Introduction
Because of the importance of restriction enzymes for cutting DNA in specific locations, restriction recognition sites are important reference points in a DNA sequence. When an organism's genome is sequenced, it is a relatively simple matter to map the location of restriction sites using computer software that compares series of nucleotides with recognition sequences of known restriction endonucleases. However, the locations of restriction sites in an unsequenced DNA fragment must be determined empirically. During this lab, you will learn how to infer the locations of restriction sites from agarose electrophoresis data and to construct a simple restriction map for a recombinant plasmid.
Relationship between gel band patterns, and number and size of fragments
The most fundamental information that can be derived from restriction digest band patterns on a gel is the size and number of fragments produced by the digest. The number of fragments is easily determined by counting the number of bands in the lane for that digest. You are already familiar with the use of a standard curve to determine the size of fragments from the distance their bands travel on a gel relative to known molecular mass standards. Closely related to the number of fragments produced in a digest is the number of times that a given restriction enzyme or combination of enzymes cuts a sequence. This depends on the form of the DNA being cut. If the DNA is linear, then the number of cuts is one less than the number of fragments. For closed circular DNA (such as a recombinant plasmid) the number of cuts is equal to the number of fragments. (Think about cutting a rubber band with scissors.)
Unfortunately, knowing the number of fragments and restriction "cut sites" tells us nothing about their ordering within the DNA sequence. Determining this requires making logical inferences from a comparison of single and double digests.
Fig. 1 Locations of cut sites with single digests of enzymes X and Y
Consider a plasmid with a circumference of 1000 bp. (We assume that basepairs have a consistent average mass and that a basepair can act as both a unit of size/mass and of length.) Restriction enzyme X cuts the plasmid twice and enzyme Y cuts the plasmid once (Fig. 1). If these digests were run on an agarose gel we would expect to see the results in Fig. 2.
Fig. 2 Gel showing fragments formed from single digests X and Y
