TABLE 2.
PCR primers and conditions evaluated in this study
| Primer seta | Sequence (5′-3′)b | Experimental |
Target | Approx product length (bp) | PCR product of agricultural field soilc |
Reference(s) | ||
|---|---|---|---|---|---|---|---|---|
| Annealing temp (°C) | Mg2+ (mM) | Native | JMP134 spiked | |||||
| 1 | ||||||||
| TfdA21F | CCTTCATCCWCTTTTCGYCG | 63.0 | 3.0 | tfdA (group 1) | 815 | − | ++ | This work |
| TfdA836R | GCTCGGCGMAGYTCACGC | |||||||
| 2 | ||||||||
| TfdA334F | CTSTGGCATTSCGACAGYTC | 60.0 | 3.0 | tfdA (group 1) | 260 | − | ++ | This work |
| TfdA608R | CSGSGTGSGTICGVYCAG | |||||||
| 3 | ||||||||
| TfdAα52F | GGCGTCGATCTGCGCAAGCC | 70.0 (65.0)d | 4.0 (3.0)d | tfdAα (group 2) | 360 | ++ | ++ | This work |
| TfdAα408R | GTTGACGACGCGCGCCGACA | |||||||
| 4 | ||||||||
| TfdAα421F | ACSGAGTTCGSIGAYATSC | 63.6 (68.0)d | 2.5 (3.0)d | tfdAα (groups 2 and 3) | 360 | ++ | ++ | Modified from references 29 and 73 |
| TfdAα779R | CAGCGGTTGTCCCACATCAC | |||||||
| 5 | ||||||||
| TfdA421dF | ACSGARTTCKSIGACATGC | 53.0 | 3.0 | tfdA-like (groups 1 to 3) | 360 | ++ | ++ | Modified from references 29 and 73 |
| TfdA778vkR | AGCGGTTGTCCCACATCAC | |||||||
| 6 | ||||||||
| TfdA421Fv | ACSGAGTTCTGYGAYATG | 59.0 | 3.0 | tfdA-like (group 1) | 360 | +− | ++ | Modified from reference 73 |
| TfdA783R | AACGCAGCGRTTRTCCCA | |||||||
| 7 | ||||||||
| Eub341F | CCTACGGGAGGCAGCAG | 55.7 | 3.0 | 16S rRNA | 190 | ++ | ++ | 49 |
| Eub534R | ATTACCGCGGCTGCTGG | |||||||
a
Numbers in primer names indicate 5′ position of binding sites relative to the reference tfdA sequence of Cupriavidusnecator JMP134 (AY365053) (primer sets 1, 2, 4, 5, and 6) and of Alphaproteobacterium RD5-C2 (AB074490) (primer set 3). F, forward; R, reverse.
b
S, C/G; Y, C/T; V, A/C/G; W, A/T; M, A/C; K, G/T; R, A/G.
c
As indicated by agarose gel electrophoresis. ++, bright band; +−, weak band; −, no band of correct size.
d
Numbers in parentheses refer to qPCR chemicals (see Materials and Methods).