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Up-to-Date Assay Design—Avoiding SNPs

PrimeTime® Pre-designed qPCR Assays

Researchers often design primers and probes for applications like RT-qPCR using sequence data from an NCBI database. However, these data do not always include all of the individual variation within a gene as the information that is available is constantly in flux. Since its inception in 1998, the list of single nucleotide polymorphisms (SNPs) in the RefSeq database at NCBI has grown to over 53 million human SNPs (Figure 1). In the human genome, SNPs are present in at least 1% of the population and occur on average once every few hundred bases. Keeping up to date with the continually increasing number of annotated SNPs is critical when designing qPCR assays, particularly when working with samples that may have different or unknown genetic backgrounds.

Number of SNPs in dbSNP

Figure 1. Increase in Known Single Nucleotide Polymorphisms (SNPs) Over Time. The rapidly increasing number of identified SNPs illustrates the advantages of assay design using current database information over premade assays that sit in storage waiting to be shipped. Updated April 2013

The Effects of SNPs on Oligonucleotide Binding and Melting Temperature

SNPs that occur in primer and probe binding sites can destabilize oligonucleotide binding. In many cases, a single SNP may not completely prevent PCR, but can often affect its efficiency. As seen in Figure 2, single mismatches can reduce the melting temperature of an oligonucleotide by several degrees Celsius. This can lead to an underestimation of the amount of gene expression in the SNP-containing sequences.

Figure 2. Single Mismatch Significantly Decreases Melting Temperature. Example shows how a single mismatch can affect melting temperature, affecting the efficiency of the PCR, and ultimately the interpretation of experimental results. This particular mismatch creates a non-standard base pairing that should not disrupt the helix. However, it substantially decreases melting temperature by over 8°C.

The Advantage of IDT Predesigned Libraries

Synthesis of PrimeTime Pre-designed qPCR Assays is performed using frequently updated information from new RefSeq releases from NCBI. Target regions are screened to avoid SNPs and sequences that are repeated elsewhere in the genome. Because each assay is synthesized only after it is ordered, there is never a stock of outdated assays that may omit SNPs only recently identified.

Are My Previously Ordered Assays OK?

Worried that your previously ordered assay ID may be out-of-date or unavailable? Compare your assay ID number to the current list at www.idtdna.com. The RefSeq version is a two digit number followed by the assay number (Figure 3). Changes in RefSeq builds do not necessarily affect every assay. If the assay number does not change between updates then the primer and probe sequences have not changed. Even if the RefSeq information has changed, the previously ordered assays can always be reordered by entering the assay ID or selecting it from your order history.

Figure 3. Understanding Your Assay ID Number.