RFLP versus a single gene to study genotypic diversity - (Jan/28/2007 )
What is the better approach to study genotypic diversity among several bacterial isolates: Digesting genomic DNA of all isolated bacterial strains with restriction enzymes and separating the fragments (RFLP) with pulsed-field gel electrophoresis (PFGA) or sequencing a single gene (e.g. an elongation factor)?
If both methods had been used and the RFLP-PFGE approach yielded a high diversity whereas sequencing the elongation factor did not, would this indicate that RFLP-PFGE yields a higher resolution? This simply indicated the single gene is not sufficiently variable, or am I wrong here?
Does anyone know a reference that compared the two methods and came to the same conclusion?
Thanks!
-christian75-
QUOTE (christian75 @ Jan 28 2007, 06:44 PM)
What is the better approach to study genotypic diversity among several bacterial isolates: Digesting genomic DNA of all isolated bacterial strains with restriction enzymes and separating the fragments (RFLP) with pulsed-field gel electrophoresis (PFGA) or sequencing a single gene (e.g. an elongation factor)?
If both methods had been used and the RFLP-PFGE approach yielded a high diversity whereas sequencing the elongation factor did not, would this indicate that RFLP-PFGE yields a higher resolution? This simply indicated the single gene is not sufficiently variable, or am I wrong here?
Does anyone know a reference that compared the two methods and came to the same conclusion?
Thanks!
If both methods had been used and the RFLP-PFGE approach yielded a high diversity whereas sequencing the elongation factor did not, would this indicate that RFLP-PFGE yields a higher resolution? This simply indicated the single gene is not sufficiently variable, or am I wrong here?
Does anyone know a reference that compared the two methods and came to the same conclusion?
Thanks!
Hello Christian,
I cant come up with a ref at the moment, but I have some general remarks. I spent my last year genotyping M. tuberculosis strains and read quite a lot on the subject.
I would say that the best method depends on your species and whats known about it. Are there many known SNPs? Then SNP typing would be nice, especially for phylogenetics. Microsatellites are good for epidemiology and could be for phylogenetic and diversity studies, but not necessarily. Sequencing one gene can be nice, but generally one gene cant tell you very much about diversity and phylogenetics. Restriction enzyme based approaches such as RFLP needs alot of DNA, is laborous and boring but could give a nice view of diversity. I personally dont like RFLPs though, because of the uncertainties. Do we have a band here etc. AFLP might be better. Needs more planning, but gives a very good view of diversity if you can get it working, as you can analyze up to 200 bands.
Cheers!
have a ref a
-Chimp-
QUOTE (Chimp @ Feb 1 2007, 12:31 AM)
QUOTE (christian75 @ Jan 28 2007, 06:44 PM)
What is the better approach to study genotypic diversity among several bacterial isolates: Digesting genomic DNA of all isolated bacterial strains with restriction enzymes and separating the fragments (RFLP) with pulsed-field gel electrophoresis (PFGA) or sequencing a single gene (e.g. an elongation factor)?
If both methods had been used and the RFLP-PFGE approach yielded a high diversity whereas sequencing the elongation factor did not, would this indicate that RFLP-PFGE yields a higher resolution? This simply indicated the single gene is not sufficiently variable, or am I wrong here?
Does anyone know a reference that compared the two methods and came to the same conclusion?
Thanks!
If both methods had been used and the RFLP-PFGE approach yielded a high diversity whereas sequencing the elongation factor did not, would this indicate that RFLP-PFGE yields a higher resolution? This simply indicated the single gene is not sufficiently variable, or am I wrong here?
Does anyone know a reference that compared the two methods and came to the same conclusion?
Thanks!
Hello Christian,
I cant come up with a ref at the moment, but I have some general remarks. I spent my last year genotyping M. tuberculosis strains and read quite a lot on the subject.
I would say that the best method depends on your species and whats known about it. Are there many known SNPs? Then SNP typing would be nice, especially for phylogenetics. Microsatellites are good for epidemiology and could be for phylogenetic and diversity studies, but not necessarily. Sequencing one gene can be nice, but generally one gene cant tell you very much about diversity and phylogenetics. Restriction enzyme based approaches such as RFLP needs alot of DNA, is laborous and boring but could give a nice view of diversity. I personally dont like RFLPs though, because of the uncertainties. Do we have a band here etc. AFLP might be better. Needs more planning, but gives a very good view of diversity if you can get it working, as you can analyze up to 200 bands.
Cheers!
have a ref a
Hi chimp,
Thanks for your answer. My question related more to a project which has already been dome and is now in the review process. A referee asked for such a reference. I think you will not find such a paper because it depends pretty much on the gene you are looking at. Generally, however the RFLP methos covers the entire. So if you find a higher diversity with RFLPs and not with a single gene analysis (as is the case when comparing our studywith anotherone), then I would rather believe the RFLPs than the single gene, because in the latter you do not know apriori (if you haven't studied it) how the genetic variability of this locus is. If it is high in other actinomycete species does not necessarily mean it is also high for the species studied in our case. Do you agree with me or do yoou think I am wrong with this oppinion?
Cheers, Christian
-christian75-
I do agree that RFLPs probably gives a better view of phylogenetics than single gene studies, as long as you generally have quite a lot of bands (say >5). But there is of course no easy way to compare these data with single gene studies without e.g. carrying out single gene sequencing of some of your RFLP typed isolates.
Good look with the reviewers!
-Chimp-
QUOTE (Chimp @ Feb 8 2007, 10:59 PM)
I do agree that RFLPs probably gives a better view of phylogenetics than single gene studies, as long as you generally have quite a lot of bands (say >5). But there is of course no easy way to compare these data with single gene studies without e.g. carrying out single gene sequencing of some of your RFLP typed isolates.
Good look with the reviewers!
Good look with the reviewers!
Dear Chimp,
Thank you very much for your help. I found an article that actually tested this, but unfortumatley with another gene. They support our assessment with experimental data. Of course you cannot necessarily transfer such results from one species to another, but in general I think we may be right. Thanks again.
Cheers,
Christian
-christian75-