TY  - BOOK
AU  - Schmidt,M.
AU  - Schwarzwaelder,K.
AU  - Bartholomae,C.
AU  - Zaoui,K.
AU  - Ball,C.
AU  - Pilz,I.
AU  - Braun,S.
AU  - Braun,S.
AU  - Von Kalle,C.
TI  - High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR)
KW  - DNA
KW  - MOLECULAR MARKER
KW  - RETROVIRUS VECTOR
KW  - STREPTAVIDIN
N2  - 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)
UR  - https://drive.google.com/file/d/1YR75gpkSKlqOKg61v6GhkdcVpVxyjIqH/view?usp=drivesdk
ER  -