[compost_tea] Re: The soil metagenome: Methods review

From: Steve Diver <steved_at_ncat.org>
Date: Thu, 19 Aug 2004 21:00:17 -0000
The additional notes you've provided are much
appreciated, Elaine.

Yes, let us make this a discussion.

For many of us, it is also necessary to have a basic
introduction to these molecular methods!! 

Some applications are more practical than others.

More later,

Steve Diver



--- In compost_tea_at_yahoogroups.com, soilfoodweb@a... wrote:
> You have to understand what is being measured, exactly, in order to
know if
> the tests used in molecualr biology measures what you want measured.
>
> What is the question you want answered?  Can these molecular methods
do what
> you want? 
>
> Let me review what the methods in Steve's e-mail are about.  All of
these
> involve methods to assess nucleic acids, sequences of nucleic acids,
and genetic
> relationships between organisms.
>
> rDNA sequencing
> DNA extraction and PCR analysis
> real-time PCR analysis
> PCR (Polymerase Chain Reaction)
> gel electrophoresis
> gene expression
> phylogenetic mapping
>
> rDNA means ribosomal DNA.  In eucaryotes, ribosomes are housed in the
> mitochondria.  You have mitochondrial nucleic acids in eucaryotes,
which are more
> highly conserved than the genomic DNA, as I understand. 
>
> Procaryotes do not have mitochondria, of course, so they don't have
this
> additional source of nucleic acid material.  Our ability to
understand genetic
> relationships is therefore much more limited in bacteria and virus,
since this
> additional set of nucleic acids is not present in procaryotes.
>
> DNA extraction is of course, just extracting DNA from any material. 
> Extracting DNA from mitochodria would be rDNA.  Otherwise, as DNA is
translated and
> multiplied, it is RNA, since cells use different nucleic acids in
producing the
> messages inside cells than for making the material of the genome. 
>
> Extracting DNA from soil was a major problem for many years, and
still messes
> certain people up, as they haven't paid attention to the fact that clay
> adsorbs nulceic acids quite well.  ANY protein will be rapidly
adsorbed on clay
> surfaces.  If the DNA you extract is sucked up by this material, the
researcher
> can, and it has happened, come to the comclusion that soil has low
diversity. 
> Not the case - the methods were faulty. 
>
> Once you extract DNA, you have to multiply it.  In order to work
with DNA,
> you need lots of it.  But you have to get the sequences the same as
were in the
> original material.  So, PCR is used to copy the sequences of DNA so
where you
> have one strand of DNA, you now have millions. 
>
> That's all that PCR is: a replication process.  Polymerase is an
enzyme that
> copies nucleic acid sequences.  But, if you extract DNA from all
cells in the
> soil, if you have one individual of one species, you will have only
one DNA
> sequence of that kind.  And if you replicate each strand of the 600
million DNA
> strands (the typical number of bacteria in a healthy soil, each
bacterium
> presumably having one genome) by a million times, you still only
have one strand
> of that DNA per 600 million strands.  Will that strand be detected
in the
> analysis of the sequences?
>
> Now, as we want to have our PCR product rapidly, we have moved to
real-time
> PCR, which has reduced the time for the whole multiplication process
to take
> place.  But when we hurry things, guess what gets lost? 
>
> So, real-time diversity?  Or actual diversity?  When we to gel
> electrophoresis of the products from PCR, are we likely to assess
the full diversity of the
> DNA strands?  Not if we have hurried things along. 
>
> Going fast allows us to get an idea of the dominant species, not the
full
> diversity.  Can there be a problem here, when interpreting this
information?  If
> you gloss over the details, yes, conclusions can be made that are
entirely
> inappropriate.  Care must be exercised in conclusions that are made. 
>
> Gene expression.  Genetic material is not always expressed.  For
example, you
> may have the genetic material which allows you to play the violin
like a
> virtuoso, but if you have never picked up a violin, that genetic
material will
> never be expressed.  Yes, it's a bit more complicated than that, as
practice is
> involved, but I think you get what I mean.  Genetic material is not
always
> used, not always expressed.
>
> So, for example, when people want to use fatty acid expression to
assess
> bacterial species diversity, they often incorrectly interpret the
data, because
> bacteria do not always express the fatty acids of interest.  Think
of the errors
> that are likely to ocurr. 
>
> But can assessing fatty acids gives us a good idea of diversity? 
>
> Show me data where full diversity has been assessed, and compare
that to
> fatty acid information.  Good correlation, or not?  Sometimes....
sometimese not. 
> So, when is the method going to be good, and when isn't it?  I am
not aware
> of such analysis.  If anyone is aware of such analysis, I'd love to
get that
> paper!  But until then, use fatty acid analysis to make conclusions
about
> diversity with a solid grain of salt.
>
> OK, back to the nucleic acid world.
>
> Molecular biologists have started to use RNA as a method of assessing
> organism diversity.  EXCEPT, RNA is only produced when a gene is
being expressed. 
> So, mRNA (messaenger RNA), rRNA (ribosomal RNA), whatever, are only
produced
> when DNA is being copied and translated. 
>
> So, can RNA ever be a method to assess diversity?  No, most of the
genes in
> any genome are not being expressed at any time. 
>
> Can you use RNA probes to tell you if a bacterium is present, or
not?  What
> if the RNA probe you are using is for an enzyme that is not being
expressed by
> that bacterium at this moment?  You are in trouble. 
>
> So, for E. coli, for example, if E. coli is not active, it does not
express
> beta-galactosidase, does not make the RNA that is what the molecular
probe
> attaches to.  Is this a problem?  Yes, if you need to know if any E.
coli is
> present. 
>
> But maybe we only need to know about ACTIVE E. coli, in which case
the RNA
> probe would be fine.
>
> Well, I could go on, but this is enough.  Probably too much.  I am
always
> happy to discuss this with folks, since there is more that I could
learn about
> these things. 
>
> But make it a discussion.  No attacks on the people trying to
understand or
> share knowledge.  And, as always, if I have made any errors of
omission or made
> a mistake in my information, a gentle correction is the most
reasonable way
> to start a discussion about any point. 
>
> Elaine R. Ingham
> Soil Foodweb Inc., Corvallis, Oregon




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Received on Thu Aug 19 2004 - 17:44:25 EDT

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