Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 88, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/51543
Keywords
Genetics; Issue 88; gene therapy; integrome; integration site analysis; LAM-PCR; retroviral vectors; lentiviral vectors; AAV; deep sequencing; clonal inventory; mutagenesis screen
Categories
Funding
- Deutsche Forschungsgemeinschaft [SPP1230]
- Bundesministerium fur Bildung und Forschung
- European Commission
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Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM-PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3'-and 5'-sequences adjacent to the integrated lentiviral vector.
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