Abstract
BackgroundMelting 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.ResultsA 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 inter-assay coefficient of variation; 0.11% and 0.14% for parallel triplex and 0.19% and 0.12% for antiparallel duplex. Measurements of Tm and fluorescence intensity over time and multiple runs showed great time and light stability of the ATTO fluorophores. The variance on Tm determinations was significant lower on the LightCycler platform compared to UV absorbance measurements, which enable discrimination of DNA structures with very similar Tm. Labelling of DNA probes with ATTO fluorophore increased Tm of antiparallel duplexes significantly, but not Tm of parallel triplexes.ConclusionsWe have established a novel pH independent FRET pair with high fluorescence signals on the LightCycler platform for both antiparallel duplex and parallel triplex formation. The method has been thoroughly validated, and is characterized by an excellent accuracy and reproducibility. This FRET pair is especially suitable for ΔTm and Tm determinations of pH dependent parallel triplex formation.
Highlights
Melting temperature of DNA structures can be determined on the LightCycler using quenching of FAM
We describe a novel pH-independent fluorescence resonance energy transfer (FRET)-based ATTO fluorophore pair using non-linked oligonucleotides to study the thermal stability of Watson-Crickbased DNA antiparallel duplex and Hoogsteen-based DNA parallel triplex
Antiparallel duplex The ATTO495-ATTO647N FRET pair designed for Tm determinations of antiparallel duplex formation was pH independent from pH 5.5 to 7.5 (Figure 3a), whereas the fluorescence level of the FAM-Cy5 FRET pair changed greatly with pH (Figure 3b)
Summary
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. 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. The stability of mixed probes kept at 4°C and used for melting point determination was evaluated using 36 independent capillaries and running six new capillaries after 0, 4, 24, 48, 120 and 192 hours. Evaluation of fluorophore stability over time and after multiple runs was conducted using 24 independent capillaries leaving 12 capillaries at 4°C and 12 capillaries at room temperature and rerunning all capillaries after four hours, 1, 2, 5, 8, 20, 28, 57 and 141 days
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
