Re: 16SRNA sequencing to determine differences between bacteria species

If the bacteria are unculturable (and the vast majority are, this is
why culture-independent bacterial community work has become
commonplace), there are other options short of doing next-gen 16S
amplicon seq.

As you're probably aware, three passages of subculturing is the
typical standard for isolation.

Some options if you can't obtain a pure culture / you're interested in
the unculturable portion of your community at hand:

1. You could make a 16S clone library. PCR amplify 16S and then clone
your PCR product. Sequence as many or few clones as necessary to
answer your question. Advantages: you can look at nearly full-length
16S sequences. Disadvantages: to elucidate diversity, you may have to
sequence a lot of clones. Not the case if you just want to ID the most
common members of a community.

2. Alternatively, Denaturing Gradient Gel Electrophoresis (DGGE)
involves PCR-amplifying a 16S fragment, adding a GC clamp in the
process. The PCR products (16S fragments with clamp) are run on an
arcylimide gel with a urea-formamide gradient. The PCR products are
separated by sequence as they denature in a sequence-specific manner
through the increasing gradient along the length of the gel. The gel
is stained with EtBr. Banding patterns can be used for comparison
directly (pretty crude), and bands can be excised from the gel, re-
amplified via PCR, and sequenced. Advantages: gives a (somewhat crude)
community profile without cloning. Disadvantages: limited sensitivity
- members constituting <5-10% of the community will likely be missed.
Also, you need to use acrylimide, which is a neurotoxin. The gradient
needs to be optimized for your project. And you need a way of casting
the gradient gel, and running a vertical gel at an elevated
temperature (say 60°C). It's all stuff that could be homegrown (the
gel running rigs are basically glorified fish tanks with a heater and
power supply) but would require work. And of course protection from
and disposal of the hazardous reagents.

Cheers,

Anthony

On Dec 3, 11:45 pm, Koz <kozovsk...@gmail.com> wrote:
> Cool. Thanks so much for this info.
> I'll share results as soon as I get something interesting :)
>
> Alexander
>
> On Dec 1, 11:16 pm, Nathan McCorkle <nmz...@gmail.com> wrote:
>
>
>
> > (Specifically Patrik's response):
> > On Oct 6, 12:00 am, Nathan McCorkle <nmz...@gmail.com> wrote:
>
> > > I was thinking that colony PCR is really quite easy, are there
> > > universal primers for 16S? I could get some in the mail in a few days
> > > for not more than $5-$15... not sure if I really need that level
> > > though, since I think this Prof is a "very very fair" grader.
>
> > JGI typically uses universal primers 27F/1391R for bacterial 16S rRNA:http://my.jgi.doe.gov/general/protocols/SOP_16S18S_rRNA_PCR_Library_C...
> > The Wikipedia page actually has a table with universal primers as
> > well:http://en.wikipedia.org/wiki/16S_ribosomal_RNA#Universal_Primers
> > 16S is such a common target in microbiology, that chances are somebody
> > at your lab may have some primers available that you could use. Ask
> > around among the grad students and postdocs...
>
> > On Thu, Dec 1, 2011 at 11:15 PM, Nathan McCorkle <nmz...@gmail.com> wrote:
> > > See this recent thread, you'll need lab access and tools to perform PCR:
>
> > > How to perform bacterial ID (I am trying to determine a Bacillus species)
> > >http://groups.google.com/group/diybio/browse_thread/thread/449334a66c...
>
> > > On Thu, Dec 1, 2011 at 9:58 PM, Koz <kozovsk...@gmail.com> wrote:
> > >> Hi ,
>
> > >> I want to do a fairly simple test and determine specific species of
> > >> bacteria that exist on different surfaces.
> > >> The agar plate/phenotype test does not give me enough precise data, so
> > >> I was wondering if there is a relatively inexpensive way to sequence
> > >> microorganisms from a collection of swabs?
> > >> Pretty new to this and have very limited access to basic lab equipment
> > >> so supposedly I would need to send this out.
> > >> Any suggestions?
>
> > >> Alexander
>
> > >> --
> > >> You received this message because you are subscribed to the Google Groups "DIYbio" group.
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>
> > > --
> > > Nathan McCorkle
> > > Rochester Institute of Technology
> > > College of Science, Biotechnology/Bioinformatics
>
> > --
> > Nathan McCorkle
> > Rochester Institute of Technology
> > College of Science, Biotechnology/Bioinformatics

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