Detection and Direct Genomic Sequencing of Multiple Rare Unknown Flanking DNA in Highly Complex Samples

Abstract

By identifying the sequence of retro- and lentiviral integration sites in peripheral blood leukocytes, the clonal composition and fate of genetically modified hematopoietic progenitor and stem cells could be mapped in vitro and in vivo. Previously available methods have been limited to the analysis of mono- or oligoclonal integration sites present in high copy numbers. Here, we perform characterization of multiple rare retroviral and lentiviral integration sites in highly complex DNA samples. The reliability of this method results from nontarget DNA removal via magnetic extension primer tag selection (EPTS) preceding solid-phase ligation-mediated PCR. EPTS/LM-PCR allowed the simultaneous direct genomic sequencing of multiple proviral LTR-flanking sequences of retro- and lentiviral vectors even if only 1 per 100 to 1000 cells contained the provirus. A primer walking "around" the integration locus demonstrated the adaptability of EPTS/LM-PCR to study unknown flanking DNA regions unrelated to proviruses. The technique is fast, inexpensive, and sensitive in minimal samples. It enables studies of retro- and lentiviral integration, viral vector tracking in gene therapy, insertional mutagenesis, transgene integration, and direct genomic sequencing that until now have been difficult or impossible to perform.

Rapid detection of retroviral vector integration sites in colony-forming human peripheral blood progenitor cells using PCR with arbitrary primers

“…Importantly, both integrations could be detected in one reaction using a cocktail of the primers FP2 and FP4 (Figure 2b). In order to validate these results, we furthermore analyzed retroviral integration sites in these HT1080 cell line clones with a different method, an LM-PCR modified after Rosenthal and Jones 14 and Schmidt et al 7 After extensive studies with three different restriction enzymes to create a large variety of amplification permissive restriction fragment length polymorphisms, we found a total of three different retroviral integration sites in the two HT1080 cell line clones. These vector integrations were identical to those detected by two-step PCR with FP2, FP4 or FP5 as arbitrary primers (Table 1), thus confirming the reliability and sensitivity of the two-step PCR.…”

“…5,6 The other approach is a ligation-mediated PCR (LM-PCR), which involves ligating a short adapter oligonucleotide to the unknown flanking DNA allowing PCR amplification. [7][8][9] Both these methods require numerous DNA preparation steps before amplification, which may limit their sensitivity. Therefore, the aim of this study was to establish a sensitive and rapid method to characterize retroviral vector integration sites.…”

Retroviral Insertional Mutagenesis Can Contribute to Immortalization of Mature T Lymphocytes

“…Ligation-mediated polymerase chain reaction (LM-PCR) was performed as previously described (20). Genomic DNA was prepared, using the DNeasy Blood & Tissue Kit (Qiagen).…”

Gadd45β is a pro-survival factor associated with stress-resistant tumors

“…For the analysis of apoptosis, highmolecular-weight DNA was extracted from 1 Â 10 7 tumor cells cultured for 3 h, as described (Heinrichs and Deppert, 2003). Retroviral integration sites were isolated using a modified protocol of a previously described method (Schmidt et al, 2001). Details are available upon request.…”