PCR and qPCR
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Design efficient PCR and qPCR primers and probes using online tools

We recommend 2 tools for choosing qPCR primers and probes (see the sidebar, Highlights of the IDT Primer Selection Tools).

If you are working with human, mouse, or rat targets, IDT has already designed qPCR assays across these transcriptomes. We use a sophisticated design engine that screens these sequences for the best possible assays and eliminates the need for you to design and analyze primer and probe sequences. Select from this collection of assays using the PrimeTime® Predesigned qPCR Assay Database.

If the predesigned human, mouse, or rat assays are not suitable for your project, we recommend using the PrimerQuest® Tool. This tool provides the most flexibility in customizing assay designs for amplification of any nucleic acid sequence. One of the most frequently used IDT programs, the PrimerQuest Tool is powerful because its design engine incorporates calculations from our cutting-edge thermodynamics research, and because it allows you to customize approximately 45 different parameters, ranging from primer, probe, or amplicon criteria to target regions.

Highlights of IDT primer selection tools

PrimeTime® Predesigned qPCR Assay Database
Select primers/probes for qPCR (human, mouse, rat transcriptome)

  • Assays are guaranteed to have >90% qPCR efficiency
  • Assay selection incorporates bioinformatic calculations that manage factors such as:
    • Cross-react searches to avoid off-target amplification
    • Recognition of splice variants
    • Secondary structure predictions
  • Orders include primers and probe sequences

PrimerQuest® Tool
Design primers or assays for PCR, qPCR, or sequencing (any species)

  • Software allows customization of ~45 parameters, which can produce qPCR assay designs:
    • With specific primer, probe, or amplicon criteria
    • Across a specified location
    • Around a fixed primer or probe location
  • Design algorithm includes multiple checks to reduce primer-dimer formation
  • Software provides flexible sequence entry and batch entries (up to 50 sequences)

To view Selected Citations from peer-reviewed journals for each of these tools, visit www.idtdna.com/PrimeTime.

The PrimerQuest Tool in 4 steps

The PrimerQuest Tool is the program of choice for designing qPCR primers/probes, sequencing oligonucleotides, and custom primers. Here is how to select the best PCR/qPCR assays for your experiments:

Step 1: Enter sequence(s)

The PrimerQuest Tool can perform batch analyses with up to 50 sequences that are each longer than 80 bases. You have 3 options for submitting your target sequence(s):

  • Enter manually using FASTA format
  • Download using Genbank or Accession ID
  • Upload in an Excel® file

Step 2: Choose your design

The PrimerQuest Tool offers 4 design options that are based on algorithms specific for common experimental setups (Figure 1). By default, your results return the 5 best primer or assay designs.

  • PCR (2 primers)
  • qPCR (2 primers + probe; for use in 5′ nuclease assays)
  • qPCR (2 primers; for use with intercalating dyes)
  • Custom (see Step 3 for additional information)
PrimerQuest Tool Design Options

Figure 1. Design buttons in the PrimerQuest® Tool.

Step 3 (Optional): Customize your design parameters

Selecting Show Custom Design Parameters allows you to adjust reaction conditions (e.g., primer, Na+, and Mg2+ concentrations), force use of a particular primer or probe sequence, and/or specify design regions. Alternatively, if you originally chose PCR or qPCR designs, you may click on Customize Assay Design from your Results page to adjust design parameters (Figure 2).

This step provides an overview of how to customize your primer designs:

  1. Select your basic Design Parameters.

    As mentioned above, default settings for some parameters vary depending on the requirements of different applications.

    Hint: Use these radio buttons to view the default parameter settings for different designs and to compare IDT’s recommended baseline settings for different applications.

  2. Customize at least 1 criterion (Table 1) used to design your primers and probe.

    Hints: Click on any criterion name to open a window with a definition of the criterion.
    If needed, select a Design Parameter radio button again to reset all the criteria back to default settings.

    Criteria Description
    Results—Results to Return The number of assay designs to view after clicking the Get Assays button (default: 5)
    Note: You cannot vary this parameter when doing a batch analysis.
    Reaction Conditions—Divalent Salt (Mg2+) (mM) The millimolar concentration of divalent salt cations (usually MgCl2) in the reaction (range: 0–600 mM)
    Primer Criteria—Primer Tm (°C) Minimum, optimum, and maximum melting temperature for a primer oligo (range 45–75°C)
    Primer Criteria—Primer GC (%) Minimum, optimum, and maximum percentage of G and C bases in the primer (range: 20–80%)
    Amplicon Criteria—Amplicon Size (bp) The minimum, optimum, and maximum size for the desired amplicon

    Table 1. Overview of custom design parameters. This table contains descriptions of the most commonly customized parameters. Some criteria will only be visible for relevant Design Parameters. For example, probe parameters are only visible when designing oligos for qPCR with 2 primers and a probe.

  3. Click the Get Assays button at the bottom of the web page.
  4. If no assays are found, click on adjust parameters to modify criteria based on the information on the results page, and then click the Get Assays button again.

Critical primer design parameters

There are ~45 customizable design parameters in the PrimerQuest® Tool. Additional, fixed parameters exist to ensure the best primer or assay designs are generated. Here are short descriptions of a few important primer design parameters that are not variable:

  • Poly-base runs are restricted to 3 consecutive, repeat bases or less to avoid polymerase slippage during primer extension.
  • The difference in the Tm of the forward and reverse primers is always ≤3° to optimize reaction efficiencies.
  • Probes cannot have a G base at the 5′ end, because G bases partially quench most green/yellow dyes.

Step 4: Select and order assays

A schematic of your sequence is near the top of each Results page with the location of the assays (i.e., amplicons) depicted by green bars (Figure 2). The assay sets are listed below the schematic with the best assays at the top of the list. The list also includes important details about the primers or primers/probe, which can be downloaded as an Excel file, if you are logged into your IDT account. Clicking on View Assay Details provides your submitted sequence along with highlights for the location of the primers or primers/probe.

If you are doing a batch analysis, you will find a dropdown menu to view the top 5 assays for each sequence (Figure 2B).

PrimerQuest Results Page (1 seq)

A. Results page for single sequence design.

PrimerQuest Results Page (Batch)

B. Results page for batch sequence design.

Figure 2. Representative results from the PrimerQuest® Tool.

Use the following steps to select your assays:

  1. (Recommended) Test primer sequences for cross reactivity with other known sequences using NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
  2. Use one of the following methods to select assays to add to your cart:
    1. Click on the box(es) next to the set number(s) that you wish to order.
    2. Click on the green assay bar(s) in the schematic.
    3. For batch analyses, click on the Select Top Result for All Sets box above the schematic.
  3. Click the Add Selected Assays to Cart button.
  4. Choose options (e.g., synthesis scale, purification options, fluorescent dyes, and quenchers) as needed, and follow screens to checkout.

More complex designs?

The PrimerQuest Tool makes it easy to design basic and highly customized primers and probes for PCR and qPCR. Now that you understand the fundamentals of using this design tool, look for future articles from IDT about experiments requiring more complex assay design considerations, such as splice-specific, multiplexing, SNPs, and CNV designs.

If you need additional assistance with designing primers, contact our technical support scientists at applicationsupport@idtdna.com.

Product focus: qPCR Reagents—everything but your sample

All the reagents you need for successful qPCR assays are available through IDT.

Related reading

Exon numbering—not as easy as 1, 2, 3…
Exon numbering and location data can differ across various software tools, including with NCBI's gene database. For example, exons within alternatively spliced transcripts are sometimes individually numbered, with no consistent gene-based numbering system across these transcripts for identifying exons. Here, we describe how IDT exon location information gives each exon a unique number and how that compares with the NCBI naming system.

Tips for using BLAST to locate PCR primers
Need a quick way to find the location of primers within a gene or the expected size of the resultant PCR product? In this tip, we show you how to get this information using BLAST.

DECODED 4.2—Special qPCR Issue
An entire DECODED issue dedicated to qPCR topics, including primer and probe design, handling primer and probe oligonucleotides, qPCR using intercalating dyes, multiplex qPCR, and more.

Additional resources: Videos

This is PrimerQuest
This video provides a quick overview of the PrimerQuest Tool.

When you need to customize PCR and qPCR primer designs
Watch this webinar to see a presentation about using the live PrimerQuest® Tool to design custom qPCR assays. The first half of this talk covers the basic design process, while the second half covers questions and advanced design scenarios.

Go to the IDT Video Library to learn more about the PrimerQuest Tool and Real-time PCR Tool.

Author: Maureen Young, PhD, is a senior scientific writer at IDT.

© 2015, 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.

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