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Assay Design Tools for qPCR

IDT offers a group of qPCR design tools to suit your specific application needs. Whether you want to quickly order a predesigned assay, or desire assistance with designing assays for a challenging target, IDT has a design tool that can help. With three different programs to choose from, it is important to know which will provide the optimal qPCR assay for your needs.

Each of the three design programs requires only the NCBI RefSeq number, which can be used to pull the target sequence from NCBI. If you are working with unannotated sequences, the freely available RealTime and PrimerQuestSM programs can also design assays for a user-specified target.

Key Features of Assay Design Tools

PrimeTime® qPCR Program

  • Optimal design for human, mouse, and rat mRNA targets
  • Incorporates bioinformatics accounting for factors such as SNPs, BLAST searches to avoid cross-reaction and off-target amplification, and recognition of splice variants

RealTime PCR Program

  • Designs specifically for qPCR, with some customization possible
  • Optimal design for targets that are not human, mouse, or rat
  • Can be used to effectively design assays for use with intercalating dyes such as SYBR® Green I (Molecular Probes, Inc.)

PrimerQuestSM PCR Primer Design

  • Highly customizable option for difficult qPCR designs

PrimeTime® qPCR

The PrimeTime qPCR program is designed to specifically complement the PrimeTime Pre-designed qPCR Assays. If your target is human, mouse, or rat, this program offers the highest level of bioinformatics analysis, including avoidance of known SNPs, BLAST search to avoid cross reaction and off-target amplification, and recognition of splice variants. This program is regularly updated with current sequence information from NCBI, so it effectively designs assays with up-to-date bioinformatics. In this era of daily growth in genomic knowledge, having current sequence data is crucial.

Figure 1. The PrimeTime® qPCR Design Tool Instantly Provides Optimal Assays for Human, Mouse, or Rat.

RealTime PCR

The RealTime PCR program provides effective qPCR assay design through a user-friendly interface for targets other than human, mouse, and rat. This program allows for customization at many levels, including the ability to select the specific exon junctions to be targeted. For additional flexibility, the qPCR reaction conditions can be customized within this program, as well as the primer, probe, and amplicon parameters. If no customization is needed, this program can quickly draw on current NCBI data and recommend an assay for your qPCR target.

Figure 2. The RealTime PCR Program Allows for qPCR Design and Customization.

PrimerQuestSM

PrimerQuest is a useful tool for qPCR assay designs with non-standard requirements. This program is not specific for qPCR design like our other two programs, but if your design requires more demanding customization, this highly flexible program can be of great use. PrimerQuest designs can be customized in many ways, such as directing the assay towards certain areas of your target or by specifying primer or probe sequences. IDT Technical Support can also offer assistance with this program to help you meet your specific design challenges.

Figure 3. Some of the qPCR Parameters Customizable With the Highly Flexible PrimerQuestSM Program.

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