Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability
Single and dual-labeled fluorescent oligodeoxynucleotides are used in many molecular biology applications. We investigated the effects of commonly used fluorescent dyes and quenchers on the thermodynamic stability of a model probe–target DNA duplex. We demonstrate that those effects can be significa...
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Veröffentlicht in: | Biochemical and biophysical research communications 2005-02, Vol.327 (2), p.473-484 |
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creator | Moreira, Bernardo G. You, Yong Behlke, Mark A. Owczarzy, Richard |
description | Single and dual-labeled fluorescent oligodeoxynucleotides are used in many molecular biology applications. We investigated the effects of commonly used fluorescent dyes and quenchers on the thermodynamic stability of a model probe–target DNA duplex. We demonstrate that those effects can be significant. Fluorescent dyes and quenchers were attached to the probe ends. In certain combinations, these groups stabilized the duplex up to 1.8
kcal/mol and increased
T
m up to 4.3
°C. None of the groups tested significantly destabilized the duplex. Rank order of potency was, starting with the most stabilizing group: Iowa Black RQ
∼
Black Hole 2
>
Cy5
∼
Cy3
>
Black Hole 1
>
QSY7
∼
Iowa Black FQ
>
Texas Red
∼
TAMRA
>
FAM
∼
HEX
∼
Dabcyl
>
TET. Longer linkers decreased stabilizing effects. Hybridizations to targets with various dangling ends were also studied and were found to have only minor effects on thermodynamic stability. Depending on the dye/quencher combination employed, it can be important to include thermodynamic contributions from fluorophore and quencher when designing oligonucleotide probe assays. |
doi_str_mv | 10.1016/j.bbrc.2004.12.035 |
format | Article |
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kcal/mol and increased
T
m up to 4.3
°C. None of the groups tested significantly destabilized the duplex. Rank order of potency was, starting with the most stabilizing group: Iowa Black RQ
∼
Black Hole 2
>
Cy5
∼
Cy3
>
Black Hole 1
>
QSY7
∼
Iowa Black FQ
>
Texas Red
∼
TAMRA
>
FAM
∼
HEX
∼
Dabcyl
>
TET. Longer linkers decreased stabilizing effects. Hybridizations to targets with various dangling ends were also studied and were found to have only minor effects on thermodynamic stability. Depending on the dye/quencher combination employed, it can be important to include thermodynamic contributions from fluorophore and quencher when designing oligonucleotide probe assays.</description><identifier>ISSN: 0006-291X</identifier><identifier>EISSN: 1090-2104</identifier><identifier>DOI: 10.1016/j.bbrc.2004.12.035</identifier><identifier>PMID: 15629139</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Base Sequence ; Dangling ends ; DNA - chemistry ; DNA - drug effects ; DNA - genetics ; DNA - metabolism ; DNA melting temperature ; DNA thermodynamics ; Fluorescent Dyes - chemistry ; Fluorescent Dyes - pharmacology ; Fluorescent probes ; Nucleic Acid Denaturation - drug effects ; Quencher ; Real-time PCR ; Spectrum Analysis ; Temperature ; Thermodynamics</subject><ispartof>Biochemical and biophysical research communications, 2005-02, Vol.327 (2), p.473-484</ispartof><rights>2004 Elsevier Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c385t-c80bb2d7faa9438d736ba613631206a66d35bdcb3921fc0169bb7bccee3a08383</citedby><cites>FETCH-LOGICAL-c385t-c80bb2d7faa9438d736ba613631206a66d35bdcb3921fc0169bb7bccee3a08383</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0006291X04028219$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15629139$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Moreira, Bernardo G.</creatorcontrib><creatorcontrib>You, Yong</creatorcontrib><creatorcontrib>Behlke, Mark A.</creatorcontrib><creatorcontrib>Owczarzy, Richard</creatorcontrib><title>Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability</title><title>Biochemical and biophysical research communications</title><addtitle>Biochem Biophys Res Commun</addtitle><description>Single and dual-labeled fluorescent oligodeoxynucleotides are used in many molecular biology applications. We investigated the effects of commonly used fluorescent dyes and quenchers on the thermodynamic stability of a model probe–target DNA duplex. We demonstrate that those effects can be significant. Fluorescent dyes and quenchers were attached to the probe ends. In certain combinations, these groups stabilized the duplex up to 1.8
kcal/mol and increased
T
m up to 4.3
°C. None of the groups tested significantly destabilized the duplex. Rank order of potency was, starting with the most stabilizing group: Iowa Black RQ
∼
Black Hole 2
>
Cy5
∼
Cy3
>
Black Hole 1
>
QSY7
∼
Iowa Black FQ
>
Texas Red
∼
TAMRA
>
FAM
∼
HEX
∼
Dabcyl
>
TET. Longer linkers decreased stabilizing effects. Hybridizations to targets with various dangling ends were also studied and were found to have only minor effects on thermodynamic stability. Depending on the dye/quencher combination employed, it can be important to include thermodynamic contributions from fluorophore and quencher when designing oligonucleotide probe assays.</description><subject>Base Sequence</subject><subject>Dangling ends</subject><subject>DNA - chemistry</subject><subject>DNA - drug effects</subject><subject>DNA - genetics</subject><subject>DNA - metabolism</subject><subject>DNA melting temperature</subject><subject>DNA thermodynamics</subject><subject>Fluorescent Dyes - chemistry</subject><subject>Fluorescent Dyes - pharmacology</subject><subject>Fluorescent probes</subject><subject>Nucleic Acid Denaturation - drug effects</subject><subject>Quencher</subject><subject>Real-time PCR</subject><subject>Spectrum Analysis</subject><subject>Temperature</subject><subject>Thermodynamics</subject><issn>0006-291X</issn><issn>1090-2104</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkE1r3DAQhkVoSTZp_0AORaeeandGsmUbeglpPgqhpdBCb0If40SL195Kdun--2rZhdya04jheV9GD2OXCCUCqo_r0troSgFQlShKkPUJWyF0UAiE6hVbAYAqRIe_zth5SmsAxEp1p-wMa5XXslux7zd9T25OfOp5PyxTpORonLnfUfrAfy80uieK-WlGz70ZH4cwPnIafU6M_PPXK-6X7UB_eZqNDUOYd2_Y694Mid4e5wX7eXvz4_q-ePh29-X66qFwsq3nwrVgrfBNb0xXydY3UlmjUCqJApRRysvaemdlJ7B3-budtY11jkgaaGUrL9j7Q-82TvnONOtNyLcPgxlpWpJWjawQRfciiE0rQao6g-IAujilFKnX2xg2Ju40gt4b12u9N673xjUKnY3n0Ltj-2I35J8jR8UZ-HQAKMv4Eyjq5ELWSj7EbF77Kfyv_x8fM5Fy</recordid><startdate>20050211</startdate><enddate>20050211</enddate><creator>Moreira, Bernardo G.</creator><creator>You, Yong</creator><creator>Behlke, Mark A.</creator><creator>Owczarzy, Richard</creator><general>Elsevier Inc</general><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>7TM</scope><scope>7X8</scope></search><sort><creationdate>20050211</creationdate><title>Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability</title><author>Moreira, Bernardo G. ; You, Yong ; Behlke, Mark A. ; Owczarzy, Richard</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c385t-c80bb2d7faa9438d736ba613631206a66d35bdcb3921fc0169bb7bccee3a08383</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Base Sequence</topic><topic>Dangling ends</topic><topic>DNA - chemistry</topic><topic>DNA - drug effects</topic><topic>DNA - genetics</topic><topic>DNA - metabolism</topic><topic>DNA melting temperature</topic><topic>DNA thermodynamics</topic><topic>Fluorescent Dyes - chemistry</topic><topic>Fluorescent Dyes - pharmacology</topic><topic>Fluorescent probes</topic><topic>Nucleic Acid Denaturation - drug effects</topic><topic>Quencher</topic><topic>Real-time PCR</topic><topic>Spectrum Analysis</topic><topic>Temperature</topic><topic>Thermodynamics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Moreira, Bernardo G.</creatorcontrib><creatorcontrib>You, Yong</creatorcontrib><creatorcontrib>Behlke, Mark A.</creatorcontrib><creatorcontrib>Owczarzy, Richard</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemical and biophysical research communications</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Moreira, Bernardo G.</au><au>You, Yong</au><au>Behlke, Mark A.</au><au>Owczarzy, Richard</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability</atitle><jtitle>Biochemical and biophysical research communications</jtitle><addtitle>Biochem Biophys Res Commun</addtitle><date>2005-02-11</date><risdate>2005</risdate><volume>327</volume><issue>2</issue><spage>473</spage><epage>484</epage><pages>473-484</pages><issn>0006-291X</issn><eissn>1090-2104</eissn><abstract>Single and dual-labeled fluorescent oligodeoxynucleotides are used in many molecular biology applications. We investigated the effects of commonly used fluorescent dyes and quenchers on the thermodynamic stability of a model probe–target DNA duplex. We demonstrate that those effects can be significant. Fluorescent dyes and quenchers were attached to the probe ends. In certain combinations, these groups stabilized the duplex up to 1.8
kcal/mol and increased
T
m up to 4.3
°C. None of the groups tested significantly destabilized the duplex. Rank order of potency was, starting with the most stabilizing group: Iowa Black RQ
∼
Black Hole 2
>
Cy5
∼
Cy3
>
Black Hole 1
>
QSY7
∼
Iowa Black FQ
>
Texas Red
∼
TAMRA
>
FAM
∼
HEX
∼
Dabcyl
>
TET. Longer linkers decreased stabilizing effects. Hybridizations to targets with various dangling ends were also studied and were found to have only minor effects on thermodynamic stability. Depending on the dye/quencher combination employed, it can be important to include thermodynamic contributions from fluorophore and quencher when designing oligonucleotide probe assays.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>15629139</pmid><doi>10.1016/j.bbrc.2004.12.035</doi><tpages>12</tpages></addata></record> |
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subjects | Base Sequence Dangling ends DNA - chemistry DNA - drug effects DNA - genetics DNA - metabolism DNA melting temperature DNA thermodynamics Fluorescent Dyes - chemistry Fluorescent Dyes - pharmacology Fluorescent probes Nucleic Acid Denaturation - drug effects Quencher Real-time PCR Spectrum Analysis Temperature Thermodynamics |
title | Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability |
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