Skip directly to site content Skip directly to page options Skip directly to A-Z link Skip directly to A-Z link Skip directly to A-Z link
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

Main Article

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).

Main Article

Page created: March 30, 2011
Page updated: March 30, 2011
Page reviewed: March 30, 2011
The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
file_external