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When Designing PCR Assays, Always Check For SNPs

SNP Impact on PCR Efficiency

Single nucleotide polymorphisms (SNPs) play an extremely valuable role in evolving species diversity and as a tool for individual and species identification. SNP profiles can also serve as a powerful diagnostic tool for certain diseases. In the human genome, common 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. Performing PCR using primers that overlie SNP sites can have a dramatic impact on the reaction, and can undermine obtaining accurate data. The presence of SNPs can influence primer and probe Tm, efficient polymerase extension, and even target spec­ificity. It is therefore critical to assess whether PCR primer and probe candidate sequences contain identified SNPs, and to avoid design­ing primers over SNPs when possible.

SNPs—What Are They?

Single base positions within a stretch of DNA that differ in sequence in individuals or a population define distinct alleles or mutations and are known as a single nucleotide polymorphisms or “SNPs”. They can occur within coding or noncoding sequences. The effect of a SNP can be variable. When they occur within coding sequences, they do not always affect the translated amino acid sequence—that is, some SNPs are silent. However, even when SNPs do not occur in coding regions, they may still affect gene expression by altering gene splicing, transcription factor binding, or mRNA degradation.

Most individual human beings share 99.9% of the same DNA sequences. Of the 0.1% difference between them, over 80% are due to SNPs [1]. Other sequence variations include insertions, deletions and transpositions. SNPs occur at different frequencies and within different populations.

Increasing Number of Known SNPs

Due to burgeoning genome studies, the sequence landscape has dramatically changed, with new SNPs being identified at record levels. Since its inception in 1998, the list of identified SNPs in the dbSNP reference database at NCBI (http://www.ncbi.nlm.nih.gov/snp) has grown from just a few million in 2000 to over 53 million human SNPs in 2012, and this number continues to sharply increase (Figure 1). Due to their effect on primer Tm, the importance of knowing whether your assay designs overlie SNPs has increased with the large number of SNPs being identified.

Figure 1. Rapidly Increasing Identification of New SNPs. Data shown is through build 137 of NCBI dbSNP.

How SNPs Can Affect Your qPCR

Effect on Tm. Underlying SNPs can affect qPCR data. SNPs that occur in primer and probe binding sites can destabilize oligonucleotide binding and reduce target specificity. As seen in Figure 2, single mismatches can reduce the Tm of an oligonucleotide by as much as 5–18°C. The degree of effect on Tm depends on the mismatch position, type of mismatch (e.g., A/A, A/C, G/T), as well as the surrounding environment/sequence [2]. The free, online IDT OligoAnalyzer® tool allows researchers to set mismatches and then calculate Tm. Access it at www.idtdna.com/scitools. Also see the Ask Alex FAQ in this issue, which outlines the steps to calculate mismatch Tm using the OligoAnalyzer tool.


Figure 2. A Single Mismatch Can Significantly Decrease Primer/Probe Melting Temperature. The example shows how a single mismatch can alter melting tempera­ture, affecting the efficiency of the PCR, and ultimately the interpretation of experimental results. These particular mismatches create non-standard base pairing that should not disrupt the helix. However, a single mismatch can substantially decrease melting temperature—by over 8°C. The screen shots show output from the free IDT OligoAnalyzer® tool available at www.idtdna.com/scitools.

Effect on qPCR amplification. In many cases, a single SNP may not completely prevent amplification, but can cause inefficient an­nealing and amplification [3]. This can lead to a delayed shift in Cq and underestimation of the amount of gene expression or even copy number loss in the SNP-containing sequences.

Using a modified single-base extension assay, Wu and colleagues [4] investigated how the type and position of a mismatch affected extension efficiency during the initial PCR cycle. They concluded that mismatches within the last 3–4 bases of the 3’ end of the primer blocked primer extension. Wu et al. attribut­ed the low extension efficiency to reduced binding of the DNA polymerase. While other research groups have contested this finding, describing a similar affinity of DNA poly­merase for correctly paired and mispaired du­plexes [5], Lefever and colleagues [3] confirm and extend the results from Wu et al.

Positional effects. In their article entitled SNPs and other mismatches reduce qPCR assay performance [3], Lefever and colleagues demonstrated that the impact of a given mis­match correlated with its distance from the 3’ primer end. Those mismatches closest to the 3’ end—typically within the last 5 bases—had the most dramatic effect on amplification. Mismatches present at the very 3’ end had the strongest shift of Cq from perfect matches, altering Cq by as much as 5–7 cycles (a 32- to 128-fold difference, dependent on the master mix used; Figure 3).


Figure 3. Mismatches at the 3’ End of Primers Reduce qPCR Performance. The data show the difference in Cq (ΔCq) between perfect match and mismatch primers as a function of the position of a single mismatch, using 5 different master mixes (A, B, C, D, and E). p values were calculated using one-way analysis of variance (ANOVA). The shift due to a SNP at the 3’ end of a primer varies up to 7 Cq, representing a 128-fold change in gene expression, dependent on the master mix used. (Data adapted from Lefever et al. [3], with permission of the publisher.)

Base composition effects. Lefever and col­leagues additionally showed that reactions containing purine/purine and pyrimidine/pyrimidine mismatches at the 3’ most position in the primer produced larger ΔCq values (mis­match vs. perfect match) and reduced end-point fluorescence values, with A/G and C/C showing the largest Cq differences compared to perfect matches [3].

Their data demonstrated that the shift in Cq between a perfect matched oligo/target and one with a single mismatch got smaller with increasing distance of the mismatch from the 3’ end. Single mismatches located more than 5 nucleotides from the 3’ end could still have a moderate effect on qPCR amplification. Further experiments by this group showed that the reduction in Tm and shift in Cq were exacerbated when SNPs occur in both primers (forward and reverse) or when more than one mismatch occurs within a given primer.

Safeguarding Your Experiments

Researchers often use primers and probes for RT-qPCR using assays that may have been validated with sequences obtained prior to the 1000 Genomes project. Although it is tempting to use a published or “lab-validated” design, given the amount of new sequence information available, it is important to make sure that primer and probe designs are not within regions containing SNPs. The following are tips for ensuring your assays avoid SNPs:

  • If the “rs” number of the SNP is known, check SNP information in NCBI dbSNP (http://www.ncbi.nlm.nih.gov/snp).
  • To obtain an up-to-date list of possible SNPs in your sequence, click on the relevant sequence match from the BLAST search to go to the nucleotide page (or access the nucleotide page directly, using the RefSeq number for the gene of inter­est). On the nucleotide page, click on SNP under the Related information menu on the right-hand side of the page.
  • If a SNP is identified, check whether the fre­quency of the SNP (minor allele frequency, or MAF) is relevant in your population.
  • When genotyping, if there are relevant SNPs adjacent to your SNP of interest, avoid allele dropout by using mixed bas­es (Ns) or inosines in the primer or probe to cover the adjacent site(s).
  • Since genomic information is constantly in flux, it is important to recheck previ­ously used primer and probe sequences.

IDT PrimeTime® Assays Are Designed to Avoid SNPs

Having to manually check whether your primers contain SNPs can be cumbersome. An alternative is to select assays where SNP screening is done for you. IDT has developed a sophisticated qPCR assay design engine that checks all designs against up-to-date SNP and intron/exon junction locations.

Design of PrimeTime Predesigned 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 recently identified SNPs.

References

  1. Piazza A. (2012) Theory of evolution and genetics. In: Fasolo A Ed. (2012) The Theory of Evolution and Its Impact. Italy:Springer-Verlag. p. 119.
  2. Owczarzy R, Tataurov AV, et al. (2008) IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucl Acids Res, 36 (suppl 2): W163–169.
  3. Lefever S, Pattyn F, et al. (2013) Single-nucleotide polymorphisms and other mismatches reduce performance of quantitative PCR assays. Clin Chem,doi: 10.1373/clinchem.2013.203653.
  4. Wu J-H, Hong P-Y, and Liu W-T. (2009) Quan­titative effects of position and type of single mismatch on single base primer extension. J Microbiol Methods, 77:267–275.
  5. Huang MM, Arnheim N, and Goodman MF. (1992) Extension of base mispairs by Taq DNA polymerase: implications for single nucleo­tide discrimination in PCR. Nucleic Acids Res, 20:4567–4573.


Author: Ellen Prediger, PhD, is the Director of Scientific Communication at IDT. We also acknowledge con­tributions from Steve Lefever and Jo Vandesompele, Ghent University, Belgium.