A novel FRET pair for detection of parallel DNA triplexes by the LightCycler.

BMC Biotechnology
Uffe V SchneiderGorm Lisby

Abstract

Melting temperature of DNA structures can be determined on the LightCycler using quenching of FAM. This method is very suitable for pH independent melting point (Tm) determination performed at basic or neutral pH, as a high throughput alternative to UV absorbance measurements. At acidic pH quenching of FAM is not very suitable, since the fluorescence of FAM is strongly pH dependent and drops with acidic pH.Hoogsteen based parallel triplex helix formation requires protonation of cytosines in the triplex forming strand. Therefore, nucleic acid triplexes show strong pH dependence and are stable only at acidic pH. This led us to establish a new pH independent fluorophore based measuring system on the LightCycler for thermal stability studies of parallel triplexes. A novel LightCycler FRET pair labelled with ATTO495 and ATTO647N was established for parallel triplex detection with antiparallel duplex as a control for the general applicability of these fluorophores for Tm determination. The ATTO fluorophores were pH stable from pH 4.5 to 7.5. Melting of triplex and duplex structures were accompanied by a large decrease in fluorescence intensity leading to well defined Tm and high reproducibility. Validation of Tm showed low intra- and...Continue Reading

References

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Citations

Mar 1, 2013·Artificial DNA, PNA & XNA·Shuji IkedaAkimitsu Okamoto
Mar 9, 2011·Biopolymers·Yong YouRichard Owczarzy
Apr 6, 2019·Nucleic Acids Research·Imee M A Del MundoKaren M Vasquez
Sep 7, 2019·Biochimica Et Biophysica Acta. Molecular Cell Research·Imee M A Del MundoGuliang Wang

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Methods Mentioned

BETA
gene knock-out
FRET
PCR
fluorescence resonance
Fluorescence

Software Mentioned

SAS
LightCycler

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