Hill Lab

Microbial ecology and molecular diagnostics

Universal primers for amplification of archaeal thermosome sequences

Detailed information on the universal thermosome PCR are available in Chaban & Hill. 2012. ISME Journal 6:430-439.

Primers amplify a region of the thermosome subunit genes of Archaea corresponding to nucleotides 142-858 of the Group II chaperonin (alpha subunit) of Methanococcus maripaludis strain S2. The universal primers have been used to amplify thermosome sequences from individual isolates and from complex communities in metagenomic studies (clone libraries and direct pyrosequencing of amplicons).

As with the cpn60 primer sets, optimal amplification across the widest taxonomic and G+C content range is achieved using a primer cocktail. We recommend a 7:1 molar ratio of JH0175/JH0178 to JH0268/JH0269. Each primer pair can also be used independently if desired, and depending on the application.

A standard thermosome PCR:

1 × PCR reaction buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl)

2.5 mM MgCl2

200 μM dNTP

400 nM of each forward and reverse primer(s)

2.5 U Taq DNA Polymerase

template DNA (1.0 ng DNA)

H2O to a final volume of 50 μl

Typical thermocycling parameters (based on Eppendorf Mastercycler or BioRad myIQ):

98°C for 3 min, 40 cycles of [30 sec at 98°C, 1 min at 54°C and 1 min at 72°C], 72°C for 10 minProduct size is ~750 bp

Primer sequences:

JH0175 (forward) 5'-GGI CCI MRR GGI ITI GAY AAR ATG-3'

JH0178 (reverse) 5'-GCI AII TCR TCI ATI CCY TTY TG-3'

JH0268 (forward) 5'-GGC CCG AAG GGC ATG GAC AAG ATG-3'

JH0269 (reverse) 5'-GGC ATG TCG TCG ATG CCC TTC TG-3'

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