Table 2: Primer sets and conditions used for 16S rRNA and AmoA genes amplification and cloning of ammonia-oxidizing prokaryotes. An initial denaturation (94°C for 5 min) and final elongation (72°C for 20 min) steps were performed to all reactions. (a)Touchdown 0.5°C per cycle, from at 68°C to 60°C; (b)+1 sec per cycle. Final elongation = 5 minutes. (b)New: reverse primer, specifically targeting Thaumarchaeota (thaum922r: 5′-TTG TGG TGC TCC CCC GCC-3′). f: forward; r: reverse primer; (c)primers contained 40-nucleotide-long GC-sequence at the 5′ end for Denaturing Gradient Gel Electrophoresis [31].

Target gene/groupPrimer pairCyclesDenaturationAnnealingElongationReference
°Cs°Cs°Cs 

Archaea 
16S rRNA
arc344f(c)
arc915r
30946068(a)607290Stahl and Amann (1991) [24]

Thaumarchaea 
16S rRNA
arc21f
thaum922r(b)
30946062607280DeLong (1992) [32]
This study

AOB 16S rRNACTOf-mix(c)
CTOr
35923057607245(b)Kowalchuk et al. (1997) [33]

AOB amoA amoA-1f(c)
amoA-2r
35946060907290Rotthauwe et al. (1997) [34]

AOA amoA Arch-amoAF(c)
Arch-amoAR
30944553607260Francis et al. (2005) [35]