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/group | Primer pair | Cycles | Denaturation | Annealing | Elongation | Reference | |||
|---|---|---|---|---|---|---|---|---|---|
| °C | s | °C | s | °C | s | ||||
| Archaea 16S rRNA |
arc344f(c)
arc915r |
30 | 94 | 60 | 68(a) | 60 | 72 | 90 | Stahl and Amann (1991) [24] |
|
| |||||||||
| Thaumarchaea 16S rRNA |
arc21f thaum922r(b) |
30 | 94 | 60 | 62 | 60 | 72 | 80 | DeLong (1992) [32] This study |
|
| |||||||||
| AOB 16S rRNA | CTOf-mix(c)
CTOr |
35 | 92 | 30 | 57 | 60 | 72 | 45(b) | Kowalchuk et al. (1997) [33] |
|
| |||||||||
| AOB amoA |
amoA-1f(c)
amoA-2r |
35 | 94 | 60 | 60 | 90 | 72 | 90 | Rotthauwe et al. (1997) [34] |
|
| |||||||||
| AOA amoA | Arch-amoAF(c)
Arch-amoAR |
30 | 94 | 45 | 53 | 60 | 72 | 60 | Francis et al. (2005) [35] |