Table 1.
Gene | Name and sequence of the oligonucleotide primer* | PCR product size | PCR conditions** |
---|---|---|---|
mcrB | LMCRB: 5′- TWYCARGGHYTVAAYGC -3′ RMCRB: 5′- CCDCCDCCDCCRTARAT -3′ |
~392 bp | 96°C–30 s; 56°C–30 s; 72°C–40 s 39 cycles |
mcrG | LMCRG1: 5′-CAYCCDCCDYTNGADGARATGGA-3′ RMCRG1: 5′-TCRAACATYANWCCRTYYTCRTC-3′ |
~356 bp | 96°C–30 s; 56°C–30 s; 72°C–35 s 39 cycles |
mtaB | LMTAB: 5′- CARGCHAAYACYGCMATGTT -3′ RMTAB: 5′- CYTGDGGRTCYCKGTA -3′ |
~436 bp | 96°C–30 s; 56°C–30 s; 72°C–40 s 39 cycles |
mtbA | LMTBA: 5′- TTCTCCCTTGCMCAGCA -3′ RMTBA: 5′- ACWGGRTCVAGRTTWCC -3′ |
~413 bp | 96°C–30 s; 55°C–30 s; 72°C–40 s 39 cycles |
IUPAC code: A (adenine), C (cytosine), G (guanine), T (thymine), R (A or G), Y (C or T), W (A or T), K (G or T), M (A or C), D (A or G or T), H (A or C or T), V (A or C or G), N (A or C or G or T).
PCR conditions were specified for Taq DNA polymerase (Qiagen). The applicability of other (high fidelity) polymerases [i.e., Phusion High-Fidelity DNA Polymerase (Thermo Scientific) and KAPA polymerase (Kapa Biosystems)] was also confirmed.