一种用于 LightCycler 检测平行 DNA 三链体的新型 FRET 对。
A novel FRET pair for detection of parallel DNA triplexes by the LightCycler.
机构信息
QuantiBact Inc, Department of Clinical Microbiology, Hvidovre Hospital, Kettegaards Alle 30, 2650 Hvidovre, Denmark.
出版信息
BMC Biotechnol. 2010 Jan 27;10:4. doi: 10.1186/1472-6750-10-4.
BACKGROUND
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.
RESULTS
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 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.
CONCLUSIONS
We 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 DeltaTm and Tm determinations of pH dependent parallel triplex formation.
背景
可以在 LightCycler 上使用 FAM 的猝灭来确定 DNA 结构的熔点。该方法非常适合在碱性或中性 pH 下进行 pH 独立熔点 (Tm) 测定，是一种替代紫外吸光度测量的高通量方法。在酸性 pH 下，FAM 的猝灭效果不是很好，因为 FAM 的荧光强烈依赖于 pH，并且随着 pH 的降低而降低。基于 Hoogsteen 的平行三联体螺旋形成需要三联体形成链中的胞嘧啶质子化。因此，核酸三联体显示出强烈的 pH 依赖性，仅在酸性 pH 下稳定。这导致我们在 LightCycler 上建立了一种新的 pH 独立荧光团测量系统，用于研究平行三联体的热稳定性。
结果
建立了一种新的 LightCycler FRET 对，用 ATTO495 和 ATTO647N 标记，用于平行三联体检测，以反平行双链作为这些荧光团用于 Tm 测定的通用性的对照。ATTO 荧光团在 pH 4.5 到 7.5 之间稳定。三联体和双链结构的熔解伴随着荧光强度的大幅下降，导致明确的 Tm 和高重复性。Tm 的验证显示出低的内和间试验变异系数；平行三联体为 0.11%和 0.14%，反平行双链为 0.19%和 0.12%。随着时间和多次运行的 Tm 和荧光强度测量表明，ATTO 荧光团具有很好的时间和光稳定性。与紫外吸光度测量相比，LightCycler 平台上 Tm 测定的方差显著降低，这使得能够区分 Tm 非常相似的 DNA 结构。用 ATTO 荧光团标记 DNA 探针显著增加了反平行双链的 Tm，但不增加平行三联体的 Tm。
结论
我们在 LightCycler 平台上建立了一种新的 pH 独立的 FRET 对，用于反平行双链和平行三联体形成，具有高荧光信号。该方法已得到彻底验证，具有出色的准确性和可重复性。该 FRET 对特别适用于 pH 依赖性平行三联体形成的ΔTm 和 Tm 测定。
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