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 ( ) and extension time ( 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 pairPositive controls in °C ext
in s
Product length in bp
ExpectedObserved

arc-NirA-f, -rHalorubrum lacusprofundi
Haloarcula marismortui
Halogeometricum borinquense
6275660–750700
700
700, 350
arc-NirB-f, -rThermococcus sibiricus 6490680700
arc-Nos-f, -rHalogeometricum borinquense
Halorubrum lacusprofundi
Pyrobaculum calidifontis
6475910–1030950
950
1050
arc-Nif-f, -rMethanotorris igneus
Methanosarcina acetivorans
6060360–415400
400
arc-Nred-f, -rHalogeometricum borinquense 6490760–1030several bands