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

Detection of a change in DNA as revealed by the appearance or disappearance of a restriction site (an additional restriction site is present in the DNA segment shown on the left, which is absent in the segment on the right)
Fig. 40.6. Detection of a change in DNA as revealed by the appearance or disappearance of a restriction site (an additional restriction site is present in the DNA segment shown on the left, which is absent in the segment on the right).

The results of digestion of genomic DNAs of four hypothetical diplold individuals (1-4 shown in A : two homozygous—I & 2 and two heterozygous—3 & 4, for restriction sites and an insertion) with three enzymes (individually and in combination), followed by hybridization with a specific probe and autoradiography. Note the polymorphisms in three of the four cases due to different enzymes
Fig. 40.7. The results of digestion of genomic DNAs of four hypothetical diplold individuals (1-4 shown in A : two homozygous—I & 2 and two heterozygous—3 & 4, for restriction sites and an insertion) with three enzymes (individually and in combination), followed by hybridization with a specific probe and autoradiography. Note the polymorphisms in three of the four cases due to different enzymes.
The genomic DNA (DNA extracted from any organism), when subjected to digestion with a restriction endonuclease and electrophoresed, may give rise to a continuous array of different sizes of fragments, which may be difficult to identify in eukaryotes. Therefore specific cloned sequences of DNA can be used to identify 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 with restriction endonucleases. Therefore, these variations are termed as 'Restriction Fragment Length Polymorphisms (RFLPs)'. These polymorphic markers are being used for genetic mapping in human beings, mice, fruitfly and in a number of crop plants. They (RFLPs) make an area of research, which is receiving considerable attention and excitement and will continue to be a thrust area for some time.

An RFLP can be demonstrated using the following experimental procedure (i) Extract and purify DNA from several individuals which may differ in some respects among themselves, (ii) Digest DNA samples with a restriction endonuclease.
(iii) Subject each DNA digest to gel electrophoresis separately, but on the same gel slab, (iv) The DNA fragments of different lengths resulting from digestion can be separated by gel electrophoresis, but the differences in the distribution patterns of fragments in several DNA samples due to digestion can not be detected directly. 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.

Electrophoresis is therefore, followed by the following steps, (v) Fragments are transferred from gel to solid support such as nitrocellulose filters—Southern blotting (see Genetic Engineering and Biotechnology 1.  Recombinant DNA and PCR (Cloning and Amplification of DNA)). (vi) The filters with small fragments will be hybridized with radioactively labelled DNA probes (non-radioactive probes labelled with biotin or digoxigenin are also sometimes used), so that the presence of individual fragments, fully or partially homologous to the probe, can be detected by autoradiography 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 preferred 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/or will appear at different locations in their corresponding autographs, which will be called an RFLP at the phenotypic level. Figure 40.6 shows how the presence of an additional site will produce two fragments and its absence will produce only one fragment. In Figure 40.7, results of a hypothetical experiment are shown, where RFLPs are detected by using four plants (which differ by point mutations as well as by insertions) and three enzymes individually. Enzymes can also be used in combination to give double or triple digests.
Detection of a change in DNA as revealed by the appearance or disappearance of a restriction site (an additional restriction site is present in the DNA segment shown on the left, which is absent in the segment on the right)
Fig. 40.6. Detection of a change in DNA as revealed by the appearance or disappearance of a restriction site (an additional restriction site is present in the DNA segment shown on the left, which is absent in the segment on the right).

The results of digestion of genomic DNAs of four hypothetical diplold individuals (1-4 shown in A : two homozygous—I & 2 and two heterozygous—3 & 4, for restriction sites and an insertion) with three enzymes (individually and in combination), followed by hybridization with a specific probe and autoradiography. Note the polymorphisms in three of the four cases due to different enzymes
Fig. 40.7. The results of digestion of genomic DNAs of four hypothetical diplold individuals (1-4 shown in A : two homozygous—I & 2 and two heterozygous—3 & 4, for restriction sites and an insertion) with three enzymes (individually and in combination), followed by hybridization with a specific probe and autoradiography. Note the polymorphisms in three of the four cases due to different enzymes.

Once a large number of RFLPs are available in a species, the parents, F1 and F2's.can be used to study their inheritance and linkage relationship and genetic linkage maps can be prepared. For preparation of RFLP linkage maps, often computer programmes are used; one such programme in MAPMAKER. Such linkage maps have been prepared by using about one thousand RFLPs each in maize and tomato. The RFLPs have also been assigned to specific chromosomes using aneuploids involving specific chromosomes in several crops. Details of these RFLP maps in several crops are given in Table 40.1. Efforts are also being made to study linkage of RFLPs with morphological and economic traits so that they can be used for practical plant breeding (see next section).

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