Quantification of the detrimental effect of a single primer-template mismatch by real-time PCR using the 16S rRNA gene as an example

Abstract

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.

FIG. 1.

Comparison of the 16S rRNA gene copy numbers in Pseudomonas aeruginosa PAO1 (A), Agrobacterium tumefaciens C58 (B), and Sinorhizobium meliloti 1021 (C), estimated by real-time PCR using the 341F-534R or the mispaired forward and reverse primers in combination with the 534R or 341F primers, respectively. Error bars show standard errors (n = 3). Mean values were compared by using a t test. Significantly different gene copy numbers (P < 0.05) are indicated by different letters.

FIG. 2.

Quantification of 16S rRNA gene copy number with primers 341F-534R in plasmids containing mutagenic 16S rRNA having a base conversion in the forward or reverse primer extension sequences. Error bars show standard errors (n = 3). Mean values were compared by using a t test. Significantly different gene copy numbers (P < 0.05) are indicated by different letters. N.D.: not determined.