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16s rRNA sequencing to determine bacteria present


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#1 Hallm_7

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Posted 11 March 2005 - 03:26 PM

Has anyone here ever done a community diversity study by using 16s rRNA sequencing? My lab has just begun to look into it to determine bacterial populations in soil and water. However, none of us have ever done it, and was hoping someone here could give advice or point us to a website or book that could help.

I have a couple of specific questions: when you are cloning the amplified fragment from PCR into a vector, do you use a kit (such as Invitrogen's Topo TA Cloning Kit) or do you use your own ligase and vector?

Secondly, let's say that there are 100 species of bacteria in a sample. When we amplify the 16s rRNA and all species form a ~1400 bp sequence, how will we separate the 100 difference fragments from each other to clone into a vector? If you had 100 fragments of 1400 bp each, and you then cloned them into a vector, wouldn't you need to eventually separate clones of each of the 100 species from each other? Maybe I'm misundrestanding the procedure here, but I just don't see at which point there will be one PCR fragment per colony for every species present in the sample.

Any direction or help would be GREATLY appreciated!

#2 phage434

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Posted 13 March 2005 - 08:19 PM

Well, the procedure is to PCR with universal 16S primers, to produce a mixed set of amplified 16S products, a different sequence from each organism present.

Then, the PCR product is cloned into a standard E. coli vector. The vector is transformed into E. coli, and antibiotic is used to select transformants with the vector present.

Single colonies of the resulting transformants are streaked out on plates. Each of these is a descendant of a single E. coli cell, containing a single plasmid. So, each of the colonies has a clonal population of identical cells containing identical plasmids. Each colony has a *different* plasmid, containing a different 16S amplified sequence.

When you prepare plasmid DNA from the cells in a single colony, you get a sequence corresponding to a single 16S sequence of an environmental isolate. By sequencing a large number of different colonies, you get a general idea of the diversity of the species in your environment.

#3 Hallm_7

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Posted 14 March 2005 - 07:43 PM

Thanks a lot for the help! That definitely helped clear up some things.




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