16S Primers - Frequently Asked Questions

1. Where is the 16 Primers database described?

   ES Wright et al. (2014) "Exploiting Extension Bias in PCR to Improve Primer Specificity in Ensembles of Nearly Identical DNA Templates." Environmental Microbiology, doi:10.1111/1462-2920.12259.

2. What genera are available in the 16S Oligos tool?

   All 1,943 archaeal and bacterial named genera in the RDP II database (10.30) have primer sets available.

3. Have you tested all of these primer sets?

   No, we have only tested a subset of these primers to verify their performance. As with any new set of primers, it is recommended to experimentally verify that they work as expected by optimizing the PCR conditions. However, the primer sets we have tested worked as expected at the default annealing temperature (64°C) and reagent conditions (Bio-Rad's iQ SYBR Green Supermix).

4. What is the difference between using the 3'-end Model and not?

   Three primer set options are available for each genus:

   • 16S Primers With Taq 3'-end Model:

     Here primers were designed with a model of elongation efficiency for mismatches near the 3'-end of the primer. These primers make use of Taq's extension bias to increase specificity to the target genus.

   • 16S Primers Without 3'-end Model:

     Here primers were designed without regard for placement of the mismatches near the 3'-end of the primer. These primers should be used if you intend to use an exo-nuclease positive polymerase (i.e. a DNA polymerase other than Taq, such as PFU or KOD).

   • 16S Primers With Induced Mismatch:

     Primers were designed with a single mismatch at the 6^th position from their 3'-end. This additional mismatch can increase specificity in difficult sequence discrimination scenarios. However, since these primers are mismatched to sequences in the target genus, it is necessary to ensure that they work as expected with a standard temperature gradient experiment using the target template.

5. How do I interpret the output page?

   After selecting a genus the output page displays the top 5 primer sets with highest specificity to that genus. The primers are listed along with their starting E. coli positions, and respective coverage of the target group. An approximate amplicon length is given based on a consensus sequence formed from sequences belonging to the genus.

6. How do I use these primers?

   Primer sequences can be ordered from many online companies, including Integrated DNA Technologies (IDT). We used the primers in an equimolar mix at 400nM final concentration, with an annealing temperature of 64°C, and denaturation, annealing, and elongation step times of 30 seconds. PCR conditions were based on the ionic concentrations in Bio-Rad's iQ SYBR Green Supermix.

7. How do I verify the specificity of the primers in silico?

   All non-target genera with greater than 0.1% predicted relative efficiency are listed with mismatches highlighted. Links to the RDP's ProbeMatch and Silva's TestProbe are provided that will automatically search the sequence repositories using first foward and reverse primer sequences that are listed. These tools enable independent confirmation of the primer set's in silico specificity to the target group.