Quantification of the detrimental effect of a single primer-template mismatch by real-time PCR using the 16S rRNA gene as an example.
We investigated the effects of internal primer-template mismatches on the efficiency of PCR amplification using the 16S rRNA gene as the model template DNA. We observed that the presence of a single mismatch in the second half of the primer extension sequence can result in an underestimation of up to 1,000-fold of the gene copy number, depending on the primer and position of the mismatch.
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INRA, University of Burgundy, Soil and Environmental Microbiology, CMSE, 17 Rue Sully, B.P. 86510, 21065 Dijon Cedex, France.
Martin-Laurent
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Philippot
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D020137:Base Pair Mismatch / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics*; D018628:Gene Dosage / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics
D017931:DNA Primers; D012336:RNA, Ribosomal, 16S