TABLE 2.
PCR primers
| Primer set | Model gene(s) | Primer (5′-3′)e | Optimal annealing temp used (°C)c | Characteristicf | Reference or source |
|---|---|---|---|---|---|
| tfdA* | R. eutropha JMP134/Burkholderia sp. strain RASC | F, AACGCAGCGRTTRTCCCAa R, ACGGAGTTCTGYGAYATGa | 58 | Unspecific binding; qPCR not possible | 59 |
| tfdA-intern | R. eutropha JMP134 | F, GCATACGACGCGCTGCCTCG R, CTTCGGCCACCGGAAGGCCT | 65 | Used for probe preperation for Southern blot hybridization | This work |
| tfdA** | Sequence alignment of 23 known tfdA genes | F, GAGCACTACGCRCTGAAYTCCCGa,b R, CTTCGGCCACCGGAAGGCCT | 64 | One band on agarose gel (approx. 220 bp); qPCR possible | This work |
| tfdAα* | Alignment of RD5-C2, HWK12, and HW13 (tfdAα genes) | F, CCGGCGTCGATCTGCGCAAG R, GTTGACGACGCGCGCCGA | NDd | No clear band on the agarose gel; fluffy PCR products | 51 |
| tfdAα** | Alignment of HW13, HWK12, and BTH (tfdAα genes) | F, ACSGAGTTCKSCGACATGCGa R, GCGGTTGTCCCACATCAC | 65 | One band (approx. 350 bp); qPCR may be possible | 25 |
| cadA | HW13 (cadA gene) | F, AAGCTGCARTTTGARAAYa | 58 | No PCR product | 24 |
| R, MGGATTGAAGAAATCCTGRTAa |
a
R, G/A; Y, T/C; S, G/C; K, G/T; M, A/C.
b
AGC clamp was added to the 5′ end of the primer for the PCR/DGGE (5′-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGG-3′).
c
The optimal annealing temperature is based on gradient ramping (58 to 69°C).
d
ND, no optimal annealing temperature could be determined.
e
F, forward; R, reverse.
f
qPCR, quantitative PCR.