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SYBR® green dye and PrimeTime™ probe qPCR assays

Overview

qPCR for gene expression analysis can be done with primers and intercalating dyes such as SYBR® Green (Thermo Fisher) or with primers and fluorescent probes such as PrimeTime™ qPCR probes. Understanding the key benefits of each method will help you design the best assay for your research experiment.

pipette tips used in genotyping analysis

Basics of quantitative PCR (qPCR)

Quantitative PCR, also known as real-time PCR, is a typical PCR reaction that amplifies a target sequence from a sample of DNA or RNA, but instead of assaying the amount of PCR product as an end-point value, qPCR assays monitor and measure the amount of PCR product produced in each cycle of amplification based on a fluorescent signal. The fluorescence is produced using intercalating dyes, such as SYBR® Green dye (Thermo Fisher) or using 5’ nuclease probes such as PrimeTime™ qPCR Probes. The accumulation of a PCR amplicon in each cycle is plotted on a graph where the x-axis has the cycle number, and the y-axis plots the relative fluorescence, a measure of the amount of PCR amplicons after subtracting any background fluorescence. qPCR reactions can be coupled with reverse transcriptase (RT) to quantify the abundance of an RNA transcript, which is sometimes referred to as RT-qPCR, qRT-PCR, or real-time RT-PCR. These assays are used to assess gene expression, a key measure for understanding biological systems, including development, splice variant-specific gene expression, or disease progression. Real-time PCR can also be used in genotyping applications, such as identifying copy number variations, determining frequency of SNPs in populations, multi-species variations, or association studies to determine genes relevant to specific traits or diseases.

Intercalating dye or primer assays

qPCR assay with intercalating SYBR green dye and primers
Figure 1. PrimeTime™ qPCR primer assay. Primer pairs (blue arrows) designed to amplify the target sequence (gray) anneal to the complementary regions at the beginning and end of the target. As DNA polymerase (green arrow) extends from the primer to turn the single-stranded target into double-stranded DNA, an intercalating dye such as SYBR® green (Thermo Fisher) attaches to the double-stranded regions and releases its fluorescent signal. The intensity of fluorescence correlates with the amount of double-stranded DNA.

The number of amplicons created during PCR amplification can be measured using intercalating dyes such as SYBR® Green (Thermo Fisher), SYTO® (Life Technologies), EvaGreen® (Biotium), and LCGreen® (Idaho Technology, Inc.). These are sometimes called primer-only assays (Figure 1). These dyes do not fluoresce until they bind to double-stranded DNA, and the amount of fluorescence is proportional to the relative amount of PCR amplicons present after extension.

Although intercalating dyes are inexpensive, there can be drawbacks to their use. If the reaction has issues with mispriming of non-specific sites, cross-reacting genes, or the accumulation of primer-dimer products, intercalating dyes will bind to the incorrect double-stranded DNA along with any actual desired PCR amplicon. It is important to confirm that the PCR reaction produces only one single PCR product, which can be done by melt curve analysis and gel electrophoresis. Another drawback is that the dyes are non-specific, so there is no way to multiplex the assays, a key for genotyping applications.

For more information on melt curve analysis, see the DECODED™ online article, Explaining multiple peaks in qPCR melt curve analysis. Another important resource for understanding whether your qPCR amplicon includes non-specific sequences is the free online uMeltSM software (University of Utah) that predicts melt curves and their derivatives.

5’ nuclease or probe-based assays

The second and more specific method of quantifying the accumulation of the PCR amplicons is called a probe-based or 5’ nuclease assay, which is also known as a PrimeTime™ or TaqMan® assay (Thermo Fisher) (Figure 2). These assays include a fluorescently labeled oligonucleotide probe in addition to the forward and reverse PCR primers. The combination of probe and primers increases the specificity for the amplification reaction since all three must bind to the correct target for fluorescence signal to be generated. In addition, probe-based assays allow users to combine multiple primer and probe sets in one reaction. These multiplex qPCR assays include unique 5′ fluorophores for each of the probes, and therefore, the wavelength of fluorescence signal indicates which transcript is present and its relative abundance. For more information on selecting the different dyes for multiplex assays, see Recommended dye combinations for multiplex qPCR. Of course, multiplex PCR assays need to have real-time or qPCR instruments and software capable of recognizing the different fluorophores. IDT has a Multiplex Dye Selection Tool that offers guidance on what fluorophores are recommended for use with many qPCR instruments, and also alternative fluorophores.

PrimeTime qPCR probes are 5' nuclease probes with a 5′ fluorophore and a 3′ quencher that are designed to anneal between the two PCR primers. Since the quencher and fluorophore are in proximity, no fluorescence is recorded. During annealing, the probe and PCR primers attach to their complementary sequences in the target region. During extension/elongation, Taq DNA polymerase uses its exonuclease activity to hydrolyze or degrade the probe. Once hydrolyzed, the 5’ fluorophore is released from the quencher. Without a quencher in proximity, the fluorescence is recordable by the qPCR instrument. The amount of fluorescence correlates with the amount of PCR products that are synthesized.

It is important that probe length is short so the quencher is close enough to absorb the fluorescence energy. Longer probes will have high background fluorescence since the quencher is less effective. For more information on strategies to design qPCR probes with low background, see How to design primers and probes for qPCR and PCR, Double-quenched probes increase qPCR sensitivity and precision, and Improve assays with customizable oligos and probes containing cost-effective locked nucleic acids.

qPCR assay with primers and probe with fluorophore and quencher
Figure 2. PrimeTime™ qPCR Probe Assays. In addition to the primer pair (blue arrows), these assays include a 5′ nuclease probe with a dye on the 5′ end (orange) and quencher on the 3′ end (dark blue). During extension or elongation, the exonuclease activity of DNA polymerase (green arrow) degrades the probe, which releases the fluorophore from the quencher. Once separated, the real-time PCR system records the intensity of the fluorescence, which correlates with the amount of PCR amplicons created in each cycle of amplification.

Converting primer-only assays to probe-based assays

SYBR Green and other types of intercalating dyes are more cost-effective for the researcher than 5′ nuclease probe assays but can pose issues if the primers form primer-dimers, bind to non-specific sites, or anneal to cross-reacting genes. PrimeTime qPCR Primer Assays include predesigned assays for human, mouse, or rat. These assays have been designed with advanced bioinformatics to optimize the assay to avoid these issues. The primer pair is designed and premixed for real-time PCR, which makes the reaction setup easy. In addition, the primer sequences are identical to a coordinating PrimeTime qPCR Probe Assays, which means you can easily transition from primer assays to 5′ nuclease assays to increase the assay specificity or establish a new multiplex assay. For more information on converting assay design, see SYBR to 5’ nuclease assays.

 

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Real-time qPCR guide: Design, validation, analysis, and troubleshooting

Familiarize yourself with critical parameters of qPCR assay design. This complete 62-page guide covers every aspect of probe-based qPCR assays.

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qPCR applications

Gene expression

Gene expression is the conversion of heritable, genetic information into RNA or protein. Studying differences and changes in gene expression are important measures for understanding biological systems, including normal development and disease progression.

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Genotyping

Genotyping is the process of determining the DNA sequence—the genotype—at specific positions within a gene of an individual. Sequence variations can be used as markers in linkage and association studies to determine genes relevant to specific traits or disease.

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Digital PCR

Digital PCR (dPCR) includes the same reagents found in a typical qPCR assay and amplifies the target in a similar manner, but dPCR divides the reaction into nano-sized droplets or wells prior to amplification. The partitions are so small that either 1 or 0 templates are in each partition. After amplification, the number of fluorescent wells or droplets represents the total number of templates found in the original sample. 

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Products for qPCR

PrimeTime qPCR Probe and Primer Assays

PrimeTime qPCR Probe Assays are 5′ nuclease assays that include a library of predesigned assays for human, mouse, and rat sequences. Custom assays may be created for these or other species using the PrimerQuest Tool. Predesigned and custom assays are available in tubes or plates.

PrimeTime qPCR Primer Assays for use with intercalating dyes, such as SYBR® Green dyes, include predesigned assays for human, mouse, or rat sequences. Custom assays may be created for these and other species using the PrimerQuest Tool. Predesigned and custom assays are available in tubes or plates.

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Affinity Plus qPCR Probes

Use Affinity Plus™ qPCR Probes for SNP genotyping, transcript variant identification, and more sensitive target detection in challenging samples (FFPE tissue, biofluids). The Affinity Plus bases used in these qPCR probes are locked nucleic acid monomers. When incorporated into a probe, they impart heightened structural stability, leading to increased hybridization melt temperature (Tm).

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rhAmpSeq amplicon sequencing system

The rhAmpSeq™ system enables highly accurate amplicon sequencing on Illumina® next generation sequencing (NGS) platforms. Whether you are investigating thousands of targets or a few, the fast and easy rhAmpSeq workflow generates NGS-ready amplicon libraries for deep, targeted resequencing.

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