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Titolo:
Quantifying DNA-protein interactions by double-stranded DNA arrays
Autore:
Bulyk, ML; Gentalen, E; Lockhart, DJ; Church, GM;
Indirizzi:
Harvard Univ, Grad Biophys Program, Boston, MA 02115 USA Harvard Univ Boston MA USA 02115 ad Biophys Program, Boston, MA 02115 USA Harvard Univ, Sch Med, Dept Genet, Boston, MA 02115 USA Harvard Univ Boston MA USA 02115 ch Med, Dept Genet, Boston, MA 02115 USA Affymetrix, Santa Clara, CA 95051 USA Affymetrix Santa Clara CA USA 95051Affymetrix, Santa Clara, CA 95051 USA
Titolo Testata:
NATURE BIOTECHNOLOGY
fascicolo: 6, volume: 17, anno: 1999,
pagine: 573 - 577
SICI:
1087-0156(199906)17:6<573:QDIBDD>2.0.ZU;2-E
Fonte:
ISI
Lingua:
ENG
Soggetto:
DENSITY OLIGONUCLEOTIDE ARRAYS; ECORI RESTRICTION ENDONUCLEASE; ESCHERICHIA-COLI; CRYSTAL-STRUCTURE; COGNATE DNA; BAM-HI; RECOGNITION; BINDING; GENOME; SEQUENCE;
Keywords:
dsDNA arrays; restriction enzymes; DNA-protein interactions;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Agriculture,Biology & Environmental Sciences
Life Sciences
Citazioni:
47
Recensione:
Indirizzi per estratti:
Indirizzo: Church, GM Harvard Univ, Grad Biophys Program, Boston, MA 02115 USA Harvard Univ Boston MA USA 02115 Program, Boston, MA 02115 USA
Citazione:
M.L. Bulyk et al., "Quantifying DNA-protein interactions by double-stranded DNA arrays", NAT BIOTECH, 17(6), 1999, pp. 573-577

Abstract

We have created double-stranded oligonucleotide arrays to perform highly parallel investigations of DNA-protein interactions. Arrays of single-stranded DNA oligonucleotides, synthesized by a combination of photolithography and solid-state chemistry, have been used for a variety of applications, including large-scale mRNA expression monitoring, genotyping, and sequence-variation analysis. We converted a single-stranded to a double-stranded array by synthesizing a constant sequence at every position on an array and then annealing and enzymatically extending a complementary primer. The efficiency of second-strand synthesis was demonstrated by incorporation of fluorescently labeled dNTPs (2'-deoxyribonucleoside 5'-triphosphates) and by terminal transferase addition of a fluorescently labeled ddNTP. The accuracy of second-strand synthesis was demonstrated by digestion of the arrayed double-stranded DNA (dsDNA) on the array with sequence-specific restriction enzymes. We showed dam methylation of dsDNA arrays by digestion with Dpnl, which cleaves when its recognition site is methylated. This digestion demonstratedthat the dsDNA arrays can be further biochemically modified and that the DNA is accessible for interaction with DNA-binding proteins. This dsDNA array approach could be extended to explore the spectrum of sequence-specific protein binding sites in genomes.

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Documento generato il 27/10/20 alle ore 05:18:33