Highly sensitive products for digital PCR

Precise quantification of target nucleic acids

Product Spotlight: Digital PCR (dPCR) is a sensitive experimental technique to measure the amount of a target DNA or cDNA in a complex sample. Using primers and probes with low background is essential for digital PCR applications such as rare allele detection, liquid biopsy, and small sample sizes. Learn how IDT Real-Time PCR can be used in your dPCR applications.

Quantitative PCR (qPCR) can identify the relative or absolute amount of a target, but in some cases the number of target DNAs falls below the limit of detection even after optimizing the assay. In addition, quantitation of data in qPCR requires the establishment of standard curves or evaluation of internal reference genes, which takes extra time to develop. dPCR offers an alternative approach that can have a lower limit of detection, provide quantitative results without the need for standard curves, and, depending on the dPCR platform, be amenable to multiplexing.

A typical dPCR workflow has similarities to qPCR since the same target-specific reagents are used to set up the initial assay. The downstream steps of the workflow for dPCR differ from standard qPCR though. Digital PCR reactions are divided into nanoliter reactions either by creating small droplets in an oil emulsion or by partitioning the reaction into micro-molded, nanoliter-sized plastic compartments in a plate. In essence, the 20,000 or so partitions become individual qPCR reactions. After dilution and partitioning, the target DNA or cDNA is statistically only found either 1 or 0 times per partition, which is why it is referred to as "digital" PCR. 

After partitioning, if the target is present, it is amplified in the presence of an intercalating dye or 5’ nuclease probe. During amplification, the intercalating dyes or 5' fluorophores from the probe are activated to release the fluorescent signal. The partitions that do not contain a target DNA or cDNA do not produce any amplicons, so no fluorescence is created. Each partition is then analyzed at the end point of the reaction. The number of positive wells or droplets directly represents the total number of starting targets in the sample.

IDT offers the highest quality of oligos and probes to use for digital PCR applications whether you are using 5’ nuclease probes or intercalating dyes to assess the number of target DNAs in your sample.  

PrimeTime™ qPCR Primer Assays for intercalating dye dPCR

When using intercalating dyes such as SYBR® (Life Technologies) or EvaGreen® (Biotium) for dPCR applications, assessing the specificity of the reaction is essential. Intercalating dyes release fluorescence when they bind to any double-stranded DNA including non-specific amplicons and/or primer-dimers; therefore, poorly designed primer sets can result in skewed results. IDT PrimeTime qPCR Primer Assays include predesigned assays for human, mouse, or rat; or you can create your own custom assay for other species using the PrimerQuest™ Tool. In concordance with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, the primer sequences are provided in our predesigned assays [1]. The predesigned qPCR assays have been assessed to avoid off-target amplification, reduce secondary structures, and reduce primer-dimer formation. In addition, the same primer pair is used in the complementary predesigned PrimeTime qPCR Probe Assay, providing an economical alternative to evaluate the primers before purchasing the corresponding probe-based assay.

PrimeTime qPCR Probe Assays for dPCR

A common approach is to verify your experiment with an IDT PrimeTime Primer Assay and then transition to a 5’ nuclease probe assay to increase the specificity. IDT offers a variety of different fluorophore/quencher combinations in regular probes as well as probes and primer sets that are synthesized in GMP conditions for when you are ready to transition your assay from research to clinical diagnostics.

IDT also offers PrimeTime qPCR Probe Assays that include a primer pair and fluorescently-labeled 5’ nuclease probe, dried and shipped in either tubes or plates. These assays are available predesigned or custom with a variety of 5’ fluorophore/quencher combinations. Since background fluorescence can detract from properly assigning each partition as positive or negative, IDT offers ZEN™ and TAO™ Double-Quenched Probes with a variety of different 5’ fluorophores, such as FAM, SUN™ (an equivalent to VIC® from ThermoFisher), HEX, TET, and Cy® 5 (Cytiva). The internal ZEN or TAO quencher reduces the overall background, which increases the sensitivity of the reaction. For data comparing background fluorescence of ZEN and TAO Double-Quenched Probes to standard probes in regular qPCR assays, see PrimeTime qPCR Probes.

Affinity Plus™ qPCR Probes with locked nucleic acids for genotyping

Using dPCR for genotyping single nucleotide variations (SNVs) requires the use of two different probes with different fluorophores that are specific for each allele and forward and reverse primers. IDT offers Affinity Plus qPCR Probes with the addition of locked nucleic acids, which allows you to design shorter probes that will anneal to areas with high AT base-pair content or anneal to areas with closely spaced SNVs. Locked nucleic acids also have tighter affinity for their target, making it easier to discriminate between two or more alleles. When evaluating rare alleles, the shorter probes using Affinity Plus bases can be an asset to make clear genotyping calls.

IDT Affinity Plus qPCR Probes with different fluorophores were used to genotype a single locus of CYP2C19*17 (rs12248560), which is an SNV that can alter the metabolism of various drug compounds depending on the genotype. Patient populations will have either A, T, or C at the locus, and in order to demonstrate the multiplex capacity, a fourth G allele was added using synthetic gBlocks™ Gene Fragments. As shown in Figure 1, the 4 Affinity Plus qPCR Probes were used to perform single-plex dPCR reactions (column 1–4) and then in 4-plex (column 5). No loss of sensitivity was seen in the 4-plex reactions in comparison to the single-plex reactions. The sensitivity of Affinity Plus qPCR Probes allows single nucleotide differences to be distinguished for quad-allele genotyping experiments.

Quad-plex genotyping digital PCR results with locked nucleic acids
Four-color Affinity™ Plus qPCR Probe multiplex assay for the discrimination of a quad-allele SNV target on the Absolute Q™ dPCR instrument (Combinati). Four Affinity Plus qPCR Probes were used to interrogate each of the 4 possible alleles for CYP2C19*17 (rs12248560). For this study, gBlocks Gene Fragments containing each of the 4 target sequences were combined to create a pooled sample. Each assay was tested individually using the pooled control gBlocks fragments to demonstrate specificity (columns 1–4). The assays were then combined into a single-tube, 4-plex assay (column 5). The data for this assay clearly demonstrates that there is no loss of sensitivity in the 4-plex reaction in comparison to single-plex assays. Data courtesy of Combinati, Inc. 


For more information about IDT dPCR products, please download our brochure,

Probes and assays for digital PCR

 

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References

  1. Bustin SA, Benes V, et al. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem, 55(4):611–622.

Published Nov 20, 2020