Table 2.
In vitro evaluation of primer pairs by real-time PCR. DNA extracted from archaeal owners of the target gene served as positive control template during the optimization of annealing temperature (T ann) and extension time (t ext).Expected lengths of the amplification product refer to distances between primer binding sites in the archaeal sequences that were used during primer design. Observed product lengths were determined by agarose gel electrophoresis of the actual PCR products.
| Primer pair | Positive controls | T ann in °C |
t
ext
in s |
Product length in bp | |
|---|---|---|---|---|---|
| Expected | Observed | ||||
| arc-NirA-f, -r |
Halorubrum lacusprofundi
Haloarcula marismortui Halogeometricum borinquense |
62 | 75 | 660–750 | 700 700 700, 350 |
| arc-NirB-f, -r | Thermococcus sibiricus | 64 | 90 | 680 | 700 |
| arc-Nos-f, -r |
Halogeometricum borinquense
Halorubrum lacusprofundi Pyrobaculum calidifontis |
64 | 75 | 910–1030 | 950 950 1050 |
| arc-Nif-f, -r |
Methanotorris igneus
Methanosarcina acetivorans |
60 | 60 | 360–415 | 400 400 |
| arc-Nred-f, -r | Halogeometricum borinquense | 64 | 90 | 760–1030 | several bands |