It’s easy to design a custom TaqMan single nucleotide polymorphism or SNP Assay using the tools online, but we often get users asking us, “Do we need to do anything to our target sequence before submitting it into the Custom TaqMan Assay Design Tool?”
Great question! Let’s find out.
You can definitely analyze and mask your target sequence to improve the likelihood of obtaining a highly-specific and well performing assay design. We call this “preparing your target sequence.” You should always prepare your target before entering it into the Custom TaqMan Assay Design Tool, which is the tool that designs the SNP assay itself.
Let’s take a look at our lab book.
First, we recommend masking non-target SNPs in the sequence that you are submitting. Masking is the process of substituting an “N” for an existing base. The custom SNP assay design pipeline will not design a primer or probe that spans a masked base. The pipeline will move upstream or downstream to another area of the sequence for assay design. Why is it important to avoid designing an assay over a SNP that is not your intended target? Assays that have probes or primers overlapping a SNP will have inefficient hybridization when the polymorphism is present, potentially producing an inaccurate genotyping call. We definitely want to avoid that!
The other important thing to mask in your target sequence is repeats. Repeats are patterns of DNA sequence that occur in multiple copies throughout the genome. Why do we want to avoid designing to non-unique regions of the genome? Assays that are designed in regions that contain repeats are likely to produce non-specific amplification. Remember, we want our assay to detect our SNP in our unique region of the genome- not bind to other areas of the genome!
The last step in preparing your genome is checking whether your target sequence is unique in your organism of study. We call this “genome QC”. Using public databases and tools, such as UCSC BLAT or NCBI BLAST, you can check whether there are any regions of your target sequence that are similar to sequences in the reference database for that particular organism. If you find any sequences that are similar, you should mask those regions with Ns. This will ensure that your assay is only detecting your target region containing your SNP of interest. You do not want to detect other regions in the genome.
Thanks for asking TaqMan about target sequence preparation! By properly preparing your target sequence, you can enhance the quality of your custom TaqMan SNP assay performance.
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Submit your question at https://www.thermofisher.com/ask