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

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

  • Manfred Schmidt - , German Cancer Research Center (DKFZ), University Medical Center Freiburg, University of Freiburg (Author)
  • Kerstin Schwarzwaelder - , German Cancer Research Center (DKFZ) (Author)
  • Cynthia Bartholomae - , German Cancer Research Center (DKFZ) (Author)
  • Karim Zaoui - , University Medical Center Freiburg (Author)
  • Claudia Ball - , National Center for Tumor Diseases Dresden, Environmental Monitoring and Endocrinology (Research Group), National Center for Tumor Diseases (NCT) Heidelberg (Author)
  • Ingo Pilz - , University of Freiburg (Author)
  • Sandra Braun - , German Cancer Research Center (DKFZ) (Author)
  • Hanno Glimm - , National Center for Tumor Diseases Dresden, National Center for Tumor Diseases (NCT) Heidelberg (Author)
  • Christof von Kalle - , German Cancer Research Center (DKFZ), Cincinnati Children's Hospital Medical Center (Author)

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 (Fig. 1 and Box 1).

Details

Original languageEnglish
Pages (from-to)1051-1057
Number of pages7
JournalNature methods
Volume4
Issue number12
Publication statusPublished - Dec 2007
Peer-reviewedYes

External IDs

PubMed 18049469