To answer this question let’s do a thought experiment. You have two bacterial whole genomes which differ in one base pair. Obviously these genomes are different, so there is certainly a sense in which these are different bacteria. The question now becomes, is the difference significant?

If the difference happened to inactivate the production of a lethal bacterial toxin in one of the two bacteria then the difference could be highly significant. It could mean that the one which produced the toxin was a lethal pathogen while the other was entirely benign. On the other hand, if the difference changed no codon in a housekeeping gene it would mean that there would be no detectable difference in behaviour between these two bacteria.

A restriction digest could pick up the difference, if an enzyme was used whose restriction site was changed by the single difference. But this would certainly not be likely to be the case for any one enzyme, and there could well be no known enzymes which would detect the difference. Sequencing, however, would in principle reveal the difference between the two bacteria.

So the answer to the question is, restriction digestion can be used to identify bacteria only in those cases where you already know a lot about the different bacteria which you want to identify. For anything else you need to sequence, and even that may not tell you what you want to know.

To answer this question let’s do a thought experiment. You have two bacterial whole genomes which differ in one base pair. Obviously these genomes are different, so there is certainly a sense in which these are different bacteria. The question now becomes, is the difference significant?

If the difference happened to inactivate the production of a lethal bacterial toxin in one of the two bacteria then the difference could be highly significant. It could mean that the one which produced the toxin was a lethal pathogen while the other was entirely benign. On the other hand, if the difference changed no codon in a housekeeping gene it would mean that there would be no detectable difference in behaviour between these two bacteria.

A restriction digest could pick up the difference, if an enzyme was used whose restriction site was changed by the single difference. But this would certainly not be likely to be the case for any one enzyme, and there could well be no known enzymes which would detect the difference. Sequencing, however, would in principle reveal the difference between the two bacteria.

So the answer to the question is, restriction digestion can be used to identify bacteria only in those cases where you already know a lot about the different bacteria which you want to identify. For anything else you need to sequence, and even that may not tell you what you want to know.

Basil gave you a good answer. In my opinion restriction digest is given you only limited information. 16S rRNA sequence is more secure and not much more work and cost, And in some cases you may have to do it after doing the restriction digest to be sure. So why not do it in the first place..

restriction digests are certainly not enough. They can be useful incertain circumstances, for example if you're screening isolates etc and want to group them based on restriction digestion patterns (I'd recommend using multiple profiles for each, with different enzymes), in addition to other criteria, e.g. morphology, physiology etc. It can be useful in certain cases, especially if isolates have been isolated using selective and/or differential media, and the aim is to 'confirm' identity so to speak. Even then you can't be 100% certain, if you want that, sequencing is the way to go.

You can amplify 16S Ribosomal RNA and do cheap sequencing of your PCR product (~10$) and then blast it to match to known organisms. From that you can say that based on commonly acceptable 16S based tree you have the organism you have. We know 16S has a fairly good resolution and is widely used. However, the species question is the one of semantics until better way to characterize bacteria is developed.

The question is not complete enough to a good and accurate answer. Mark, what do you pretend to identify, and what do you mean by identification. Do you want to recognize differences between two sorts of clones derived from the same original strain? (example: you introduced a suicidal plasmid in one of them and expect to see some effects on genomic DNA; or you induced another kind of mutation) Or are you working with sample isolates which you want to identify and eventually taxonomically characterize? If this is the case a restriction will only allow you to group bacteria. Identification, or a close identification is then obtained by sequencing the 16S rDNA of a representative strain. Even then it is not garanteed that will actually identify it.. since you need a phylogenetically close sequence present in the database. If that's not the case, don't despare. be happy. You may well be on your way to a nice systematics paper. Basil's answer is quite complete, especially if yo're doing a screening of mutants or strains of the same species :). Have a nice day!

I would like to isolate specific bacterium in saltern ponds. Is it a good idea to use restriction digestion to screen bacterium before sending them for sequencing? Would a restriction digest with more fragment be better or more reliable than ones with little fragment?

Thank you for your help sirs! Have a nice day!

If it is a good idea to screen isolates before sequencing 16S rDNA? Either by RFLP, RAPD or even total soluble protein profile, yes, since you will end up with several isolates which are in fact the same as others. Depending on how many isolates you have, you may end up by saving a considerable amount of time and money with this approach. This is especially true if you are dealing with more than 50 isolates per sample, and even more so if you intend to persue with taxonomic characterization if you stumble upon potentially new organisms. About more bands vs small bands. More bands is ok, if it doesn't become too messy, therefore compromising the profile analysis. Take care!

thank you Romeu! that was very helpful! thank you everyone! cheers!

sequencing is needed. by restriction analysis, you can see that something differs from something other, but you dont know what species are you looking at. the best way is to amplify 16S with eubacterial primers and let it sequenced. Then blast it live happily ever after:).

restriction enzymes method is not sufficient, use biochemical test and also DNA sequencing

We study gut bacteria, and we use both types. tRFLP using restriction enzymes can be helpful to see differences in the community between two samples, and it can suggest some of the species that might be present. It is good for doing lots of samples because it is much cheaper than sequencing. But if you want to really know what you have, you need to sequence. You could potentially use it to screen samples if you can predict the peak you want.

in order to identify a bacterial, you need to look at the genotypic and also phenotypic characteristics of the bacteria. In this case, you need to conduct some informative biochemical tests and DNA sequencing.