Primer Dimer

Primer dimer formation is a common problem when primers are not checked for self-annealing. Figure 1 is a chromatogram displaying data collected using a primer that had not been checked for self-annealing. Most portions of the sequence is readable but of poor quality. Observe the short, intense region in the first twenty bases of the sequence. A primer dimer will itself become a template for the reaction from which extension will occur. This region is most likely sequence generated from the self-annealed primer.

Figure 1. Chromatogram of sequence reaction displaying large primer dimer peaks at the beginning of the sequence.

The first step in troubleshooting the sequence in Figure 1 was to check the primer sequence for self-annealing using Oligo Analyzer 3.0. The strongest primer dimer predicted to form is shown in Figure 2.

Figure 2. Predicted self-annealing structure for sequencing primer.

The stop point of the sequence can be adjusted to analyze a specific region. Figure 3 is a chromatogram displaying only the first twenty bases of the sequencing reaction in figure 1. The sequence read from this chromatogram is 5'-ACTGTCGAAAACGCATA-3'. When compared to the predicted primer dimer structure an exact match to the sequence is found.

Figure 3. Chromatogram displaying shortened read length.

A second primer was designed and used with the same template. The sequence results (Figure 4) show strong, well resolved peaks and the abscence of non-specific binding.

Figure 4. Chromatogram displaying data from sequence reaction with new primer.