Right Nomar, messy is what I meant. I have an old paper that you might wish to follow (it works, I promise). Look at this one:

STRUCTURAL ORGANIZATION OF THE GENOME OF THE ZYGOMYCETE ABSIDIA-GLAUCA - EVIDENCE FOR HIGH REPETITIVE DNA CONTENT

By: WOSTEMEYER, J; BURMESTER, A

CURRENT GENETICS Volume: 10 Issue: 12 Pages: 903-907 Published: 1986

Best wishes and good luck - Johannes.

In order to estimate the GC content of a plant genome or any genome.

you can use the following online tool (link: http://www.sciencebuddies.org/science-fair-projects/project_ideas/Genom_GC_Calculator.shtml)

to estimate the GC % as well as all other nucleotide percentages seperately, by applying the DNA sequence into the tool.

All d best

Regards

Murugan N

I don't use PCR products. My sample is genomic DNA, not sequences. I have seen this website, but thanks, anyways.

If your plant genome is already available in the databank, you can check in the website itself. or otherwise you tell me your plant name i will check and let you know.

Just download this perl script, save it in .pl extention and run it.

I am not sure if I understand you correctly. You want to measure the G/C-content experimentally, not just to analyse pre-existing sequences?

I am pretty sure you can estimate GC content using melting. This paper looks at a yeast chromosome sequence, and does it the other way - predicting melting temperature from GC content - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC310056/?page=4

When I was supervised by the author of this paper about 10 years ago, he showed me some unpublished data where he had use "first derivative thermal denaturation profiles of nuclear DNA" to estimate variation of GC content within a genome.

One thing I think about this is that GC content varies throughout genomes, and varies at different scales of organisation, so I wonder whether your choice of sequence fragment length may make subtle differences in the denaturing profile you get.

Yes, of course measuring melting profiles will do the job. And of course the medium fragment length matters. Therefore, I strongly recommend to control fragment length. Maybe, you decide to chop the DNA down to 600 bp in length, which can nicely be done by ulrasonic treatment. With some luck you will be able to find classes of DNA with distinguishable G/C. Sometimes melting curves do not show the resolution, that we dream of, however.

I also recommend that you accompany the very valuable melting results by CsCl density gradients. This can easily be done with fragments of the same size in the presence of bisbenzimide. This lowers density and increases the differences between A/T- and G/C-rich fragments. If there are satellites in your genome, you will see separate bands in your gradients. You can easily measure G/C by standardizing the gradients with DNA of known G/C. E.coli (50 % GC), Staph. aureus (33 % GC), and Streptomyces lividans (72 % GC). This simple approach that I used for characterisation of fungal DNAs reduces GC-measurements to determining positions of fluorescent bands in the gradient by a simple ruler, and to drawing a calibratuion line on millimeter paper.

Good luck!

@Johannes Wöstemeyer. Yes, I want to estimate the GC content experimentally, not with pre-existing NGS sequences. My sample's genome has not been sequenced yet. Your method seems interesting, but I have never tried that approach before. Do you have any published paper with that protocol?

Also, I have a sheared genomic DNA sample from 100 to 600 bp long. But since each fragment from this pool may contain different GC content, since each may come from different genomic regions with different GC content, i.e. isochores, measuring the melting curves would probably show a complex pattern, maybe a messy plot? I have not tried this before.

Right Nomar, messy is what I meant. I have an old paper that you might wish to follow (it works, I promise). Look at this one:

STRUCTURAL ORGANIZATION OF THE GENOME OF THE ZYGOMYCETE ABSIDIA-GLAUCA - EVIDENCE FOR HIGH REPETITIVE DNA CONTENT

By: WOSTEMEYER, J; BURMESTER, A

CURRENT GENETICS Volume: 10 Issue: 12 Pages: 903-907 Published: 1986

Best wishes and good luck - Johannes.

@Johannes Wöstemeyer: Thanks a lot!