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003			MX-MdCICY
005			20250625160141.0
040			_cCICY
090			_aB-15924
245	1	0	_aHigh-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR)
490	0		_vNature Methods, 4(5), p.1051-1057, 2007
520	3		_aIntegrating 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).
650	1	4	_aDNA
650	1	4	_aMOLECULAR MARKER
650	1	4	_aRETROVIRUS VECTOR
650	1	4	_aSTREPTAVIDIN
700	1	2	_aSchmidt, M.
700	1	2	_aSchwarzwaelder, K.
700	1	2	_aBartholomae, C.
700	1	2	_aZaoui, K.
700	1	2	_aBall, C.
700	1	2	_aPilz, I.
700	1	2	_aBraun, S.
700	1	2	_aBraun, S.
700	1	2	_aVon Kalle, C.
856	4	0	_uhttps://drive.google.com/file/d/1YR75gpkSKlqOKg61v6GhkdcVpVxyjIqH/view?usp=drivesdk _zPara ver el documento ingresa a Google con tu cuenta: @cicy.edu.mx
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