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The G + C content in mol% of DNA is the proportion of guanine and cytosine with respect to the total amount of nucleotides in the genome. This characteristic is widely used in taxonomic descriptions of prokaryotic microorganisms.

Traditionally, this value was obtained through laborious techniques such as thermal denaturation of genomic DNA, and DNA separation by CsCl buoyant density. More recently other techniques are used, such as HPLC separation or calculation by real-time PCR according to the protocol of Gonzalez and Saiz-Jimenez (2002).

The availability of genomic sequences is making it increasingly easy to estimate the value of the G + C mol% content from these sequences using freely available software such as GGDH, and to obtain more accurate values ​​than in the laboratory.

Necessary material

An active culture, genomic DNA or genomic sequence of the strain.

Genomic DNA must be delivered in cold-storage and be of good quality (A260 / A280 between 1.8 and 2). We require more than 2 micrograms of DNA at a concentration of 40 ng/µl resuspended in ultrapure water or in TE buffer.

Methodology

From an active culture or genomic DNA, the value of the G+C mol% content is calculated by real time PCR. At CECT, the DNA is denatured in a StepOnePlus ™ thermocycler, Real-Time PCR System from Applied Biosystems, with the StepOne ™ software program.

Bibliography

Gonzalez, J.M. and Saiz-Jimenez, C. (2002). A fluorimetric method for the estimation of G+C mol% content in microorganisms by thermal denaturation temperature. Environ Microbiol 4, 770–773