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Titolo:
Optimization of an oligonucleotide microchip for microbial identification studies: a non-equilibrium dissociation approach
Autore:
Liu, WT; Mirzabekov, AD; Stahl, DA;
Indirizzi:
Northwestern Univ, Dept Civil Engn, Environm Hlth Engn Program, Evanston, IL 60208 USA Northwestern Univ Evanston IL USA 60208 n Program, Evanston, IL 60208 USA Argonne Natl Lab, Ctr Mechanist Biol, Argonne, IL 60439 USA Argonne Natl Lab Argonne IL USA 60439 chanist Biol, Argonne, IL 60439 USA
Titolo Testata:
ENVIRONMENTAL MICROBIOLOGY
fascicolo: 10, volume: 3, anno: 2001,
pagine: 619 - 629
SICI:
1462-2912(200110)3:10<619:OOAOMF>2.0.ZU;2-X
Fonte:
ISI
Lingua:
ENG
Soggetto:
16S RIBOSOMAL-RNA; HYBRIDIZATION PROBES; ESCHERICHIA-COLI; DNA; ECOLOGY; ARRAYS; QUANTIFICATION; ORGANIZATION; MUTATIONS; DATABASE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Agriculture,Biology & Environmental Sciences
Life Sciences
Citazioni:
31
Recensione:
Indirizzi per estratti:
Indirizzo: Liu, WT Natl Univ Singapore, Dept Civil Engn, Blk E1A,07-03,1 Engn Dr 2, Singapore117576, Singapore Natl Univ Singapore Blk E1A,07-03,1 Engn Dr 2 Singapore Singapore 117576
Citazione:
W.T. Liu et al., "Optimization of an oligonucleotide microchip for microbial identification studies: a non-equilibrium dissociation approach", ENVIRON MIC, 3(10), 2001, pp. 619-629

Abstract

The utility of a high-density oligonucleotide microarray (microchip) for identifying strains of five closely related bacilli (Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus medusa and Bacillus subtilis) wasdemonstrated using an approach that compares the non-equilibrium dissociation rates ('melting curves') of all probe-target duplexes simultaneously. For this study, a hierarchical set of 30 oligonucleotide probes targeting the 16S ribosomal RNA of these bacilli at multiple levels of specificity (approximate taxonomic ranks of domain, kingdom, order, genus and species) was designed and immobilized in a high-density matrix of gel pads on a glass slide. Reproducible melting curves for probes with different levels of specificity were obtained using an optimized salt concentration. Clear discrimination between perfect match (PM) and mismatch (MM) duplexes was achieved. Bynormalizing the signals to an internal standard (a universal probe), a more than twofold discrimination (> 2.4x) was achieved between PM and 1-MM duplexes at the dissociation temperature at which 50% of the probe-target duplexes remained intact. This provided excellent differentiation among representatives of different Bacillus species, both individually and in mixtures of two or three. The overall pattern of hybridization derived from this hierarchical probe set also provided a clear 'chip fingerprint' for each of these closely related Bacillus species.

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Documento generato il 23/01/20 alle ore 03:54:58