results demonstrated that these two protocols are complementary to each other and that they are better employed in combination for maximal cloning efficiency. These protocols are easy-to-use, reliable and efficient, and are readily applicable to large-scale cloning of insertion sites of provirus and other integrated DNA elements, as well as for detection and cloning of differential insertions unique to drug-resistant cells.