DMS footprinting
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Dimethyl
sulphate (DMS) is one of the oldest and most versatile chemical reagents used
to probe RNA structure.
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It
was introduced for RNA structure mapping in 1980. This early method allowed
detection of methylation by DMS at N7 of guanosine and N3
of cytidine nucleotide because of modification facilitate cleavage of the
chain.
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The
sites of the methylation were determined by polyacrylamide gel electrophoresis
(PAGE) analysis of end-labelled RNA.
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Analogous
method was used to footprint the binding of protein to DNA.
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DMS
footprinting is performed by binding the protein to its end labelled DNA target
then attacking the DNA protein complex with DMS.
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DMS
footprinting follows a similar principle as fingerprinting except that the DNA
methylation agent DMS, instead of DNase.
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DMS
foot printing which starts in the same ways as DNase footprinting with end
DMS footprinting (Dimethyl
sulphate)
In vitro DNA
protein interaction
DMS
footprinting starts with end labelling of DNA and the protein
↓
DNA-protein
complex is methylated with DMS (Mild treatment) i.e. only one methylation event
occurs per DNA molecule.
↓
Protein is dislodge
(removed)
↓
DNA treated
with piperalin (baser amine) which removes methylated purines.
↓
Creating a purine site
↓
Gel autoradiography to detect labelled
DNA (Increases protein destroys the DNA double helix such that it makes the
base corresponding more vulnerable to methylation)
Application:
-
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DMS
footprinting has proven to be tremendously versitable method and has been
applied to large fraction known structure RNS`s.
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Initial
application of the method concern.
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