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Agarose and RNA
Name: Angela
Status: Other
Grade: Other
Location: AK
Country: United States
Date: April 2008
Question:
Why is the RNA denatured when run through agarose gels,
but DNA is typically not denatured while it is run?
Replies:
DNA is typically double-stranded, and RNA is typically single-stranded. When
you refer to DNA being denatured, it means the double strands separate --
which usually requires temperatures near boiling. Gels are not usually run
at this high a temperature. Thus, DNA that starts double-stranded will stay
that way, and RNA that starts single-stranded will also stay that way. When
running plasmid DNA (plasmids are rings instead of linear pieces of DNA),
you often will cut it first using an enzyme, but this is not the same as
denaturing.
Another take on this has to do with the shape of DNA strands. In solution,
they coil up on each other like a ball of yarn. In order to migrate through
a gel, a large biomolecule must uncoil. When proteins uncoil, they often
lose their function -- this is called denaturing. Because DNA's function
isn't shape dependent like proteins, when DNA uncoils it isn't referred to
as 'denaturing'.
Hope this helps,
Burr
Good question, Angela. RNA is single-stranded, but it is possible for small areas
to base pair within the same strand. For example, if there is a stretch of GGGG,
and then, say, twenty bases down the strand a stretch of CCCC, the G's can pair
with the C's, forming what is called a hairpin loop. In other words, there is
"intramolecular base pairing," to use the fancy terminology. This RNA will run
differently in a gel than an RNA of the same size that does not have any
intramolecular base pairing. Adding something to denature the RNA prevents this
type of base pairing, so that the RNA runs "true" in a gel, according to its
overall length.
Since DNA is double stranded to begin with, there is no need to worry about this
occuring.
Paul Mahoney, Ph.D.
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Update: June 2012
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