Restriction map

From Wikipedia, the free encyclopedia

In molecular biology, restriction maps are used to compare relatedness of two different species at the molecular level. "Restriction mapping" is one of the three methods used by scientists, the other two methods being: DNA-DNA hybridization, and DNA or RNA sequence analysis.

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 by electrophoresis 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 that does 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.

[edit] Method

For linear DNA fragments, the positions of the restriction sites can be determined by carrying out a digest with one enzyme, then another enzyme, and then a double digest. 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. For demonstrative purposes, an EcoRI and HindIII restriction mapping experiment will be considered. The resulting samples and a DNA ladder are subsequently run using electrophoresis, 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, as they are all parts of the same original fragment. If fragment sizes do not properly add up, there are two likely problems. In one case, some of the smaller fragments may have ran 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. Continuing the above example, the fragments all add up to 4 kb and the initial fragment was 4kb long. The fragments produced by the EcoRI digest were: 800 bp and 3200 bp. The HindIII digest produced fragments of 2800 bp, 200 bp, and 1000 bp. The double digest produces fragments of 800, 2000, 200, and 1000 bp. By making a diagram and placing the restriction sites in the places that satisfy all of the digests you have inferred the placement of the restriction sites relative to each other.

  800       EcoRI|           2000                           HindIII|  200    HindIII|   1000

---

4 kb

The 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 purrified via rapid denaturation and renaturation.

In this technique all of the linear non supercoiled DNA is denatured and the stands are separated, when the temperature (or salt condition affecting backbone stabilization, higher salt=more backbone stabilization) is lowered quickly enough the separated 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.

The purified plasmid is ready for restriction enzyme digests. Once digested the plasmid is linear (if there was a restriction site in it). If there was one restriction cut site there will be one fragment, because the plasmid started off as circular. 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 in 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 and 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.

Sources:

 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..