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Nat Methods. 2007 Dec;4(12):1051-7.

High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR).

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1
Department of Translational Oncology, National Center for Tumor Diseases, German Cancer Research Center, Im Neuenheimer Feld 350, 69120 Heidelberg, Germany. manfred.schmidt@nct-heidelberg.de

Abstract

Integrating vector systems used in clinical gene therapy have proven their therapeutic potential in the long-term correction of immunodeficiencies. The integration loci of such vectors in the cellular genome represent a molecular marker unique for each transduced cell and its clonal progeny. To gain insight into the physiology of gene-modified hematopoietic repopulation and vector-related influences on clonal contributions, we have previously introduced a technology--linear amplification-mediated (LAM) PCR--for detecting and sequencing unknown DNA flanking sequences down to the single cell level (Supplementary Note online). LAM-PCR analyses have enabled qualitative and quantitative measurements of the clonal kinetics of hematopoietic regeneration in gene transfer studies, and uncovered the clonal derivation of non-leukemogenic and leukemogenic insertional side effects in preclinical and clinical gene therapy studies. The reliability and robustness of this method results from the initial preamplification of the vector-genome junctions preceding nontarget DNA removal via magnetic selection. Subsequent steps are carried out on a semisolid streptavidin phase, including synthesis of double complementary strands, restriction digest, ligation of a linker cassette onto the genomic end of the fragment and exponential PCR(s) with vector- and linker cassette-specific primers. LAM-PCR can be adjusted to all unknown DNA sequences adjacent to a known DNA sequence. Here we describe the use of LAM-PCR analyses to identify 5' long terminal repeat (LTR) retroviral vector adjacent genomic sequences.

PMID:
18049469
DOI:
10.1038/nmeth1103
[Indexed for MEDLINE]
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