Restriction map

A restriction map is a map of known restriction sites within a sequence of DNA. Restriction mapping requires the use of restriction enzymes. In molecular biology, restriction maps—along with DNA-DNA hybridization, and DNA or RNA sequence analysis—are used to determine the genetic relationships between two or more subjects, often different species, at the molecular level.

Restriction enzymes

In restriction maps, the same restriction enzymes are used as in recombinant DNA technology. Each type of restriction enzyme recognizes a specific sequence of a few nucleotides and cleaves DNA wherever such sequences are found in the genome. The DNA fragments found after the treatment can be separated via electrophoresis gel and compared to restriction fragments derived from the DNA of another species. Two samples of DNA with similar maps for the locations of restriction sites will produce similar collections of fragments. In contrast, two genomes that have diverged extensively since their last common ancestor will have a very different distribution of restriction sites, and the DNA will not match closely in the sizes of restriction fragments.

Because so many fragments are obtained from the genome of a cell, restriction mapping is more practical for comparing smaller segments of DNA, usually a few thousand nucleotides long. Several laboratories have used restriction maps to compare mitochondrial DNA (mtDNA) for eukaryotic organisms (as opposed to prokaryotes), which is relatively small. There is the added benefit that mtDNA changes by mutation about ten times faster than the nuclear genome, which makes it possible to sort out phylogenetic relationships between very closely related species, or even between different populations of the same species.

Method

The length of restriction recognition sites varies: The enzymes EcoRI, SacI and SstI each recognize a 6 base-pair (bp) sequence of DNA, whereas NotI recognizes a sequence 8 bp in length, and the recognition site for Sau3AI is only 4 bp in length. Length of the recognition sequence dictates how frequently the enzyme will cut in a random sequence of DNA. Enzymes with a 6 bp recognition site will cut, on average, every 4⁶ or 4096 bp; a 4 bp recognition site will occur roughly every 256 bp. Different restriction enzymes can have the same recognition site - such enzymes are called isoschizomers: Look at the recognition sites for SacI and SstI - they are identical. In some cases isoschizomers cut identically within their recognition site, but sometimes they do not. Isoschizomers often have different optimum reaction conditions, stabilities and costs, which may influence the decision of which to purchase. Restriction recognitions sites can be unambiguous or ambiguous: The enzyme BamHI recognizes the sequence GGATCC and no others - this is what is meant by unambiguous. In contrast, HinfI recognizes a 5 bp sequence starting with GA, ending in TC, and having any base between (in the table, "N" stands for any nucleotide) - HinfI has an ambiguous recognition site. XhoII also has an ambiguous recognition site: Py stands for pyrimidine (T or C) and Pu for purine (A or G), so XhoII will recognize and cut sequences of AGATCT, AGATCC, GGATCT and GGATCC. The recognition site for one enzyme may contain the restriction site for another: For example, note that a BamHI recognition site contains the recognition site for Sau3AI. Consequently, all BamHI sites will cut with Sau3AI. Similarly, one of the four possible XhoII sites will also be a recognition site for BamHI and all four will cut with Sau3AI. For linear DNA fragments, the positions of restriction sites in the fragment can be determined by carrying out digests with two different restriction endonucleases. The fragment of interest must contain sites for the chosen restriction enzymes.

The experimental procedure first requires 3 aliquots of the purified DNA fragment. Digestion is then performed with each enzyme. One aliquot receives one enzyme, another aliquot receives the other enzyme, and the final aliquot receives both enzymes (i.e. the double digest). The resulting samples are subsequently run on an electrophoresis gel, typically on agarose gel.

The first step following the completion of electrophoresis, is to add up the sizes of the fragments in each lane (Moffatt 2006). The sum of the individual fragments should equal the size of the original fragment, and each digests fragments should also sum up to be the same size as each other. If fragment sizes do not properly add up, there are two likely problems. In one case, some of the smaller fragments may have run off the end of the gel. This frequently occurs if the gel is run too long. A second possible source of error is that the gel was not dense enough and therefore was unable to resolve fragments close in size. This leads to a lack of separation of fragments which were close in size. If all of the digests produce fragments that add up one may infer the position of the REN (restriction endonuclease) sites by placing them in spots on the original DNA fragment that would satisfy the fragment sizes produced by all three digests.

Example

The purified plasmid is ready for restriction enzyme digests. Once digested the plasmid is linear. The linear DNA can be run on a gel alongside a standard marker (fragments of known sizes).

This technique can be used to check for the pressence of an insert in a plasmid vector by digesting with the same enzyme used to insert the gene to be cloned(Dale, Von Schantz, 2003). This will generate two fragments. Vectors are designed to have certain restriction sites n certain places so during cloning the plasmid is not cut more than once, so ligation is possible. It is also possible to check that the insert was oriented properly(Dale, Von Schantz, 2003). If you know of a restriction site placed towards one end of the insert you can determine the orientation by observing the size of the fragments in the gel.

For example, if you have a recombinant plasmid anThe following is an explanation of using restriction mapping for studying plasmids. It would be necessary to perform some restriction digests and do some mapping when working with plasmids as vectors for cloning.

To do the restriction mapping procedure it is necessary to have a pure sample of the unit of DNA you are studying (Dale, Von Schantz, 2003). In the case of studying a transformed or untransformed plasmid the plasmid can be purified via rapid denaturation and renaturation (see d you know that there is a HindIII site in your insert 2 kb in (consider a 3 kb insert). There is also a HindIII in the multicloning site in your vector, but the insert was cloned using EcoRI. The ends of the insert would be cut with EcoRI excising the insert, but HindIII will not excise the insert. You know from an earlier digest with EcoRI that the plasmid is 5 kb long and that the insert is 3 kb long. Now you do your digest with HindIII. You run the resulting fragments on a gel alongside the standard marker, and you observe 2 fragments. One fragment is 2 kb long and the other is 6 kb long. The 2 kb fragment is part of the insert, the 6 kb fragment is the remaining insert plus the whole plasmid vector. That means that your 2 kb fragment comes from the end of your insert furthest from the multi-cloning site, and the remaining 1 kb of your insert is adjacent to the multicloning site. If the insert orientation were opposite this you would observe fragments of 7 kb and 1 kb.

Restriction mapping is a useful tool for selecting recombinant colonies that turned out the way you wanted.

Appendix

Rapid Denaturation and Renaturation: In this technique the DNA mixture is heated. The DNA in the mixture is denatured (strands separated). The linear supercoiled DNA is subject to tangling and staying denatured when the temperature is lowered quickly after heating. In other words, the strands come back together in a disordered fashion, basepairing randomly. The circular supercoiled plasmids' strands will stay relatively closely aligned and will renature correctly. Therefore, the linear DNA will form an insoluble aggregate and the supercoiled plasmids will be left in solution. This can be followed by phenol extraction to remove proteins and other molecules.

See also

* Vector NTI - bioinformatics software used among other things to predict restriction sites on a DNA vector

References

*Moffatt, B (2006). Course Notes Biology 208.Waterloo, University of Waterloo.
*Dale, J, & von Schantz, M (2003). From Genes to Genomes.West Sussex:John Wiley & Sons Ltd..