ArticleBMC biotechnology2009
Evaluation of the impact of single nucleotide polymorphisms and primer mismatches on quantitative PCR.
Article in BMC biotechnology, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 45 papers.
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Who cites it
45 citing papers in PubMed.
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- CRISPR-Cas systems for diagnosing infectious diseases.Methods (San Diego, Calif.) · 2022Review
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- Technical considerations in PCR-based assay design for diagnostic DNA methylation cancer biomarkers.Clinical epigenetics · 2022Article
- Dominant mutations in the severe acute respiratory syndrome coronavirus-2 genome challenge polymerase chain reaction detection.Clinical and translational discovery · 2022Article
- Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform.Biosensors & bioelectronics · 2022Article
- Pairing of Parental Noroviruses with Unequal Competitiveness Provides a Clear Advantage for Emergence of Progeny Recombinants.Applied and environmental microbiology · 2021Article
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
3 authors.
Funding
No grant is acknowledged in the PubMed record.
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.
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