Empirical establishment of oligonucleotide probe design criteria.
Identifieur interne : 002326 ( PubMed/Corpus ); précédent : 002325; suivant : 002327Empirical establishment of oligonucleotide probe design criteria.
Auteurs : Zhili He ; Liyou Wu ; Xingyuan Li ; Matthew W. Fields ; Jizhong ZhouSource :
- Applied and environmental microbiology [ 0099-2240 ] ; 2005.
English descriptors
- KwdEn :
- MESH :
- chemical , genetics : Oligonucleotide Probes.
- genetics : Shewanella.
- standards : Research Design.
- Base Pair Mismatch, Nucleic Acid Hybridization, Oligonucleotide Array Sequence Analysis, Species Specificity.
Abstract
Criteria for the design of gene-specific and group-specific oligonucleotide probes were established experimentally via an oligonucleotide array that contained perfect match (PM) and mismatch probes (50-mers and 70-mers) based upon four genes. The effects of probe-target identity, continuous stretch, mismatch position, and hybridization free energy on specificity were tested. Little hybridization was observed at a probe-target identity of < or =85% for both 50-mer and 70-mer probes. PM signal intensities (33 to 48%) were detected at a probe-target identity of 94% for 50-mer oligonucleotides and 43 to 55% for 70-mer probes at a probe-target identity of 96%. When the effects of sequence identity and continuous stretch were considered independently, a stretch probe (>15 bases) contributed an additional 9% of the PM signal intensity compared to a nonstretch probe (< or =15 bases) at the same identity level. Cross-hybridization increased as the length of continuous stretch increased. A 35-base stretch for 50-mer probes or a 50-base stretch for 70-mer probes had approximately 55% of the PM signal. Little cross-hybridization was observed for probes with a minimal binding free energy greater than -30 kcal/mol for 50-mer probes or -40 kcal/mol for 70-mer probes. Based on the experimental results, a set of criteria are suggested for the design of gene-specific and group-specific oligonucleotide probes, and the experimentally established criteria should provide valuable information for new software and algorithms for microarray-based studies.
DOI: 10.1128/AEM.71.7.3753-3760.2005
PubMed: 16000786
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pubmed:16000786Le document en format XML
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<affiliation><nlm:affiliation>Environmental Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6038, USA.</nlm:affiliation>
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<author><name sortKey="Wu, Liyou" sort="Wu, Liyou" uniqKey="Wu L" first="Liyou" last="Wu">Liyou Wu</name>
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<author><name sortKey="Li, Xingyuan" sort="Li, Xingyuan" uniqKey="Li X" first="Xingyuan" last="Li">Xingyuan Li</name>
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<author><name sortKey="Fields, Matthew W" sort="Fields, Matthew W" uniqKey="Fields M" first="Matthew W" last="Fields">Matthew W. Fields</name>
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<author><name sortKey="Zhou, Jizhong" sort="Zhou, Jizhong" uniqKey="Zhou J" first="Jizhong" last="Zhou">Jizhong Zhou</name>
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<term>Nucleic Acid Hybridization</term>
<term>Oligonucleotide Array Sequence Analysis</term>
<term>Oligonucleotide Probes (genetics)</term>
<term>Research Design (standards)</term>
<term>Shewanella (genetics)</term>
<term>Species Specificity</term>
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<front><div type="abstract" xml:lang="en">Criteria for the design of gene-specific and group-specific oligonucleotide probes were established experimentally via an oligonucleotide array that contained perfect match (PM) and mismatch probes (50-mers and 70-mers) based upon four genes. The effects of probe-target identity, continuous stretch, mismatch position, and hybridization free energy on specificity were tested. Little hybridization was observed at a probe-target identity of < or =85% for both 50-mer and 70-mer probes. PM signal intensities (33 to 48%) were detected at a probe-target identity of 94% for 50-mer oligonucleotides and 43 to 55% for 70-mer probes at a probe-target identity of 96%. When the effects of sequence identity and continuous stretch were considered independently, a stretch probe (>15 bases) contributed an additional 9% of the PM signal intensity compared to a nonstretch probe (< or =15 bases) at the same identity level. Cross-hybridization increased as the length of continuous stretch increased. A 35-base stretch for 50-mer probes or a 50-base stretch for 70-mer probes had approximately 55% of the PM signal. Little cross-hybridization was observed for probes with a minimal binding free energy greater than -30 kcal/mol for 50-mer probes or -40 kcal/mol for 70-mer probes. Based on the experimental results, a set of criteria are suggested for the design of gene-specific and group-specific oligonucleotide probes, and the experimentally established criteria should provide valuable information for new software and algorithms for microarray-based studies.</div>
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<Abstract><AbstractText>Criteria for the design of gene-specific and group-specific oligonucleotide probes were established experimentally via an oligonucleotide array that contained perfect match (PM) and mismatch probes (50-mers and 70-mers) based upon four genes. The effects of probe-target identity, continuous stretch, mismatch position, and hybridization free energy on specificity were tested. Little hybridization was observed at a probe-target identity of < or =85% for both 50-mer and 70-mer probes. PM signal intensities (33 to 48%) were detected at a probe-target identity of 94% for 50-mer oligonucleotides and 43 to 55% for 70-mer probes at a probe-target identity of 96%. When the effects of sequence identity and continuous stretch were considered independently, a stretch probe (>15 bases) contributed an additional 9% of the PM signal intensity compared to a nonstretch probe (< or =15 bases) at the same identity level. Cross-hybridization increased as the length of continuous stretch increased. A 35-base stretch for 50-mer probes or a 50-base stretch for 70-mer probes had approximately 55% of the PM signal. Little cross-hybridization was observed for probes with a minimal binding free energy greater than -30 kcal/mol for 50-mer probes or -40 kcal/mol for 70-mer probes. Based on the experimental results, a set of criteria are suggested for the design of gene-specific and group-specific oligonucleotide probes, and the experimentally established criteria should provide valuable information for new software and algorithms for microarray-based studies.</AbstractText>
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