High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR).
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.
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
Manfred
M
Schmidt
Kerstin
K
Schwarzwaelder
Cynthia
C
Bartholomae
Karim
K
Zaoui
Claudia
C
Ball
Ingo
I
Pilz
Sandra
S
Braun
Hanno
H
Glimm
Christof
C
von Kalle
D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D004251:DNA Transposable Elements / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
D004251:DNA Transposable Elements; D005819:Genetic Markers