Interaction of fluorescent dyes with DNA and spermine using fluorescence spectroscopy

K. Gracie, W. E. Smith, P. Yip, J. U. Sutter, D. J S Birch, D. Graham, K. Faulds

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Oligonucleotides labelled with fluorescent dyes are widely used as probes for the identification of DNA sequences in detection methods using optical spectroscopies such as fluorescence and surface enhanced Raman scattering (SERS). Spermine is widely used in surface enhanced based assays as a charge reduction and aggregating agent as it interacts strongly with the phosphate backbone and has shown to enhance the signal of a labelled oligonucleotide. The fluorescence intensity of two commonly used labels, FAM and TAMRA, were compared when spermine was added under different experimental conditions. There was a marked difference upon conjugating the free dye to an oligonucleotide, when FAM was conjugated to an oligonucleotide there was around a six fold decrease in emission, compared to a six fold increase when TAMRA was conjugated to an oligonucleotide. Dye labelled single and double stranded DNA also behaved differently with double stranded DNA labelled with FAM being a much more efficient emitter in the mid pH range, however TAMRA becomes increasingly less efficient as the pH rises. Upon addition of the base spermine, signal enhancement from the FAM labelled oligonucleotide is observed. Increasing probe concentrations of TAMRA oligonucleotide above 0.5 μM led to signal reduction most likely through quenching, either by an interaction with guanine, or through self-quenching. By using different bases for comparison, spermine and triethylamine (TEA), different affects were observed in the measured fluorescence signals. When TEA was added to FAM, a reduction in the pH dependence of fluorescence was observed, which may be useful for mid pH range assays. With the drive to increase information content and decrease time and complexity of DNA assays it is likely that more assays will be carried out in complex media such as extracted DNA fragments and PCR product. This model study indicates that dye DNA and dye spermine interactions are dye specific and that extreme care with conditions is necessary particularly if it is intended to determine the concentrations of multiple analytes using probes labelled with different dyes. This journal is

LanguageEnglish
Pages3735-3743
Number of pages9
JournalAnalyst
Volume139
Issue number15
Early online date22 May 2014
DOIs
Publication statusPublished - 7 Aug 2014

Fingerprint

Spermine
fluorescence spectroscopy
Oligonucleotides
Fluorescence Spectrometry
Fluorescence spectroscopy
Fluorescent Dyes
dye
DNA
Dyes
Coloring Agents
Assays
fluorescence
Fluorescence
assay
probe
Quenching
fold
Raman Spectrum Analysis
Single-Stranded DNA
DNA sequences

Keywords

  • interaction
  • fluorescent dyes
  • DNA
  • spermine
  • fluorescence spectroscopy

Cite this

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Interaction of fluorescent dyes with DNA and spermine using fluorescence spectroscopy. / Gracie, K.; Smith, W. E.; Yip, P.; Sutter, J. U.; Birch, D. J S; Graham, D.; Faulds, K.

In: Analyst, Vol. 139, No. 15, 07.08.2014, p. 3735-3743.

Research output: Contribution to journalArticle

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