High efficiency restriction enzyme-free linear amplification-mediated polymerase chain reaction approach for tracking...

Retroviral vectors are an efficient and widely employed means of introducing an exogenous expression cassette into target cells. These vectors have been shown to integrate semi-randomly into the cellular genome, and can be associated with genotoxicity due to impact on expression of proximate genes. Therefore, efficient and accurate integration site analysis, while quantifying contributions of individual vector-containing clones, is desirable. Linear amplification-mediated polymerase chain reaction (LAM-PCR) is a widely used technique for identifying integrated proviral and host genomic DNA junctions. However, LAM-PCR is subject to selection bias inherent in the reliance of the assay on the presence of a restriction enzyme-cutting site adjacent to a retrievable integration site, and it is further limited by an inability to discriminate prior to sequencing between the flanking genomic DNA of interest and uninformative internal vector DNA. We report a modified restriction enzyme-free LAM-PCR (Re-free LAM-PCR) approach that is less time and labor intensive compared to conventional LAM-PCR, but in contrast to some other nonrestrictive methods, compares in efficiency and sensitivity, excludes retrieval of uninformative internal vector sequences, and allows retrieval of integration sites unbiased by the presence of nearby restriction sites. However, we report that Re-free LAM-PCR remains inaccurate for quantitation of the relative contributions of individual integration site-containing clones in a polyclonal setting, suggesting that bias in LAM-PCR retrieval of integration sites is not wholly explained by restriction enzyme-related factors.