Modeling formamide denaturation of probe-target hybrids for improved microarray probe design in microbial diagnostics.
Application of high-density microarrays to the diagnostic analysis of microbial communities is challenged by the optimization of oligonucleotide probe sensitivity and specificity, as it is generally unfeasible to experimentally test thousands of probes. This study investigated the adjustment of hybr...
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Public Library of Science (PLoS)
2012-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0043862&type=printable |
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| author | L Safak Yilmaz Alexander Loy Erik S Wright Michael Wagner Daniel R Noguera |
| author_facet | L Safak Yilmaz Alexander Loy Erik S Wright Michael Wagner Daniel R Noguera |
| author_sort | L Safak Yilmaz |
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| description | Application of high-density microarrays to the diagnostic analysis of microbial communities is challenged by the optimization of oligonucleotide probe sensitivity and specificity, as it is generally unfeasible to experimentally test thousands of probes. This study investigated the adjustment of hybridization stringency using formamide with the idea that sensitivity and specificity can be optimized during probe design if the hybridization efficiency of oligonucleotides with target and non-target molecules can be predicted as a function of formamide concentration. Sigmoidal denaturation profiles were obtained using fluorescently labeled and fragmented 16S rRNA gene amplicon of Escherichia coli as the target with increasing concentrations of formamide in the hybridization buffer. A linear free energy model (LFEM) was developed and microarray-specific nearest neighbor rules were derived. The model simulated formamide melting with a denaturant m-value that increased hybridization free energy (ΔG°) by 0.173 kcal/mol per percent of formamide added (v/v). Using the LFEM and specific probe sets, free energy rules were systematically established to predict the stability of single and double mismatches, including bulged and tandem mismatches. The absolute error in predicting the position of experimental denaturation profiles was less than 5% formamide for more than 90 percent of probes, enabling a practical level of accuracy in probe design. The potential of the modeling approach for probe design and optimization is demonstrated using a dataset including the 16S rRNA gene of Rhodobacter sphaeroides as an additional target molecule. The LFEM and thermodynamic databases were incorporated into a computational tool (ProbeMelt) that is freely available at http://DECIPHER.cee.wisc.edu. |
| format | Article |
| id | doaj-art-af7ce56b4526481fb69d40d5b0eecf37 |
| institution | OA Journals |
| issn | 1932-6203 |
| language | English |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj-art-af7ce56b4526481fb69d40d5b0eecf372025-08-20T02:08:46ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-0178e4386210.1371/journal.pone.0043862Modeling formamide denaturation of probe-target hybrids for improved microarray probe design in microbial diagnostics.L Safak YilmazAlexander LoyErik S WrightMichael WagnerDaniel R NogueraApplication of high-density microarrays to the diagnostic analysis of microbial communities is challenged by the optimization of oligonucleotide probe sensitivity and specificity, as it is generally unfeasible to experimentally test thousands of probes. This study investigated the adjustment of hybridization stringency using formamide with the idea that sensitivity and specificity can be optimized during probe design if the hybridization efficiency of oligonucleotides with target and non-target molecules can be predicted as a function of formamide concentration. Sigmoidal denaturation profiles were obtained using fluorescently labeled and fragmented 16S rRNA gene amplicon of Escherichia coli as the target with increasing concentrations of formamide in the hybridization buffer. A linear free energy model (LFEM) was developed and microarray-specific nearest neighbor rules were derived. The model simulated formamide melting with a denaturant m-value that increased hybridization free energy (ΔG°) by 0.173 kcal/mol per percent of formamide added (v/v). Using the LFEM and specific probe sets, free energy rules were systematically established to predict the stability of single and double mismatches, including bulged and tandem mismatches. The absolute error in predicting the position of experimental denaturation profiles was less than 5% formamide for more than 90 percent of probes, enabling a practical level of accuracy in probe design. The potential of the modeling approach for probe design and optimization is demonstrated using a dataset including the 16S rRNA gene of Rhodobacter sphaeroides as an additional target molecule. The LFEM and thermodynamic databases were incorporated into a computational tool (ProbeMelt) that is freely available at http://DECIPHER.cee.wisc.edu.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0043862&type=printable |
| spellingShingle | L Safak Yilmaz Alexander Loy Erik S Wright Michael Wagner Daniel R Noguera Modeling formamide denaturation of probe-target hybrids for improved microarray probe design in microbial diagnostics. PLoS ONE |
| title | Modeling formamide denaturation of probe-target hybrids for improved microarray probe design in microbial diagnostics. |
| title_full | Modeling formamide denaturation of probe-target hybrids for improved microarray probe design in microbial diagnostics. |
| title_fullStr | Modeling formamide denaturation of probe-target hybrids for improved microarray probe design in microbial diagnostics. |
| title_full_unstemmed | Modeling formamide denaturation of probe-target hybrids for improved microarray probe design in microbial diagnostics. |
| title_short | Modeling formamide denaturation of probe-target hybrids for improved microarray probe design in microbial diagnostics. |
| title_sort | modeling formamide denaturation of probe target hybrids for improved microarray probe design in microbial diagnostics |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0043862&type=printable |
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