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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.
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.
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.
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.
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.
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.
仅供科研使用，不可用于诊断目的。