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.
Yin
Bin
B
University of Minnesota, Minneapolis, MN, USA. binxx002@umn.edu
Largaespada
David A
DA
D000818:Animals; D015139:Blotting, Southern; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D016678:Genome / Q000235:genetics; D006801:Humans; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics*; D016662:Virus Integration / Q000235:genetics*
D004279:DNA, Viral