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Optimization of an oligonucleotide microchip for microbial identification studies: a non-equilibrium dissociation approach
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) was demonstrated using an approach that compares the non-equilibrium dissociati...
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Published in: | Environmental microbiology 2001-10, Vol.3 (10), p.619-629 |
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creator | Liu, Wen-Tso Mirzabekov, Andrei D. Stahl, David A. |
description | 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) was demonstrated 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. By normalizing 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. |
doi_str_mv | 10.1046/j.1462-2920.2001.00233.x |
format | article |
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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. By normalizing 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. 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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.</description><subject>Bacillus - classification</subject><subject>Evaluation Studies as Topic</subject><subject>Life Sciences (General)</subject><subject>Nucleic Acid Hybridization</subject><subject>Oligonucleotide Array Sequence Analysis - methods</subject><subject>Oligonucleotide Probes - genetics</subject><subject>Phylogeny</subject><subject>RNA, Bacterial - genetics</subject><subject>RNA, Ribosomal - genetics</subject><subject>Space life sciences</subject><subject>Temperature</subject><issn>1462-2912</issn><issn>1462-2920</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><recordid>eNqNkc1O3TAQha2qVaHQN6gqr7pL8E-cOFU3FaKARGHTComN5dhOmdskDnYiLjx9nebqsmVjjzXfOSOfQQhTklNSlCebnBYly1jNSM4IoTkhjPN8-wYd7htv9zVlB-hDjJsEVrwi79EBpRVjomCH6PlmnKCHZz2BH7BvsU5nB3_8MJvO-Qmswz2Y4M09jLj1YX01oDucesMELZhVHKfZgotfscaDHzL3MEMHTYC5xxZi9AZWTo9j8NrcH6N3re6i-7i7j9DvH2e_Ti-yq5vzy9PvV5kpZM0z56wxjSYlE9wy3RpOai0YJ5aUopRF3dRGloWzshK0ZrK1TjjRGMKcILak_Ah9WX3T2IfZxUn1EI3rOj04P0dVJa-qkiSBcgXTB2MMrlVjgF6HJ0WJWnJXG7VEqpZ41ZK7-p-72ibp592MuemdfRHugk7AtxV4hM49vdpYnf28TEWSf1rlg45aDVOIC1YQImXJFvdsbUOc3HbvrsNfVaaVC3V7fa4u6jtxm_avJP8HaOOrRA</recordid><startdate>200110</startdate><enddate>200110</enddate><creator>Liu, Wen-Tso</creator><creator>Mirzabekov, Andrei D.</creator><creator>Stahl, David A.</creator><general>Blackwell Science Ltd</general><scope>BSCLL</scope><scope>CYE</scope><scope>CYI</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>200110</creationdate><title>Optimization of an oligonucleotide microchip for microbial identification studies: a non-equilibrium dissociation approach</title><author>Liu, Wen-Tso ; Mirzabekov, Andrei D. ; Stahl, David A.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4893-eedccba06253d2afc309a5230d0656849b9c864ed8751928fde5e5bc02e50d613</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Bacillus - classification</topic><topic>Evaluation Studies as Topic</topic><topic>Life Sciences (General)</topic><topic>Nucleic Acid Hybridization</topic><topic>Oligonucleotide Array Sequence Analysis - methods</topic><topic>Oligonucleotide Probes - genetics</topic><topic>Phylogeny</topic><topic>RNA, Bacterial - genetics</topic><topic>RNA, Ribosomal - genetics</topic><topic>Space life sciences</topic><topic>Temperature</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Liu, Wen-Tso</creatorcontrib><creatorcontrib>Mirzabekov, Andrei D.</creatorcontrib><creatorcontrib>Stahl, David A.</creatorcontrib><collection>Istex</collection><collection>NASA Scientific and Technical Information</collection><collection>NASA Technical Reports Server</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Environmental microbiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Liu, Wen-Tso</au><au>Mirzabekov, Andrei D.</au><au>Stahl, David A.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Optimization of an oligonucleotide microchip for microbial identification studies: a non-equilibrium dissociation approach</atitle><jtitle>Environmental microbiology</jtitle><addtitle>Environ Microbiol</addtitle><date>2001-10</date><risdate>2001</risdate><volume>3</volume><issue>10</issue><spage>619</spage><epage>629</epage><pages>619-629</pages><issn>1462-2912</issn><eissn>1462-2920</eissn><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) was demonstrated 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. By normalizing 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.</abstract><cop>Oxford, UK</cop><pub>Blackwell Science Ltd</pub><pmid>11722542</pmid><doi>10.1046/j.1462-2920.2001.00233.x</doi><tpages>11</tpages></addata></record> |
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subjects | Bacillus - classification Evaluation Studies as Topic Life Sciences (General) Nucleic Acid Hybridization Oligonucleotide Array Sequence Analysis - methods Oligonucleotide Probes - genetics Phylogeny RNA, Bacterial - genetics RNA, Ribosomal - genetics Space life sciences Temperature |
title | Optimization of an oligonucleotide microchip for microbial identification studies: a non-equilibrium dissociation approach |
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