Blending DNA binding dyes to improve detection in real-time PCR
The success of real-time PCR (qPCR) analysis is partly limited by the presence of inhibitory compounds in the nucleic acid samples. For example, humic acid (HA) from soil and aqueous sediment interferes with amplification and also quenches the fluorescence of double-stranded (ds) DNA binding dyes, t...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2017-03-01
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| Series: | Biotechnology Reports |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2215017X17300152 |
| _version_ | 1828521121340719104 |
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| author | Linda Jansson Marianne Koliana Maja Sidstedt Johannes Hedman |
| author_facet | Linda Jansson Marianne Koliana Maja Sidstedt Johannes Hedman |
| author_sort | Linda Jansson |
| collection | DOAJ |
| description | The success of real-time PCR (qPCR) analysis is partly limited by the presence of inhibitory compounds in the nucleic acid samples. For example, humic acid (HA) from soil and aqueous sediment interferes with amplification and also quenches the fluorescence of double-stranded (ds) DNA binding dyes, thus hindering amplicon detection. We aimed to counteract the HA fluorescence quenching effect by blending complementary dsDNA binding dyes, thereby elevating the dye saturation levels and increasing the fluorescence signals. A blend of the four dyes EvaGreen, ResoLight, SYBR Green and SYTO9 gave significantly higher fluorescence intensities in the presence and absence of HA, compared with the dyes applied separately and two-dye blends. We propose blending of dyes as a generally applicable means for elevating qPCR fluorescence signals and thus enabling detection in the presence of quenching substances. |
| first_indexed | 2024-12-11T19:43:10Z |
| format | Article |
| id | doaj.art-ba3bdcfc116d4c478dbd30309be3ebd7 |
| institution | Directory Open Access Journal |
| issn | 2215-017X |
| language | English |
| last_indexed | 2024-12-11T19:43:10Z |
| publishDate | 2017-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Biotechnology Reports |
| spelling | doaj.art-ba3bdcfc116d4c478dbd30309be3ebd72022-12-22T00:52:58ZengElsevierBiotechnology Reports2215-017X2017-03-0114C343710.1016/j.btre.2017.02.002Blending DNA binding dyes to improve detection in real-time PCRLinda Jansson0Marianne Koliana1Maja Sidstedt2Johannes Hedman3Applied Microbiology, Department of Chemistry, Lund University, Lund, SE-221 00, SwedenApplied Microbiology, Department of Chemistry, Lund University, Lund, SE-221 00, SwedenApplied Microbiology, Department of Chemistry, Lund University, Lund, SE-221 00, SwedenApplied Microbiology, Department of Chemistry, Lund University, Lund, SE-221 00, SwedenThe success of real-time PCR (qPCR) analysis is partly limited by the presence of inhibitory compounds in the nucleic acid samples. For example, humic acid (HA) from soil and aqueous sediment interferes with amplification and also quenches the fluorescence of double-stranded (ds) DNA binding dyes, thus hindering amplicon detection. We aimed to counteract the HA fluorescence quenching effect by blending complementary dsDNA binding dyes, thereby elevating the dye saturation levels and increasing the fluorescence signals. A blend of the four dyes EvaGreen, ResoLight, SYBR Green and SYTO9 gave significantly higher fluorescence intensities in the presence and absence of HA, compared with the dyes applied separately and two-dye blends. We propose blending of dyes as a generally applicable means for elevating qPCR fluorescence signals and thus enabling detection in the presence of quenching substances.http://www.sciencedirect.com/science/article/pii/S2215017X17300152Fluorescence quenchingHumic acidPCR inhibitionqPCRSoil |
| spellingShingle | Linda Jansson Marianne Koliana Maja Sidstedt Johannes Hedman Blending DNA binding dyes to improve detection in real-time PCR Biotechnology Reports Fluorescence quenching Humic acid PCR inhibition qPCR Soil |
| title | Blending DNA binding dyes to improve detection in real-time PCR |
| title_full | Blending DNA binding dyes to improve detection in real-time PCR |
| title_fullStr | Blending DNA binding dyes to improve detection in real-time PCR |
| title_full_unstemmed | Blending DNA binding dyes to improve detection in real-time PCR |
| title_short | Blending DNA binding dyes to improve detection in real-time PCR |
| title_sort | blending dna binding dyes to improve detection in real time pcr |
| topic | Fluorescence quenching Humic acid PCR inhibition qPCR Soil |
| url | http://www.sciencedirect.com/science/article/pii/S2215017X17300152 |
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