Plasmid Mapping
• Purpose: identifying the position of
“restriction sites” on a fragment of DNA
– Can help identify the position of a specific
gene within DNA
• Restriction enzymes (endonuclease) cut a
plasmid into smaller fragments of DNA
Gel Electrophoresis
• Procedure used to separate the DNA
fragments by their size
Gel Electrophoresis
1. Restriction enzymes “cut up” the DNA
samples into fragments
2. DNA samples placed into wells at one
end of the chamber
3. An electric current is applied to the gel –
smaller fragments move towards the
positive end faster than larger fragments
Example
• This fragment of
DNA is 7.0 kb
(kilobases) in length
• When digested with
Hind III enzyme,
two fragments result
(a 6.2 kb fragment
and a 0.8 kb
fragment)
Example
• Thus, we know there is a Hind III
restriction site 0.8 kb from one end of the
fragment
Example
• If we digest the
fragment with
another enzyme,
Sal I, two fragments
result
• Now, they are 5.8
kb and 1.2 kb in
length
Example
Example
• If the restriction sites for both enzymes are
on the same end, we’d expect the 0.8 kb
fragment to be within the 1.2 kb fragment
Example
• A double-enzyme digestion would give
three fragments (0.4, 0.8, and 5.8 kb)
Example
• However, if the restriction sites are at
opposite ends, we’d expect the 0.8 kb
fragment to be within the 5.8 kb fragment
Example
• A double-enzyme digestion would give
three fragments (0.8, 5.0, and 1.2 kb)
Example
• In order to
determine which
map is correct,
we must digest
the DNA with
both enzymes
Which is the correct model?
or
