Problematic PCR? Then you’ve come to the right place! Below are some troubleshooting tips.
SNPs—remind me what they are
In case you’re not familiar with them, single nucleotide polymorphisms (SNPs) are where single base positions within a stretch of DNA have mutated. They are the single most common type of genetic variation in humans, and the number of annotated SNPs across many species is quickly increasing. SNPs can occur within coding or noncoding regions, and their effects can be variable. So when present within coding sequence, they will not necessarily impact the resulting protein. Conversely, when SNPs occur in noncoding regions, they can still impact gene expression by altering gene splicing, transcription factor binding, or mRNA degradation.
Watching out for SNPs
With the large number of SNPs identified, it is now common when running a PCR to have primers or probes that overlie SNP sites. Such situations can influence primer and probe Tm, polymerase extension, and target specificity. Essentially, the presence of SNPs that are unaccounted for can result in inaccurate and unreliable data.
Thus, the importance of knowing whether your assay designs overlie SNPs has increased in-line with the large number of SNPs being identified. So, you need to safeguard your experiments by always double-checking that your primer/probe designs are free from SNPs:
- If the ‘rs’ number of the SNP is known, check NCBI dpSNP
- Obtain an up-to-date list of possible SNPs in your sequence
- Check whether the frequency of the SNP is relevant to your population
- When genotyping, if relevant SNPs are adjacent to the SNP of interest, avoid allele dropout by using mixed bases
- Remember, genomic information is constantly being updated. Always recheck previously checked sequences!
Don’t want to always have to check whether your primers and probes contain SNPs?
Then simply purchase predesigned qPCR assays, which are designed using frequently updated information using refSeq releases from NCBI. Target regions are screened to avoid SNPs and sequences that are repeated elsewhere in the genome. Each assay is synthesized after it’s ordered, meaning that recently identified SNPs are never omitted!
Ellen Prediger is Director, Scientific Communication, IDT, Coralville, IA, U.S.A.; http://www.idtdna.com/site.