Internal fragments of the seven loci can be amplified by PCR, using the primers listed below and chromosomal DNA as a template. PCR involved an initial denaturation of 95 °C for 3 min; 34 cycles of 95 °C for 30 s, 50 °C for 1 min, and 72 °C for 1 min; and a final extension of 72 °C for 10 min.
| Gene and function | Primer | Sequences (5'-3') | Size of amplicon used for assigning alleles |
|---|---|---|---|
| Carbamate Kinase (arcC) | arcC-F | TGTGATGAGCACGCTACCGTTAG | 465 |
| arcC-R | TCCAAGTAAACCCATCGGTCTG | ||
| Shikimate dehydrogenase (aroE) | aroE-F | CATTGGATTACCTCTTTGTTCAGC | 420 |
| aroE-R | CAAGCGAAATCTGTTGGGG | ||
| ABC transporter (gtr) | gtr-F | CAGCCAATTCTTTTATGACTTTT | 438 |
| gtr-R | GTGATTAAAGGTATTGATTTGAAT | ||
| DNA mismatch repair protein (mutS) | mutS-F3 | GATATAAGAATAAGGGTTGTGAA | 412 |
| mutS-R3 | GTAATCGTCTCAGTTATCATGTT | ||
| Pyrimidine operon regulatory protein (pyrR) | pyr-F2 | GTTACTAATACTTTTGCTGTGTTT | 428 |
| pyr-R4 | GTAGAATGTAAAGAGACTAAAATGAA | ||
| Triosephosphate isomerase (tpiA) | tpi-F2 | ATCCAATTAGACGCTTTAGTAAC | 424 |
| tpi-R2 | TTAATGATGCGCCACCTACA | ||
| Acetyl coenzyme A acetyltransferase (yqiL) | yqiL-F2 | CACGCATAGTATTAGCTGAAG | 416 |
| yqiL-R2 | CTAATGCCTTCATCTTGAGAAATAA |
Sequences for each locus must be obtained for both the forward and reverse strands, and must be 100% accurate, since even a single error will alter the allelic number obtained.