Evaluation of the impact of single nucleotide polymorphisms and primer mismatches on quantitative PCR

Abstract

BackgroundRobust designs of PCR-based molecular diagnostic assays rely on the discrimination potential of sequence variants affecting primer-to-template annealing. However, for accurate quantitative PCR (qPCR) assessment of gene expression in populations with gene polymorphisms, the effects of sequence variants within primer binding sites must be minimized. This dichotomy in PCR applications prompted us to design experiments to specifically address the quantitative nature of PCR amplifications with oligonucleotides containing mismatches.ResultsWe performed qPCR reactions with several primer-target combinations and calculated ratios of molecules obtained with mismatch oligonucleotides to the average obtained with perfect match primer pairs. Amplifications were performed with genomic DNA and complementary DNA samples from different genotypes to validate the findings obtained with plasmid DNA. Our results demonstrate that PCR amplifications are driven by probabilities of oligonucleotides annealing to target sequences. Empiric probabilities can be measured for any primer pair. Alternatively, for primers containing mismatches, probabilities can be measured for individual primers and calculated for primer pairs.ConclusionThe ability to evaluate priming (and mispriming) rates and to predict their impacts provided a precise and quantitative description of assay performance. Priming probabilities were also found to be a good measure of analytical specificity.

Natural variation in expression of genes involved in xylem development in loblolly pine (Pinus taeda L.)

“…This might be due to the fact that the SNPs were mostly present toward the 5′ end of the primers. Boyle et al (2009) have shown that SNPs present at the 5′ end of the primer do not affect the binding efficiency of the primer, and our results are in agreement with that observation. Table 3).…”

Association Genetics of Wood Physical Traits in the Conifer White Spruce and Relationships With Gene Expression

“…Transcript levels could be compared among the different genotypic classes for six of the significantly associated SNPs (after FDR correction for multiple testing) by using RT-qPCR with gene-specific primers as described in Boyle et al (2009). The assays used differentiating secondary xylem from the 30-year-old trees of the association population.…”

“…PCR mixtures were assembled using LightCycler 480 SYBR Green I Master (Roche, Basel, Switzerland) with an epMotion 5075 pipetting robot (Eppendorf, Hamburg, Germany) and amplifications used a LightCycler 480 (Roche) with conditions and melting curve analyses as described (Boyle et al 2009). The number of RNA transcript molecules was determined using the LRE methodology (Rutledge and Stewart 2008) adapted for Excel (Boyle et al 2009). Normalization was performed using GeNorm (Vandesompele et al 2002) and the reference genes ef1a (BT102965), cdc2 (BT106071), and rpl3A (BT115036).…”

“…For each SNP genotypic class, 8-25 trees were individually sampled by removing three small plugs of 1 cm 2 each of actively growing secondary xylem tissue at 1.3 m from the ground; these represented the entire annual growth produced near the end of the earlywood phase. Tissue handling and RNA extractions were performed as described for white spruce (Bedon et al 2007); complementary DNA synthesis was prepared from 500 ng of total RNA (Boyle et al 2009). PCR mixtures were assembled using LightCycler 480 SYBR Green I Master (Roche, Basel, Switzerland) with an epMotion 5075 pipetting robot (Eppendorf, Hamburg, Germany) and amplifications used a LightCycler 480 (Roche) with conditions and melting curve analyses as described (Boyle et al 2009).…”

“…Tissue handling and RNA extractions were performed as described for white spruce (Bedon et al 2007); complementary DNA synthesis was prepared from 500 ng of total RNA (Boyle et al 2009). PCR mixtures were assembled using LightCycler 480 SYBR Green I Master (Roche, Basel, Switzerland) with an epMotion 5075 pipetting robot (Eppendorf, Hamburg, Germany) and amplifications used a LightCycler 480 (Roche) with conditions and melting curve analyses as described (Boyle et al 2009). The number of RNA transcript molecules was determined using the LRE methodology (Rutledge and Stewart 2008) adapted for Excel (Boyle et al 2009).…”