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

Publikation: Beitrag in FachzeitschriftForschungsartikelBeigetragenBegutachtung

Beitragende

  • Manfred Schmidt - , Deutsches Krebsforschungszentrum (DKFZ), Universitätsklinikum Freiburg, Albert-Ludwigs-Universität Freiburg (Autor:in)
  • Kerstin Schwarzwaelder - , Deutsches Krebsforschungszentrum (DKFZ) (Autor:in)
  • Cynthia Bartholomae - , Deutsches Krebsforschungszentrum (DKFZ) (Autor:in)
  • Karim Zaoui - , Universitätsklinikum Freiburg (Autor:in)
  • Claudia Ball - , Nationales Centrum für Tumorerkrankungen Dresden, Umweltmonitoring und Endokrinologie (FoG), Nationales Zentrum für Tumorerkrankungen (NCT) Heidelberg (Autor:in)
  • Ingo Pilz - , Albert-Ludwigs-Universität Freiburg (Autor:in)
  • Sandra Braun - , Deutsches Krebsforschungszentrum (DKFZ) (Autor:in)
  • Hanno Glimm - , Nationales Centrum für Tumorerkrankungen Dresden, Nationales Zentrum für Tumorerkrankungen (NCT) Heidelberg (Autor:in)
  • Christof von Kalle - , Deutsches Krebsforschungszentrum (DKFZ), Cincinnati Children's Hospital Medical Center (Autor:in)

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

OriginalspracheEnglisch
Seiten (von - bis)1051-1057
Seitenumfang7
FachzeitschriftNature methods
Jahrgang4
Ausgabenummer12
PublikationsstatusVeröffentlicht - Dez. 2007
Peer-Review-StatusJa

Externe IDs

PubMed 18049469

Schlagworte