Determining the effect of primer mismatches on quantitative PCR accuracy and developing guidance for design of primers with sequence variations
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Although quantitative PCR (qPCR) is a powerful tool for investigating environmental systems, target gene sequences for organisms of interest often are not well known, which has resulted in few reliable primers for many applications. Additionally, the sequences of target genes found in diverse strains often contain sequence variations, and therefore, primer sets containing single or multiple primer-template mismatches are common. However, the detrimental impact of these mismatches on quantification accuracy and amplification efficiency has not been investigated thoroughly. Thus, the research ...