Blending DNA Binding Dyes to Improve Detection in Real-time PCR

Overview

Journal Biotechnol Rep (Amst)

Specialty Biotechnology

Date 2017 May 2

PMID 28459006

Citations 2

Authors

Linda Jansson

Marianne Koliana

Maja Sidstedt

Johannes Hedman

Affiliations

Soon will be listed here.

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 propose blending of dyes as a generally applicable means for elevating qPCR fluorescence signals and thus enabling detection in the presence of quenching substances.

Citing Articles

PCR inhibition in qPCR, dPCR and MPS-mechanisms and solutions.

Sidstedt M, Radstrom P, Hedman J Anal Bioanal Chem. 2020; 412(9):2009-2023.

PMID: 32052066 PMC: 7072044. DOI: 10.1007/s00216-020-02490-2.

Employing DNA binding dye to improve detection of Enterocytozoon hepatopenaei in real-time LAMP.

Ma B, Yu H, Fang J, Sun C, Zhang M Sci Rep. 2019; 9(1):15860.

PMID: 31676806 PMC: 6825238. DOI: 10.1038/s41598-019-52459-0.

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