SUN fluorophore characteristics
Fluorophore characteristics such as absorption and emission wavelengths and intensity of the emitted light are key to providing clear and accurate gene expression and genotyping experimental results. SUN fluorophore has peak excitation at 538 nm and peak emission at 554 nm, spectral characteristics that are similar to HEX and VIC dyes. In addition, most common qPCR instruments can read the emission without additional calibration. SUN dye is a new addition to the portfolio of Freedom™ Dyes available from IDT, providing you with a cost-effective, license-free alternative to VIC dye. See Freedom Dyes for more information.
Figure 1. SUN fluorophore molecular structure.
Table 1. Common fluorophore emission and excitation wavelengths.
|Texas Red® -X||598||617|
Yakima Yellow is a registered trademark of ELITechGroup; Texas Red-X is licensed from Molecular Probes, Inc., a wholly owned subsidiary of LifeTechnologies, Corp.; Cy is a registered trademark of Cytiva.
SUN probes provide strong signal intensity
Applications such as qPCR, multiplex qPCR, and digital PCR require oligonucleotide probes with strong emission spectra, especially for rare transcript detection. In qPCR assays containing primers specific to PGK1, the fluorescence signal for SUN probes after background subtraction (ΔRn) was comparable to or higher than the probes labeled with VIC or HEX dyes.
Figure 2. SUN double-quenched probes emit as much or more fluorescence than VIC- and HEX-labeled probes. qPCR assays using the same primer and probe sequences were designed to amplify PGK1 with 5 different probe configurations. The double-quenched probe, SUN/ZEN/IBFQ, provided the highest fluorescence signal, followed by the single-quenched SUN/IBFQ probe, which had comparable performance to the VIC/MGB-NFQ probe. The HEX-labeled probes had the lowest fluorescence intensity for this assay. qPCR amplification was performed using a 153 bp gBlocks™ Gene Fragment template. Each reaction was run in triplicate using PrimeTime™ Gene Expression Master Mix and data was generated using the QuantStudio™ 7 Flex Real-Time PCR System software (Thermo Fisher Scientific) with the following PCR conditions: 10 min 95°C, 40X (15 sec 95°C, 1 min 60°C).
SUN probes perform comparable to VIC probes in qPCR
SUN fluorophore is a molecular equivalent to VIC dye and provides comparable or better signal intensity compared to dyes with similar excitation and emission spectra. To investigate SUN fluorophore performance, qPCR was performed on 2 different target genes, GUSB and PGK1. The assays used identical primers, but to directly compare 5’ nuclease probes, one was labeled with a 5’ SUN dye and the other was labeled with a 5’ VIC fluorophore. The SUN-labeled probe had excellent signal intensity, performing comparably to VIC-labeled probes for both gene targets. In part A, the lower starting fluorescence value of the SUN labeled probe is primarily due to quencher configuration. When the background levels of fluorescence were subtracted (Figure 3, part B), the Cq values were equivalent, and the resulting standard curves for both probes had R2 values of 0.999.
These data clearly demonstrate that the 5’ SUN-labeled probe performs comparably in gene expression studies and can outperform VIC-labeled probes in fluorescent intensity.
Figure 3. PrimeTime SUN-labeled probes deliver comparable performance to VIC/MGB-NFQ probes. A double-quenched SUN/ZEN/IBFQ probe was compared to a VIC/MGB-NFQ probe in a qPCR assay for GUSB and PGK1. gBlocks Gene Fragments with sequences identical to GUSB and PGK1 were analyzed over 6 sequential 10-fold dilutions (102–107 copies). Normalized fluorescence values were plotted (A) including and (B) subtracting background fluorescence. Cq values were comparable, and the final R2 value for both standard curves equaled 0.999. Each reaction was run in triplicate using the IDT PrimeTime Gene Expression Master Mix, and data was generated using the QuantStudio™ 7 Flex Real-Time PCR System software (Thermo Fisher) with the following PCR cycling conditions: 10 min 95°C, 40X (15 sec 95°C, 1 min 60°C).
SUN probes are available in many different configurations
SUN probes can be purchased as individual PrimeTime qPCR probes or as part of PrimeTime qPCR Probe Assays. They are available as both double-quenched and single-quenched probes in a variety of different sizes and scales for gene expression studies. The probes can be ordered with mixed bases or locked nucleic acid bases, such as Affinity Plus monomers. Locked nucleic acids significantly increase Tm for shorter probe designs. Modulating the number of Affinity Plus bases in each probe increases the accuracy of allele detection in genotyping application. See Affinity Plus qPCR Probes for more information.
5’ SUN-labeled nuclease probes provide a cost-effective option for obtaining high-quality qPCR data from your gene expression and genotyping applications.