Advanced

Blending DNA binding dyes to improve detection in real-time PCR

Jansson, Linda LU ; Koliana, Marianne; Sidstedt, Maja LU and Hedman, Johannes LU (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)
Please use this url to cite or link to this publication:
author
organization
publishing date
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
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
2017-05-09 03:00:02
@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},
  keyword      = {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},
  volume       = {14},
  year         = {2017},
}