Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples

By identifying the sequence of retro- and lentiviral integration sites in p eripheral blood leukocytes, the clonal composition and fate of genetically modified hematopoietic progenitor and stem cells could be mapped in vitro a nd in vivo. Previously available methods have been limited to the analysis of mono- or oligoclonal integration sites present in high copy numbers. Her e, we perform characterization of multiple rare retroviral and lentiviral i ntegration sites in highly complex DNA samples. The reliability of this met hod results from nontarget DNA removal via magnetic extension primer tag se lection (EPTS) preceding solid-phase ligation-mediated PCR. EPTS/LM-PCR all owed the simultaneous direct genomic sequencing of multiple proviral LTR-fl anking sequences of retro- and lentiviral vectors even if only 1 per 100 to 1000 cells contained the provirus. A primer walking "around" the integrati on locus demonstrated the adaptability of EPTS/LM-PCR to study unknown flan king DNA regions unrelated to proviruses. The technique is fast, inexpensiv e, and sensitive in minimal samples. It enables studies of retro- and lenti viral integration, viral vector tracking in gene therapy, insertional mutag enesis, transgene integration, and direct genomic sequencing that until now have been difficult or impossible to perform.