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Titolo:
Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples
Autore:
Schmidt, M; Hoffmann, G; Wissler, M; Lemke, N; Mussig, A; Glimm, H; Williams, DA; Ragg, S; Hesemann, CU; von Kalle, C;
Indirizzi:
Univ Freiburg, Dept Internal Med 1, D-79106 Freiburg, Germany Univ Freiburg Freiburg Germany D-79106 Med 1, D-79106 Freiburg, Germany James Whitcomb Riley Hosp Children, Herman B Wells Ctr Pediat Res, Sect Pediat Hematol Oncol, Indianapolis, IN 46202 USA James Whitcomb Riley Hosp Children Indianapolis IN USA 46202 IN 46202 USA Indiana Univ, Sch Med, Howard Hughes Med Inst, Indianapolis, IN 46202 USA Indiana Univ Indianapolis IN USA 46202 d Inst, Indianapolis, IN 46202 USA Univ Hohenheim, Inst Genet, D-70599 Stuttgart, Germany Univ Hohenheim Stuttgart Germany D-70599 net, D-70599 Stuttgart, Germany
Titolo Testata:
HUMAN GENE THERAPY
fascicolo: 7, volume: 12, anno: 2001,
pagine: 743 - 749
SICI:
1043-0342(200105)12:7<743:DADGSO>2.0.ZU;2-Z
Fonte:
ISI
Lingua:
ENG
Soggetto:
LIGATION-MEDIATED PCR; POLYMERASE-CHAIN-REACTION; HEMATOPOIETIC STEM-CELLS; INSERTIONAL MUTAGENESIS; AMPLIFICATION; MICE; TRANSDUCTION; METHYLATION; EXPRESSION; ADJACENT;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
30
Recensione:
Indirizzi per estratti:
Indirizzo: von Kalle, C Univ Freiburg, Dept Internal Med 1, Hugstetter Str 55, D-79106 Freiburg, Germany Univ Freiburg Hugstetter Str 55 Freiburg Germany D-79106 any
Citazione:
M. Schmidt et al., "Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples", HUM GENE TH, 12(7), 2001, pp. 743-749

Abstract

By identifying the sequence of retro- and lentiviral integration sites in peripheral blood leukocytes, the clonal composition and fate of geneticallymodified hematopoietic progenitor and stem cells could be mapped in vitro and in vivo. Previously available methods have been limited to the analysisof mono- or oligoclonal integration sites present in high copy numbers. Here, we perform characterization of multiple rare retroviral and lentiviral integration sites in highly complex DNA samples. The reliability of this method results from nontarget DNA removal via magnetic extension primer tag selection (EPTS) preceding solid-phase ligation-mediated PCR. EPTS/LM-PCR allowed the simultaneous direct genomic sequencing of multiple proviral LTR-flanking sequences of retro- and lentiviral vectors even if only 1 per 100 to 1000 cells contained the provirus. A primer walking "around" the integration locus demonstrated the adaptability of EPTS/LM-PCR to study unknown flanking DNA regions unrelated to proviruses. The technique is fast, inexpensive, and sensitive in minimal samples. It enables studies of retro- and lentiviral integration, viral vector tracking in gene therapy, insertional mutagenesis, transgene integration, and direct genomic sequencing that until now have been difficult or impossible to perform.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 04/12/20 alle ore 09:14:15