Biotechnology Index Glossary
A restriction map is a description of restriction endonuclease cleavage sites within a piece of DNA. Generating such a map is usually the first step in characterizing an unknown DNA, and a prerequisite to manipulating it for other purposes. Typically, restriction enzymes that cleave DNA infrequently (e.g. those with 6 bp recognition sites) and are relatively inexpensive are used to produce at a map.
The DNA to be restriction mapped it usually contained within a well-characterized plasmid or bacteriophage vector for which the sequence is known. In fact, there are usually multiple known restriction sites immediately flanking the uncharacterized DNA, which facilitates making the map. In the following discussion, it is assumed that the unknown DNA has been inserted into a plasmid vector, but the principles can readily be applied to other situations.
Creating a Map by Digesting DNA with Multiple Restriction Enzymes
The most straightforward method for restriction mapping is to digest samples of the plasmid with a set of individual enzymes, and with pairs of those enzymes. The digests are then "run out" on an agarose gel to determine sizes of the fragments generated. If you know the fragment sizes, it is usually a fairly easy task to deduce where each enzyme cuts, which is what mapping is all about.
To illustrate these idea, consider a plasmid that contains a 3000 base pair (bp) fragment of unknown DNA. Within the vector, immediately flanking the unknown DNA are unique recognition sites for the enzymes Kpn I and BamH I. As illustrated in the figure below, consider first seperate digestions with Kpn I and BamH I:
Digestion with Kpn I yields two fragments: 1000 bp and "big". Since there is a single Kpn I site in the vector, the presence of a 1000 bp fragment tells you that there is also a single Kpn I site in the unknown DNA and that it is 1000 bp from the Kpn I in the vector. The "big" fragment consist of the vector plus the remaining 2000 bp of the unknown.
Digestion with BamH I yields 3 fragments: 600, 2200 and "big". The "big" fragment is again the vector plus a little bit (200 bp in this case) of unknown DNA. The presence of 600 and 2200 bp fragments indicate that there are two BamH I sites in the unknown. You can deduce immediately that one BamH I site is 2800 bp (600 + 2200) from the BamH I in the vector. The second BamH I site can be in one of two positions: 600 or 2200 bp from the BamH I site in the vector. At this point, there is no way to know which of these alternative positions is correct.
The trick to determining where the second BamH I site is located is to digest the plasmid with Kpn I and BamH I together (click the diagram below with your mouse to see this effect). This so-called double digest yields fragments of 600, 1000 and 1200 bp (plus the "big" fragment). The 600 bp fragment is the same as obtained by digestion with BamH I alone. The 1000 and 1200 bp fragments tell you that Kpn I cut within the 2200 bp BamH I fragment observed when the plasmid was cut with BamH I alone. You already know where Kpn I cuts in the unknown DNA, and you therefore now know the location of the second BamH I site!
If the process outlined above were conducted with a larger let of enzymes, a much more complete map would result. In essense, single digests are used to determine which fragments are in the unknown DNA, and double digests to order and orient the fragments correctly.
Success in using this technique depends upon obtaining complete digestion of the DNA with each of the enzymes used! Partial digestion will yield fragments that are ultimately a great source of confusion. One way to avoid this problem is to add up the estimated sizes of all the fragments in each lane - if they don't sum to roughly that of the intact DNA, it is likely that digestion was not complete. One other thing to watch for is the presence of two fragments of roughly the same size, that may appear to be one fragment on an agarose gel. This situation is often suspected by observing an abnormally bright fragment on an ethidium-stained gel, or by a fragment being broader than expected.
Creating a Map by Partial Digests of End-Labeled DNA
If a fragment of DNA is labeled with a radioisotope on only one end, it can be partially digested with restriction enzymes to generate labeled fragments that directly reveal where the cleavage sites are located. Partial digestion, which is usually something to be avoided, is performed by using very small amounts of enzyme or short periods of time.
As an example of how this procedure is applied, consider the diagram to the right. The fragment of DNA to be mapped for Pst I sites is contained within a plasmid and flanked by restriction recognition sites that are not present in the fragment itself - in this case, Not I and EcoR I. The steps that might be taken to map this fragment by partial digestion are:
- Digest the plasmid to completion with EcoR I, then label the ends of the linearized plasmid with radioactive nucleotides.
- Digest the labeled DNA with Not I, run the digest on an agarose gel, and isolate the fragment of interest, which now is labeled on only one end. This DNA is the substrate to be used for partial digestion.
- Perform a partial digest the end-labeled fragment with Pst I - in addition to the full length fragment, this will generate 4 additional radiolabeled fragments.
- Seperate the labeled partial digestion products on an agarose gel, and expose the gel to Xray film (autoradiography) to visualize the sizes of the labeled fragments. By incorporating labeled molecular weight markers (not shown in diagram), the sizes of the partial digestion fragments can be deduced, and hence the positions of all the Pst I recognition sites.
A single preparation of end-labeled DNA can be used for mapping recognition sites for several different restriction enzymes, making this an efficient means of generating comprehensive maps. There are, however, at least two potential problems that can cause problems in interpretation:
- For a given enzyme, some recognition sites can be cleaved much less efficiently than others. The cause of this problem is usually not known, but it can lead one to miss mapping of certain sites.
- If is difficult to map sites near the ends of the fragment. For this reason, it is often best to perform the procedure twice, with preparations of fragment labeled at opposite ends (e.g. in the example, one preparation labeled at the EcoR I end and one labeled at the Not I end).
Using a Computer to Generate Restriction Maps
All of the techniques described above for generating a restriction map assume that you don't have the sequence of the DNA. If the sequence is known, it is a simple matter to feed that sequence into any number of computer programs, which will search the sequence for dozens of restriction enzyme recognition sites and build a map for you. One such program you can use is Mapper, available as part of the Molecular Toolkit.
Next Topic for Biology and Activity of Restriction Endonucleases: DNA Ligation
Last updated on January 19, 2000
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