Hi all,
can anyone suggest me a method-protocol to understand if a bacterial gene is on the genomic or plasmidic DNA?I have done PCR on purified DNA sample (that contains both genomic and plasmdic DNA)and I found the gene I was looking for. Now I would like to understand if this gene is located on the plasmid or on the genome. How can I do that? I was said that I can do it by Southern Blot. So, I might extract the plasmidic DNA and hybridize it with a probe containing my gene. But what about the southern blot with genomic DNA if my DNA sample contains both the genomic and plasmidic DNA? With a southern blot on this extract, I can't distinguish if the gene is on plasmid or chromosome if the probe hybridizes. How can I see if the gene is only on the chromosome and not on plasmid as well?
Thanks
Dear Silvia
In addition with Dr. Franz's suggestions,
1) Plasmid (size: 20-80 Mda) are usually conjugative and have antibiotic resistant genes and virulence genes. If the bacteria harbor such type of plasmid, you can perform conjugation experiment. Then, confirm transfer of plasmid in your transconjugant. If the plasmid transfer via conjugation and PCR positive to your target gene that confirm that the plasmid have the gene.
2) Use PFGE technique to determine the location of the gene in bacterial chromosome. Because during chromosomal DNA preparation, plasmids are removed due to excessive washing. Then perform southern blot to detect the location of your target gene using DNA probe.
Hope it helps.
Dear Silvia
The two answers already posted seem very helpful, but I think that it is worth considering the known properties of your system before deciding on the best strategy. First I would want to know what bacterium you are working on and what the gene is that you are interested in and on that basis I would assess whether the gene is likely to be on a plasmid or in the chromosome and also whether it is likely to be associated with other genes of related function. Second, it would be helpful to know if plasmids are known in this bacterium and if so what properties they have - fo example selectable markers that would allow transfer of the plasmid to be followed. This context will help you to decide what is the simplest way forward.
I will be happy to provide more feedback if you want to share more information about your system.
Thank you Christopher.
I anticipate that it is a completely new field for me. Anyway, I am working on antibiotic resistant bacterial isolates and I am interested in determining the location of resistance genes, if they are located on the chromosome or on plasmids. This is done to understand if the resistance is transferred by mobile genetic elements. I don't know actually if my isolates have plasmids or not. So the first thing I should do is to analyse the presence of them in the isolates. What I was thinking is to extract the plasmids from the isolates (if there are any), run them on a gel and try to do a Southern Blot to see if my probe (that it would be the resistance gene) hybridizes with the plasmid extract. And I would do the Southern blot also with the genomic DNA. Now, my issue is the following: I can see with the Southern the presence or not of the gene if I use the plasmids extracted. But, in case I have a positive for the plasmids and I also have a positive for the Southern with genomic extract (since this latter contains both the plasmid and chromosomal DNA) , I can t distinguish if the gene is just on the plasmid or on both chromosome and plasmid. I hopei it is clear..
Dear Sylvia
Sorry for the slow response - last week was too busy. If you are dealing with a resistance gene that is well characterised in other strains then there should be a reasonable chance that you can predict something about its genetic context - for example, is it likely to be part of a transposon or integron or associated with an IS element. This could give you can idea about how big the DNA segment will be that is duplicated if there is a copy in the plasmid and in the chromosome. If there are two or more copies then you would expect that the duplicated DNA would be in different genomic contexts so in your southern blot of total DNA, so long as you cut with restriction enzymes the do not cut in the duplicated DNA but do cut in the flanking DNA, then you would expect to see just one band if there is a single copy and two or more bands if there are multiple copies. The same logic could be used for an enzyme that cuts in the gene of interest but not in the duplicated flanks when you would expected to get two bands for a single copy of the gene and four or more if there are multiple copies.
I hope this helps. Chris
- Badges
- Science topic