Restriction Mapping

Introduction:

Restriction mapping is the process of obtaining structural information on a piece of DNA by the use of restriction enzymes.

Restriction enzymes:

Restriction enzymes are enzymes that cut DNA at specific recognition sequences called "sites." They probably evolved as a bacterial defense against DNA bacteriophage. DNA invading a bacterial cell defended by these enzymes will be digested into small, non-functional pieces. The name "restriction enzyme" comes from the enzyme's function of restricting access to the cell. A bacterium protects its own DNA from these restriction enzymes by having another enzyme present that modifies these sites by adding a methyl group. For example, E.coli makes the restriction enzyme Eco RI and the methylating enzyme Eco RI methylase. The methylase modifies Eco RI sites in the bacteria's own genome to prevent it from being digested.

Restriction enzymes are endonucleases that recognize specific 4 to 8 base regions of DNA. For example, one restriction enzyme, Eco RI, recognizes the following six base sequence:

 

	              		                       
                  5' . . . G-A-A-T-T-C . . .  3'
                  3' . . . C-T-T-A-A-G . . .  5'
	              		                       

A piece of DNA incubated with Eco RI in the proper buffer conditions will be cut wherever this sequence appears. As you can see, this site is palindromic; that is, reading the upper strand from 5' to 3' is the same as reading the lower strand from 5' to 3'. As a result, each strand of the DNA can self-anneal and the DNA forms a small cruciform structure:

Figure 1

All restriction enzyme sites are palindromic. This structure may help the enzyme to recognize the sequence that it is designed to cut.

There are hundreds of restriction enzymes that have been isolated and each one recognizes its own specific nucleotide sequence. Sites for each restriction enzyme are distributed randomly throughout a particular DNA stretch. Digestion of DNA by restriction enzymes is very reproducible; every time a specific piece of DNA is cut by a specific enzyme, the same pattern of digestion will occur. Restriction enzymes are commercially available and their use has made manipulating DNA very easy.

Restriction Mapping:

Restriction mapping involves digesting DNA with a series of restriction enzymes and then separating the resultant DNA fragments by agarose gel electrophoresis. The distance between restriction enzyme sites can be determined by the patterns of fragments that are produced by the restriction enzyme digestion. In this way, information about the structure of an unknown piece of DNA can be obtained. An example of how this works is shown below. You have isolated a clone in pBluescript (look at bacterial transformation lab again to see its restriction map). You know how big the pBluescript portion of the plasmid is (3.0 kilobases) and what restriction enzymes are present in the plasmid (because you have its restriction map from the company that sold you the plasmid). You also know that the insert is 2.0 kb long and that it is inserted the Eco RI site. Your task is to find out more information about the insert:

Figure 2

At this point, you would digest plasmid with an enzyme that you know is in the pBluescript plasmid. For example, you know that there is only one Bam HI site in pBluescript, and it is in the multiple cloning site next to the Eco RI site (figure 2). If you digest this plasmid with Bam HI, there are two possibilities: 1) There are no Bam HI sites in the insert. If this is the case, when you run this digestion on a gel you will see only one DNA fragment, and it will be 5.0 kb long (3.0 kb of pBluescript DNA and 2.0 kb of insert DNA). 2) There is a Bam HI site in the insert. If this is the case, then the enzyme will cut the circular plasmid in two places, in the pBluescript part of the plasmid and in the insert. In this case, you will end up with two fragments of DNA. One will be pBluescript with some of the insert still attached and the other will be just insert. The sizes of the two fragments (determined by electrophoresis) will tell you where the site is. These two possibilities are shown in figure 3:

Figure 3

In the second case, where there is a site in the insert, the gel might look like this:

Figure4

In this case, we learn two pieces of information: 1) that there is a Bam HI site in the insert, and 2) where the site is in relation to the one end of the insert. When the Bam HI digestion is separated on an agarose gel, the sizes of the two fragments can be determined. In the above gel, the fragments are 3.6 kb and 1.4 kb. Therefore, we know that the Bam HI site is 1.4 kb away from the right hand side of the insert (figure 5). In this way, you have "mapped" the Bam HI site:

Figure 5

By testing the insert for the presence and location of sites of many different restriction enzymes, a "restriction map" of the clone is made. This will give us important structural information on the insert.

Uses of Restriction Mapping:

Restriction map information is important for many techniques used to manipulate DNA. One application is to cut a large piece of DNA into smaller fragments to allow it to be sequenced. Genes and cDNAs can be thousands of kilobases long (megabases - Mb); however, they can only be sequenced 400 bases at a time. DNA must be chopped up into smaller pieces and subcloned to perform the sequencing. Also, restriction mapping is an easy way to compare DNA fragments without having any information of their nucleotide sequence. For example, you may isolate two clones for a gene that are 8 kb and 10 kb long. You know that they overlap, because the procedure you used to isolate them told you that they have sequences in common. A restriction map can tell you how much they overlap by:

Figure 6

From the restriction map information, you can tell which parts of the two clones are identical and which parts are different. The parts of the clones that overlap are identical. If you were interested in the sequence of this gene, you would only have to sequence the area of overlap in one of the clones, greatly reducing the amount of sequencing that you would have to do.

Procedure:

The procedure for this experiment will be identical to the procedure of the last experiment. You will digest 3 ul of your plasmid with a restriction enzyme and then electrophorese it on an agarose gel. You will then determine if the insert has a site for that enzyme and where the site is based on the results of the agarose gel. The restriction enzyme you will use will be told to you at the beginning of class.

You will set up your restriction digestion like this:

 

          14.5 ul       H2O
           2.0 ul       10X Rest. Enzyme buffer
           3.0 ul       plasmid DNA solution
           0.5 ul       Restriction Enzyme
          20.0 ul       Total

When loading a gel, you should always load a DNA marker, your uncut plasmid and the vector plasmid (the plasmid without an insert) linearized. The DNA marker and linearized vector are used to estimate the sizes of your restriction fragments. They will be pre-measured and provided on your lab bench. You will set up the above digestion and allow it to digest for 30 min. While this is occurring you will put 18 ul of H2O and 2 ul of your plasmid uncut in a tube. When the digestion is complete, add 2 ul of dye to each of the four tubes (marker, linearized vector, digested plasmid, and uncut plasmid) and then load them on an agarose gel.

 

Identifying Your Unknown

After the gel has finished running, stain it as before and take a picture. Determine the sizes of the fragments resulting from your restriction digests by graphing on semi-log paper and construct a restriction map of your clone from this data. Determine which insert you have in your plasmid from among the possible inserts. (Restriction maps of the possible inserts will be handed out in class).

 











If you have questions please send me a message at my Email address