Blending DNA binding dyes to improve detection in real-time PCR
(2017) In Biotechnology Reports 14. p.34-37- Abstract
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... (More)
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.
(Less)
- author
- Jansson, Linda LU ; Koliana, Marianne ; Sidstedt, Maja LU and Hedman, Johannes LU
- organization
- publishing date
- 2017-03-01
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Fluorescence quenching, Humic acid, PCR inhibition, qPCR, Soil
- in
- Biotechnology Reports
- volume
- 14
- pages
- 4 pages
- publisher
- Elsevier
- external identifiers
-
- scopus:85017516455
- pmid:28459006
- ISSN
- 2215-017X
- DOI
- 10.1016/j.btre.2017.02.002
- language
- English
- LU publication?
- yes
- id
- f38b5c35-9803-4666-9f27-ccef8ce53819
- date added to LUP
- 2017-05-08 14:04:46
- date last changed
- 2024-04-14 10:09:23
@article{f38b5c35-9803-4666-9f27-ccef8ce53819,
abstract = {{<p>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.</p>}},
author = {{Jansson, Linda and Koliana, Marianne and Sidstedt, Maja and Hedman, Johannes}},
issn = {{2215-017X}},
keywords = {{Fluorescence quenching; Humic acid; PCR inhibition; qPCR; Soil}},
language = {{eng}},
month = {{03}},
pages = {{34--37}},
publisher = {{Elsevier}},
series = {{Biotechnology Reports}},
title = {{Blending DNA binding dyes to improve detection in real-time PCR}},
url = {{http://dx.doi.org/10.1016/j.btre.2017.02.002}},
doi = {{10.1016/j.btre.2017.02.002}},
volume = {{14}},
year = {{2017}},
}