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Titolo:
APEX disease gene resequencing: Mutations in exon 7 of the p53 tumor suppressor gene
Autore:
Shumaker, JM; Tollet, JJ; Filbin, KJ; Montague-Smith, MP; Pirrung, MC;
Indirizzi:
Duke Univ, Dept Chem, Levine Sci Res Ctr, Durham, NC 27708 USA Duke Univ Durham NC USA 27708 m, Levine Sci Res Ctr, Durham, NC 27708 USA Baylor Coll Med, Dept Mol & Human Genet, Houston, TX 77030 USA Baylor CollMed Houston TX USA 77030 & Human Genet, Houston, TX 77030 USA
Titolo Testata:
BIOORGANIC & MEDICINAL CHEMISTRY
fascicolo: 9, volume: 9, anno: 2001,
pagine: 2269 - 2278
SICI:
0968-0896(200109)9:9<2269:ADGRMI>2.0.ZU;2-R
Fonte:
ISI
Lingua:
ENG
Soggetto:
ARRAYED PRIMER EXTENSION; DNA-SEQUENCE ANALYSIS; OLIGONUCLEOTIDE ARRAYS; SYNTHETIC OLIGONUCLEOTIDES; BIOMOLECULAR INTERACTIONS; APOLIPOPROTEIN-E; POINT MUTATIONS; HYBRIDIZATION; POLYMORPHISMS; POLYMERASE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
41
Recensione:
Indirizzi per estratti:
Indirizzo: Pirrung, MC Duke Univ, Dept Chem, Levine Sci Res Ctr, Box 90317, Durham, NC 27708 USA Duke Univ Box 90317 Durham NC USA 27708 , Durham, NC 27708 USA
Citazione:
J.M. Shumaker et al., "APEX disease gene resequencing: Mutations in exon 7 of the p53 tumor suppressor gene", BIO MED CH, 9(9), 2001, pp. 2269-2278

Abstract

Detection of mutations in disease genes will be a significant application of genomic research. Methods for detecting mutations at the single nucleotide level are required in highly mutated genes such. as the tumor suppressorp53. Resequencing of an individual patient's DNA by conventional Sanger methods is impractical, calling for novel methods for sequence analysis. Toward this end, an arrayed primer extension (APEX) method for identifying sequence alterations in primary DNA structure was developed. A two-dimensional array of immobilized primers (DNA chip) was fabricated to scan p53 exon 7 by single bases. Primers were immobilized with 200 mum spacing on a glass support. Oligonucleotide templates of length 72 were used to study individualAPEX resequencing reactions. A template-dependent DNA polymerase extensionwas performed on the chip using fluorescein-labeled dideoxynucleotides (ddNTPs). Labeled primers were evanescently excited and the induced fluorescence was imaged by CCD. The average signal-to-noise ratio (SIN) observed was 30:1. Software was developed to analyze hi.-h-density DNA chips for sequence alterations. Deletion, insertion, and substitution mutations were detected. APEX can be used to scan for any mutation (up to two-base insertions) ina known region of DNA by fabricating a DNA chip comprising complementary primers addressing each nucleotide in the wild-type sequence. Since APEX is a parallel method for determining DNA sequence, the time required to assay a region is independent of its length. APEX has a high level of accuracy, is sequence-based, and can be miniaturized to analyze a large DNA region with minimal reagents. (C) 2001 Elsevier Science Ltd. All rights reserved.

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Documento generato il 22/01/20 alle ore 09:44:05