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.
Schmidt
Manfred
M
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
Schwarzwaelder
Kerstin
K
Bartholomae
Cynthia
C
Zaoui
Karim
K
Ball
Claudia
C
Pilz
Ingo
I
Braun
Sandra
S
Glimm
Hanno
H
von Kalle
Christof
C
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