Investigation 9: Restriction Enzyme Analysis of DNA

A restriction enzyme recognizes one specific DNA sequence, usually six base pairs long, and cuts the DNA only where that exact sequence appears. Different enzymes recognize different sequences, so the same plasmid cut with two different enzymes gets sliced at different locations, producing different numbers and sizes of fragments. That is why two digests of identical starting DNA show different band patterns on a gel: the DNA is the same, but the cut sites are not. The recognition sequence is usually a palindrome, reading the same on both strands in the 5' to 3' direction, which lets the enzyme bind symmetrically and cut both strands.

To predict band sizes from a plasmid map, treat the circular plasmid like a clock face marked in base pairs. Each cut site is a position on that circle. The fragment between two consecutive cut sites is just the distance separating them, and because the plasmid is circular, the fragments must add up to the total plasmid length. With three cut sites you get three fragments: go around the circle from one cut site to the next, then the next, then back to the first, subtracting positions in order and letting the final fragment wrap around through position 0.

On a gel, smaller fragments move farther because they slip through the gel matrix more easily, so fragment sizes are read off against a ladder of known linear sizes. This investigation connects directly to gel electrophoresis technique and to the idea of using controls and standards to interpret data, which shows up across the required investigations whenever raw measurements need translating into real quantities.