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