Molecular Breakpoint Analysis of Chromosome Translocations in Cancer Cell Lines by Long Distance Inverse-PCR

With conventional cytogenetic screening by fluorescence in situ hybridization (FISH) using genomic tilepath clones, identification of genes in oncogenic chromosome translocations is often laborious, notably if the region of interest is gene-dense. Conventional molecular methods for partner identification may also suffer severe limitations; for instance, genomic PCR screening requires prior knowledge of both sets of breakpoints, while rapid amplification of cDNA ends (RACE) is not only limited to translocations causing mRNA fusion, but also fails to provide potentially relevant breakpoint data. With Long Distance Inverse (LDI)-PCR, however, it is theoretically possible to identify unknown translocation partners and to map the breakpoints down to the base pair level. Implementing LDI-PCR only requires approximate sequence information on one partner, rendering it ideal for use in combination with frontline FISH analysis. The protocol described here has been tuned for use by those wishing to identify new cancer genes in tumor cell lines.