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Titolo:
Comprehensive mutation analysis of TSC1 using two-dimensional DNA electrophoresis with DGGE
Autore:
Dabora, SL; Sigalas, I; Hall, F; Eng, C; Vijg, J; Kwiatkowski, DJ;
Indirizzi:
Brigham & Womens Hosp, Div Hematol, Boston, MA 02115 USA Brigham & Womens Hosp Boston MA USA 02115 v Hematol, Boston, MA 02115 USA Dana Farber Canc Inst, Dept Adult Oncol, Boston, MA 02115 USA Dana Farber Canc Inst Boston MA USA 02115 ult Oncol, Boston, MA 02115 USA Beth15srael Deaconess Med Ctr, Gerontol Div, Mol Genet Sect, Boston, MA 021 Beth Israel Deaconess Med Ctr Boston MA USA 02115 et Sect, Boston, MA 021 Harvard Univ, Sch Med, Boston, MA 02115 USA Harvard Univ Boston MA USA 02115 vard Univ, Sch Med, Boston, MA 02115 USA
Titolo Testata:
ANNALS OF HUMAN GENETICS
, volume: 62, anno: 1998,
parte:, 6
pagine: 491 - 504
SICI:
0003-4800(199811)62:<491:CMAOTU>2.0.ZU;2-U
Fonte:
ISI
Lingua:
ENG
Soggetto:
TUBEROUS-SCLEROSIS PATIENTS; TOF MASS-SPECTROMETRY; GENETIC-ANALYSIS; NON-PENETRANCE; PRODUCT; IDENTIFICATION; MOSAICISM; RB1; GAP;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
38
Recensione:
Indirizzi per estratti:
Indirizzo: Dabora, SL Brigham2115omens Hosp, Div Hematol, 221 Longwood Ave,LMRC 301, Boston, MA 0 Brigham & Womens Hosp 221 Longwood Ave,LMRC 301 Boston MA USA 02115
Citazione:
S.L. Dabora et al., "Comprehensive mutation analysis of TSC1 using two-dimensional DNA electrophoresis with DGGE", ANN HUM GEN, 62, 1998, pp. 491-504

Abstract

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by the development of benign tumors in multiple organs often causingserious neurologic impairment. To develop a reliable genetic test for TSC,two-dimensional electrophoresis with denaturing gradient gel electrophoresis (2D DGGE) has been developed to detect mutations in TSC1. The 23 exons of TSC1 were amplified using two rounds of PCR. In the first round, all coding regions of TSC1 were amplified in four fragments ranging in size from 7.4 kb to 9.9 kb. In the second round, 32 fragments representing 23 exons were amplified using primers designed to avoid overlapping fragments and with a GC clamp on one end to optimise melting characteristics. These exon fragments were then separated by size in the first dimension using a polyacrylamide gel, and by melting characteristics in the second dimension using a urea/formamide gradient to yield 32 distinct bands. If a mutation is present, four bands instead of one, are typically observed. During the development of this assay, we analysed 63 patient samples with known TSC1 mutations fromprior studies. These 63 patients had 68 known mutations or polymorphisms. With DGGE, all 68 of these were identified (45 point mutations, 3 small insertions, 20 small deletions) and an additional 27 single base variants werediscovered, To evaluate the assay; we analysed 19 of these samples in a blinded study. In the blinded analysis, 19/20 (95%) known mutations or polymorphisms were detected. The single missed mutation in the blinded analysis could be identified in retrospect and the assay was modified accordingly. During this study, we identified 2 new mutations (exon 8 and exon 15), a new polymorphism (intron 4), and the first variant identified in a non-coding exon (exon 2).

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Documento generato il 04/12/20 alle ore 22:32:30