Restriction Mapping

Restriction Mapping

Restriction mapping is a physical mapping technique to determine the relative location of restriction sites on a DNA fragment to give a restriction map.

It is a method to map an unknown segment of DNA. First, by breaking it into pieces and then identifying the locations of the breakpoints. This method relies upon the use of proteins termed as restriction enzymes. These enzymes cut, or digest, DNA molecules at short, specific sequences termed as restriction sites.

Restriction Cleavage and Gel Electrophoresis

If we take a  DNA sample, digest it with a specific restriction enzyme and then subject the sample to gel electrophoresis, we will notice a series of band on the gel slab or cylinder. The position of different bands will depend on DNA fragment size. Smaller the fragment, more rapidly it will move, and longer the fragment, more slowly will it move. It will mean that the fragment away from the loading site will be smaller and those close to the loading site will represent longer DNA fragments.

The gel can be calibrated by using a mixture of DNA fragments of known lengths so that the position of bands on this standard gel can be compared with bands in the experimental DNA digest. Thus determining the fragment length in each band of DNA digest.

Construction of a restriction map

The data of digestion by more than one endonucleases is useful in arranging the sites of breakage in a defined order. This is possible by several methods and one of the method involves double digests. Here, we extract each fragment that the individual digest produces, with either enzyme A or enzyme B and then cleave it with the other enzyme. Now digestion of the original DNA sample by a mixture of both the enzymes takes place to confirm the results of individual successive digests.

In the figure below we can see the result of such an exercise. Firstly, the fragment A-2100 undergoes digestion with enzyme B. The enzyme digests it into two fragments  of 1900 base pairs and 200 base pairs. Then fragment B-2500 (from individual digest with enzyme B) similarly cuts into two fragments of 1900 bp and 600 bp, suggesting overlap of A-2100  and B-25oo (Fig.1), in the region of the fragment of 1900 bp. In reciprocal digests same fragments are available.

Using this information, we can find the overlapping regions in A and B digests and find out the sites of cleavage by A and B. This will then allow us to prepare the restriction map as we can see in Figure.2.

Use of partial digests, end labelling and hybridization in restriction mapping

The technique of individual and double digests can be supplemented with other techniques for actual construction of maps. For instance, by permitting incomplete digestion, fragments longer than those obtained by complete digestion may be obtained. These will be termed as partial digest. Another useful technique involves labelling of the ends i.e., end labelling of DNA molecule before digestion. By doing this we can easily identify the fragments containing these ends, even after digestion. Some of the features of restriction map can be confirmed by nucleic acid hybridization.

The above technique will help in accurate completion of a restriction map. But will require that we have a complete set of restriction fragments which make the entire DNA region being mapped.

Once the restriction map is ready, we can compare it with the genetic map. Changes due to deletions, duplications, inversions and interchanges on the genetic map can be easily located on restriction map. But point mutations cannot be easily mapped on the restriction map. It is because the restriction sites often do not change due to mutation. In such situations one may like to determine the nucleotide sequences of individual fragments and compare them in the normal and the mutant individuals.

Sequencing of nucleotides is more laborious and cannot be easily undertaken for the whole genome. Another technique of molecular genetic markers in the form of restriction  fragment length polymorphisms (RFLPs) is helpful. It is useful in generating genetic linkage maps in human beings maize, tomato etc.

Restriction fragment length polymorphisms (RFLPs) as markers for genetic maps

The genomic DNA, after digestion with restriction endonuclease and electrophoresis, may give rise to a continuous array of different sizes of fragments. This may be difficult to identify in eukaryotes. Therefore specific cloned sequences of DNA are useful in identifying variation at the DNA level. These variations are monitored as changes in the length of defined individual DNA fragments produced by digestion of DNA sample is restriction endonucleases. Therefore these variations are termed as restriction fragment length polymorphisms.

These polymorphic markers are helpful for genetic mapping in human beings, mice, fruit fly and in a number of crop plants. They make an area of research which is receiving considerable attention and excitement and will continue to be a thrust area for sometime.

The following experimental procedure demonstrates RFLPs

1. Extract and purify DNA from several individuals which may differ in some respects among themselves.
2. Digest DNA samples with restriction endonuclease.
3. Subject each DNA digest to gel electrophoresis separately, but on the same gel slab.
4. Gel electrophoresis easily separates the DNA fragments of different lengths resulting from digestion. But we cannot detect directly the differences in the distribution patterns of fragments in several DNA samples due to digestion. This is because the number of fragments is large and the range in size is rather continuous, such that they form a continuous smear on the gel.
5. The next step is the transfer of fragments from gel to solid support such as nitrocellulose filters- Southern blotting.
6. The next step is the hybridization of filters with small fragments with radioactively labelled DNA probes.  Autoradiography can detect the presence of individual fragments, fully or partially homologous to the probe, after hybridization. Since DNA clones having repetitive DNA may hybridize with many fragments, they will give a smear on autoradiography. Therefore, unique DNA sequences are in preference as probes for detecting RFLPs.

If two or more than two individuals differ in a restriction site affecting the number and length of DNA fragments homologous to the probe, the bands may differ in number and will appear at different locations in their corresponding autographs, which will be termed as RFLP at the phenotypic level.

Once a large number of RFLPs are available in a species, the  parents, F1 and F2’s can be useful in studying their inheritance and linkage relationships. We can also prepare genetic linkage maps . For preparation of RFLP maps, often computer programs are in use; one such program is MAPMAKER.