Volume 10, Number 9—September 2004
Research
Silent Nucleotide Polymorphisms and a Phylogeny for Mycobacterium tuberculosis
Table 1
Amplification primers
| Gene/locus | Forward primer | Reverse primer | Product (bp) | Reaction conditionsa | Cycles |
|---|---|---|---|---|---|
| gyrA | gyrA-ext F 5′-ACAGACACGACGTTGCCGCC-3′ | gyrA-ext R 5′′-GTCGATTTCCCTCAGCATCTCC-3′ | 435 | 95°C for 15 min b 95°C for 15 s 68°C for 30 s 72°C for 1 min 72°C for 5 min | 30 |
| inhA | mabA-ext F 5′-TCGTAGGGCGTCAATACACC-3′ | mabA-ext R 5′-TCATTCGACCGAATTTGTTG-3′ | 605 | 94°C for 5 min 94°C for 30 s 60°C for 30 s 72°C for 30 s 72°C for 5 min | 30 |
| katG | katG-ext3F 5′-CGACGAAATGGGACAACAGT-3′ | katG-ext3R 5′-TGCATGAGCATTATCCCGTA-3′ | 1,507 | 94°C for 5 min 94°C for 30 s 60°C for 30 s 72°C for 1 min 72°C for 7 min | 30 |
| katG -ext5F 5′-TCGACTGTGCTGTTGGCGAGG-3′ | katG-ext5R 5′-CTTCGCCGACGAGGTCGTGG-3′ | 1,531 | 95°C for 15 min b 95°C for 30 s 68°C for 30 s 72°C for 1 min 72°C for 7 min | 30 | |
| oxyR-ahpC | oxyR-ext F 5′-TCGAGCTGCGACGGTGCTGG-3′ | oxyR-extR 5′-CTGCGGGTGATTGAGCTCAGG-3′ | 1,437 | 95°C for 15 min b 95°C for 30 s 72°C for 30 s 72°C for 1 min 72°C for 7 min | 30 |
| pncA | pncA-ext F 5′′-AACCAAGGACTTCCACATCG-3′ | pncA-extAR 5′-CAGAAACTGCAGCATCATCG-3′ | 1,324 | 95°C for 15 min b 95°C for 30 s 64°C for 30 s 72°C for 1 min 72°C for 7 min | 30 |
| rpoB | rpoB-46F 5′-GGCCGTGGGCACCGCTCC-3′ | rpoB 1868R 5′-CCAGCGGGGCCTCGCTACG-3′ | 1,822 | 95°C for 15 min b 95°C for 15 s 65°C for 30 s 72°C for 3 min 72°C for 10 min | 30 |
| rpoB 1711F 5′-GTGCCCTCGTCTGAGGTGGAC-3′ | rpoB 3602R 5′-AAGACCGATGCGGAGTTCATCG-3′ | 1,891 | 95°C for 15 min b 95°C for 15 s 65°C for 30 s 72°C for 3 min 72°C for 10 min | 30 | |
| rpsL | rpsL-extF 5′-GGCCGACAAACAGAACGT-3′ | rpsL-extR 5′-GTTCACCAACTGGGTGAC-3′ | 494 | 94°C for 5 min 94°C for 30 s 56°C for 30 s 72°C for 30 s 72°C for 5 min | 30 |
aA final PCR reaction volume of 25 μL was used that contained 2.5 μL of 10 x ammonium sulfate reaction buffer (Bioline, London, UK), 1.5 mmol/L magnesium chloride (Bioline); 200 μmol/L each of dATP, dTTP, dGTP, and dCTP;, 300 nmol/L of each primer pair, 0.8 units of Taq DNA polymerase (Bioline), 1 μL (≈10 ng) of template DNA and sterile distilled water. Amplification was carried out in 0.2 ml thin-wall polymerase chain reaction tubes in a DNA Thermal Cycler 9600 (Applied Biosystems, Warrington, UK). Products were purified by precipitation with 20% polyethylene detected with an ABI Prism 3700 or an ABI Prism 377 automated DNA sequencer (ABI, Warrington, UK).
bReaction performed with Hotstar Taq and reaction buffer (Qiagen, Crawley, UK).