TABLE 2.
PCR primers and conditions for conventional PCR
| Primera | Target | Sequence | Tab (°C) | MgCl2 concn (mM) | Primer concn (μM) | Tpc (°C) | Expected product size (bp) |
|---|---|---|---|---|---|---|---|
| NAH-F | Naphthalene dioxygenase | 5′-CAAAA(A/G)CACCTGATT(C/T)ATGG | 47 | 2.5 | 0.3 | 83 | 377 |
| NAH-R | 5′-A(C/T)(A/G)CG(A/G)G(C/G)GACTTCTTTCAA | ||||||
| TOD-F | Toluene dioxygenase | 5′-ACCGATGA(A/G)GA(C/T)CTGTACC | 53 | 2.0 | 0.5 | 83 | 757 |
| TOD-R | 5′-CTTCGGTC(A/C)AGTAGCTGGTG | ||||||
| TOL-F | Xylene monooxygenase | 5′-TGAGGCTGAAACTTTACGTAGA | 55 | 2.5 | 0.2 | 82 | 475 |
| TOL-R | 5′-CTCACCTGGAGTTGCGTAC | ||||||
| BPH1-F | Biphenyl dioxygenase | 5′-GGACGTGATGCTCGA(C/T)CGC | 57 | 2.0 | 0.06 | 88 | 671 |
| BPH1-R | 5′-TGTT(C/G)GG(C/T)ACGTT(A/C)AGGCCCAT | ||||||
| BPH2-F | Biphenyl dioxygenase | 5′-GACGCCCGCCCCTATATGGA | 63 | 2.5 | 0.1 | 88 | 724 |
| BPH2-R | 5′-AGCCGACGTTGCCAGGAAAAT | ||||||
| BPH3-F | Biphenyl dioxygenase | 5′-CCGGGAGAACGGCAGGATC | 62 | 1.5 | 0.1 | 87 | 570 |
| BPH4-F | 5′-AAGGCCGGCGACTTCATGAC | 63 | 1.5 | 0.4 | 87 | 452 | |
| BPH3-Rd | 5′-TGCTCCGCTGCGAACTTCC | ||||||
| RMO-F | Toluene monooxygenase | 5′-TCTC(A/C/G)AGCAT(C/T)CAGAC(A/C/G)GACG | 53 | 3 | 0.4 | 82 | 466 |
| RMO-R | 5′-TT(G/T)TCGATGAT(C/G/T)AC(A/G)TCCCA | ||||||
| RDEG-F | Toluene monooxygenase | 5′-T(C/T)TC(A/C/G)AGCAT(A/C/T)CA(A/G)AC(A/C/G)GA(C/T)GA | 52 | 3 | 0.5 | 87 | 466 |
| RDEG-R | 5′-TT(A/G/T)TCG(A/G)T(A/G)AT(C/G/T)AC(A/G)TCCCA | ||||||
| PHE-F | Phenol monooxygenase | 5′-GTGCTGAC(C/G)AA(C/T)CTG(C/T)TGTTC | 49 | 4 | 0.3 | 86 | 206 |
| PHE-R | 5′-CGCCAGAACCA(C/T)TT(A/G)TC |
a
Forward (−F) and reverse (−R) primers are indicated.
b
Ta, the annealing temperature used during real-time PCR.
c
Tp, the polymerization temperature used during real-time PCR.
d
BPH3-R is used with BPH3-F and BPH4-F.