Restriction Mapping
of Plasmid DNA
Restriction Maps
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Restriction enzymes can be used to construct
maps of plasmid DNA
Allows identification of restriction sites found in
the sequence of the plasmid
Alternative means to discover DNA sequence
without sequencing
Can be used to determine which restriction
enzymes to use for recombinant techniques
Getting the Information
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Isolate the DNA
Digest it with restriction enzymes
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Sort fragments by size using gel electrophoresis
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Breaks DNA into various size fragments
Can sort DNA fragments differing by only a single
nucleotide in length
Fragment lengths can be used to assemble map
of relative positions
Digestion
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Can do single or multiple digestions
Single digestion consists of only one enzyme in the
mixture
Multiple digestions include many or all the enzymes
desired
Step-wise process gives the most information
Digest with individual enzymes and include a multiple
digestion
Use different sizes of the fragments to construct the
map
Making the Map
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The gel will be stained and photographed after running
Distances migrated by DNA fragments will be
measured from the photograph of the gel
A standard curve will be produced by plotting distances
migrated by ladder fragments against the size of the
fragments
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Semilog graph paper must be used to obtain a linear plot
The standard curve will be use to determine fragment
sizes of the digested plasmid DNA
A circular map will be constructed from the data