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

Research output: Contribution to journalArticle

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.

Details

Authors
Organisations
External organisations
  • Swedish National Forensic Center
  • Lund University
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Medical Laboratory and Measurements Technologies

Keywords

  • Fluorescence quenching, Humic acid, PCR inhibition, qPCR, Soil
Original languageEnglish
Pages (from-to)34-37
Number of pages4
JournalBiotechnology Reports
Volume14
Publication statusPublished - 2017 Mar 1
Publication categoryResearch
Peer-reviewedYes