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Restriction Fragment Length Polymorphism RFLP
Restriction fragment length polymorphism denotes that a single restriction enzyme produces fragments of different lengths from the same stretch of genomic DNA of different strains of a species or from different related species.
RFLPs are detected as follows:
(i) large molecular weight genomic DNA is isolated from several strains or related species,
(ii) these DNAs are then digested with a selected restriction enzyme,
(iii) the fragments in these digests are separated through electrophoresis,
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(iv) the resulting gel lanes are transferred to a suitable solid support and exposed to a suitably
radio-labelled appropriate DNA probe under conditions favouring DNA: DNA hybridization (Southern hybridization),
(v) the free probes (not involved in hybridization) are removed, and finally (vi) the fragments to which the probe has hybridized are detected by filming them as distinct bands on a suitable photofilm through radioautography.
The pattern of RFLPs generated will depend mainly on the following:
(i) differences in the DNAs of selected strains/species,
(ii) the restriction enzyme used and
(iii) the DNA probe employed for Southern hybridization.
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Detectable RFLPs are generated due to the following changes in the DNAs of organisms:
(i) changes in the base sequences of recognition sites of the restriction enzymes used,
(ii) relatively large deletions and
(iii) relatively large additions in the concerned stretch of genomic DNA. A very large number of restriction enzymes is now available permitting the selection of such enzymes that would generate RFLPs.
The DNA probes may be obtained from
(i) genomic libraries,
(ii) cDNA libraries (these may be random or specific), or
(iii) chromosome specific libraries obtained from addition/substitution lines, flow-sorted chromosomes, chromosome-specific repeated sequences or micro-dissected chromosomes. Single-copy sequences (representing most likely, structural genes) are the best probes, but low-copy and even multiple-copy sequences are also used.
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Generally, probes prepared from the same species are used, but those from other species may also be employed. An RFLP is detected as a differential movement of a band on the gel lanes from different species/strains; each such band is regarded as a single RFLP locus.
It may be noted that an RFLP locus is definable only by the combination of a specific restriction enzyme with a specific DNA probe. The linkage among different RFLP loci and that between RFLP loci and oligogenes/ polygenes is readily determined by studying
(i) a set of recombinant inbreds derived from a suitable cross, or
(ii) F2 or backcross progeny from such a cross. (A suitable cross means the cross between two strains differing for the concerned RFLP loci and/or oligogenes/polygenes.)
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RFLP maps similar to the conventional linkage maps can be readily prepared, and these can be effectively integrated with the genetic maps prepared conventionally. Efficient protocols for such mappings have been developed.
The RFLP maps can be assigned to specific chromosomes/chromosome arms based on:
(i) linkage with genetic markers already assigned to specific chromosomes,
(ii) use of addition/substitution lines,
(iii) study of suitable translocation stocks,
(iv) employing monosomic/trisomic lines, or
(v) in situ hybridization to polytene chromosomes.
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RFLPs have several unique advantages:
(i) the number of RFLP loci is very large so that even very small segments of the chromosomes can be mapped; ideally every cistron could be mapped;
(ii) mapping does not necessarily depend on gene function,
(iii) even quantitative trait loci can be mapped which is virtually impossible through conventional techniques,
(iv) it is astoundingly rapid as compared to conventional linkage mapping and
(v) fewer individuals (25-50 individuals/F2 generation) need to be studied.
However,
(i) the technique is 100 to 1000 times as costly as the conventional linkage mapping.
(ii) It utilizes radioactive probes which are risky to handle and difficult to dispose off. Further,
(iii) the technique requires considerable skill and effort, and far greater time than, say, RAPDs (random amplified polymorphic DNAs) (Appendix 4.III).
RFLPs may have the following applications:
(i) identification and isolation of any gene known to be linked with an RFLP locus,
(ii) finger printing of strains/varieties for their unequivocal identification,
(iii) linkage mapping of quantitative trait loci,
(iv) identification of the most important loci affecting a quantitative trait,
(v) highly efficient indirect selection for tightly linked quantitative trait loci and even for those oligogenes a direct selection for which may either be difficult or costly,
(vi) determination of chromosome segments alteration of which is likely to yield the best results,
(vii) establishing the relationships among various strains/species,
(viii) understanding the identity and function of thus far 'mysterious' polygenes etc. RFLP maps are being generated for several crop species, e.g., more notably, maize, rice, wheat, etc.; it is hoped that the entire genome of Arabidopsis thaliana will soon be mapped to saturation or even sequenced in full.
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