Applied Biosystems design pipeline

Applied Biosystems uses a highly sophisticated oligonucleotide probe/primer design pipeline. We accomplished this by developing robust primer design algorithms, and an extensive array of bioinformatics tools and processes to automate assay design. The pipeline also integrates design details with the manufacturing process and assay quality control.

Although the pre-designed Applied Biosystems TaqMan Gene Expression Assays are widely used as determined by their frequent citations in scientific journal articles, some researchers still need to design their own assays. If a pre-designed assay is not available, consider using the Custom Assay Design Tool on the Thermofisher website to take advantage of the proprietary design algorithm and rigorous bioinformatic interrogations.

Below is a discussion  of considerations you should take into account when you design your own assay.

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Evaluation of target sequences

Oligonucleotide probe and primer (assay) design is a critical element in the experimental design process for any real-time qPCR experiment. If you choose to design an assay on your own, consider the following bioinformatics criteria that should be addressed for the successful design of a primer/probe set. 

  1. The key feature in assay design is specificity of the assay for the transcript of interest.  It is important to ensure that the fluorescent signal being detected during real time PCR is specific to the target transcript, and that there is no contribution from homologous sequences that might complicate the interpretation of the quantitative data. High target specificity is assured by comparing the sequence of the designed probe and primers to other sequences in the transcriptome from which the gene is transcribed.  Ensure sequence uniqueness with a BLAT® or BLAST® database search.  The transcript specificity problem can be difficult to tackle because of high homology between closely related genes, alternative splicing within a single gene, and the potential presence of transcribed pseudogenes. We have been able to design highly robust assays to deal with many of these specificity issues using our bioinformatics design pipeline, and continue to refine our design algorithms to tackle even the most challenging design problems.

  2. For gene expression assays, it is also important to ensure that the assay is specific for detecting cDNA and not gDNA (genomic DNA). RNA samples can often be contaminated with significant amounts of gDNA, depending on the RNA purification methods utilized. The genomic DNA contamination problem can be solved with high-quality RNA purification chemistries, use of highly active DNase and rigorous quality control of the prepared RNA samples.  During the qPCR experiment it is recommended to run a “No RT” (Reverse Transcriptase) control to determine if any of the signal is a result of gDNA contamination. When possible, TaqMan assays for gene expression should be designed across exon-exon boundaries, and thus the position of the exon boundaries within a multi-exon transcript must be determined prior to the design of the assay.   If possible, place the probe, rather than one of the primers, over the exon-exon boundary to ensure that the primers bind in two distinct exons. Placing the probe over the exon-exon boundary ensures that the fluorescent signal is only generated from templates that have correctly spliced exons.

  3. An assay should be designed in a region of unambiguous sequence that does not contain any known single nucleotide polymorphisms (SNPs) or repeat sequences.

For more information on selecting the amplicon location and ensuring specificity see Appendix B Bioinformatics tools for manually evaluating target sequences from the Custom TaqMan Assays Design and Ordering Guide.

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Primer and probe design

Once a unique “location” on the transcript of interest has been decided, the next step in creating your own TaqMan gene expression assay is to design primers and a probe.

You can do this using Applied Biosystems’ Primer Express® software. This will help you to design primers and probes based on the sequence of interest, while considering important parameters, such as melting temperature (Tm) and GC content. The software is optimized for use with TaqMan reagents and universal thermal cycling conditions. Default variables have already been standardized, thus streamlining the process.

When designing the assay, put the forward and reverse primers as close as possible to the probe while avoiding overlap. Amplicons should be kept short to ensure doubling at each PCR cycle.  Amplicons of 50–150 base pairs are recommended to promote efficient amplification. The amplicon should also span at least one non-coding sequence (intron) to prevent amplification of the target gene in genomic DNA.

G/C content is an important consideration when designing primers. Here, the last five nucleotides on the 3’ end should have no more than two G and/or C nucleotides, as this could lead to non-specific product formation. The optimal primer length is 20 bases and Tm should be kept at 58–60°C (10°C lower than that of the probe, enabling the use of universal thermal cycling parameters).

For both primers and probe, keep G/C content at 30–80%, and avoid runs of four or more G nucleotides to ensure efficient amplification.

The primers should be specific to the target gene and, if you synthesized several primer sets, you should always select the pair of primers that generates the highest signal-to-noise ratio (with no amplification of genomic DNA in the case of gene expression assays).

Once a unique “location” on the transcript of interest has been decided, the next step in creating your own TaqMan gene expression assay is to design primers and a probe.

You can do this using Applied Biosystems’ Primer Express® software. This will help you to design primers and probes based on the sequence of interest, while considering important parameters, such as melting temperature (Tm) and GC content. The software is optimized for use with TaqMan reagents and universal thermal cycling conditions. Default variables have already been standardized, thus streamlining the process.

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Selecting reagent concentrations

For most TaqMan gene expression assays, when DNA or cDNA is used as the template, a concentration of 900 nM for primers and 250 nM for the probe should provide a highly sensitive assay.

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What to do next

If designing a primer/probe assay set is not where you want to spend your time (and hope you get it right) order a predesigned TaqMan Gene Expression Assay or let us design one for you.  Use the Applied Biosystems' Custom Assay Design Tool for designing Custom TaqMan Assays and select the Custom Plus option, which offers bioinformatics analysis of target sequences, in silico quality control step, and specificity for either gene- or transcript-level detection.  If you do not have an input sequence, the assay design tool will help you to search for sequences by gene symbol or location in the genome, e.g. across a range of model species. Once designed, you can reorder a custom assay at your own convenience. All assay sequences remain entirely confidential.

Using proprietary algorithms, the Custom Plus option performs thorough checks on your sequences—such as optimal Tm requirements, GC content, buffer and salt conditions, oligonucleotide concentrations, secondary structure formation and amplicon size—to ensure the assay is successful. The in silico QC pipeline also removes designs that are not highly specific to the gene of interest or that might detect homologous genes or pseudogenes.

Applied Biosystems’ assay design guidelines provide a reliable procedure for designing your own assay, and must be followed completely for the best results.

Through our extensive experience with probe and primer design of TaqMan assays for quantitative RT-PCR, we have empirically determined the parameters useful for selecting oligonucleotide sequences that are most likely to result in successful, functional assays.  Before designing your own assay, we recommend that you check whether there is a predesigned assay that meets your needs using our comprehensive Assay Search Tool.  Our aim is to provide the most robust quantitative assays that will fit the requirements of the entire spectrum of sample types and sample preparation methodologies utilized by the broad range of users of a particular assay.