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Volume 10, Number 9—September 2004

Research

Silent Nucleotide Polymorphisms and a Phylogeny for Mycobacterium tuberculosis

Lucy Baker*, Tim Brown*, Martin Maiden†, and Francis Drobniewski*Comments to Author 
Author affiliations: *Heal Protection Agency, London, UK; †University of Oxford, Oxford, UK

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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).

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