I am trying to determine the copy number of a gene which control flower development in B napus. I have done the following works to do that.
1. Isolated mRNA from from B napus flower bud and converted to cDNA.
2. Did RT-PCR using primer situated on the 5' and 3' UTR (based on genome sequence) and cloned the RT-PCR product in pGEM T Easy vector.
3. Miniprep 4 cDNA clone and sequenced it. In the sequenced file I have seen a number single nucleotide difference among these clone.
Now I am not sure whether they are different genes or different allele in the same locus. How can I analyze my cDNA sequence data whether they are different genes or different allele. Also what further experiment I can do to confirm the copy number of this gene.
I think you may try silencing each of these sequences separately and check if you get less of its product. If they are different genes there should be no changes in the amount of product. I think it is the same gene that got some punctual mutations, since it is foreing DNA it is not as protected as genomic DNA.
I hope this article can help you
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2836558/
Regards
In order to calculate the copy number of a gene I reather use qPCR with a standard curve made with a clonned plasmid.
Dear Hassan:
If you intend to know the gene copy number, the best way to do it is running the good old Southern Blot analysis. You may use one of the cDNA clone as DNA probe. Digest your plant genomic sample using restriction enzyme, hopefully one that did cut your gene somewhere, but did not cut in the middle of your DNA probe (you may predict this using your gene sequences) and separate the digested DNA in agarose gel. Transfer the DNA profile into membrane and probe them using your DNA probe.
The expected result: If you only see single band, it probably only one copy. However, if you see multiple bands, you may suspect there are more than one copy. This can at least be one way to find out. You can also back up the initial finding with other techniques to verify the gene copy number.
In my opinion, analyzing the level of RNA expression using qPCR may not be able to estimate the gene copy number.
Hi Mahmudul,
Blasting the B napus genome and manually inspecting the Sanger traces could definitely be informative as mentioned by Andrew, and should probably be done first no matter what. Perhaps the SNPs you find even match different gene copies in the sequenced genome? But southern blotting or qPCR using a cloned plasmid standard are good complementary and rigorous methods, especially if there are different copy numbers between plants.
Keep in mind though that genomic DNA, not cDNA should be used for the qPCR method, as you’re not interested in expression levels, but gene copy number. Southern blotting could also be performed on genomic DNA in which case you don´t need an enzyme that cuts within your gene of interest.
If you think cloning is a hassle, you could get a plasmid synthesized by some commercial provider. This has actually become quite affordable. I used the method for checking copy-number variation in cancer cells. In that case I used a multi-copy element as reference gene because some of these tissues and cell lines had massive large and small duplications etc. so any reference gene could sit on a duplicated chromosome etc. Using a multi-copy element spread on multiple chromosomes, duplication of one copy doesn’t matter that much. Multicopy elements can however be hard to identify, and as you probably have no reason to expect this kind of chaos in your plant genomes, using a couple of “normal” single copy genes as reference is probably good enough. The gene fragments do not have to be longer than what you need to perform qPCR.
For the qPCR, you could either use some arbitrary concentration of plasmid standard and make dilutions for the standard curve and compare you gene of interest (GOI) with one or optimally a few reference genes. Or better, you could calculate the exact number of plasmid molecules and hence template sequences in the standard curve dilutions and get an exact number of copies of your GOI in the genome template. You anyway need some reference genes as a check that things are working ok.
Best, vegard
For nitty gritty details on qPCR quantitation (more thorough than I ever did), check out this post, specifically the answer by Jack M Gallup:
https://www.researchgate.net/post/How_to_calculate_the_copy_no_in_qPCR_of_the_samples_from_their_respective_Ct
I should also mention, as I rememberd this as I read the post linked above: It massively improves the standard curve if you spike the standard dilutions with some DNA so that all the dilutions have the same DNA concentration. This could be plasmid without insert or whatever as long as it doesn't contain template sequences. I simply performed the starndard dilution in TE buffer containing a fixed concentration of no-insert plasmid (e.g. 20 or 50 ng/ul).
You can use pPCR or directly Sanger sequence the cDNA and then BLAST the sequence and look where it matches.
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