Restriction Fragment Length Polymorphism (Rflp)
As DNA changes are not restricted to those that affect phenotype, restriction fragment length polymorphisms (RFLP) analysis is a powerful technique for the characterization of DNA at the molecular level. These markers are inherited in the same manner as genes that code for visible phenotypes. The recombination frequency between an RFLP and a detectable phenotype can be measured. Thus, genetic maps can be constructed to include both genotypic and phenotypic markers. RFLPs can thereby provide a link between genes that lie far apart. In 1980, an RFLP map was created for the human genome.
Restriction maps that result from different patterns of distribution of restriction sites in the DNA of individuals within a population of organisms are called restriction fragment length polymorphisms (RFLPs). Differences in individual base pairs between comparable sequences of any two individual chromosomes occur at a frequency of greater than 1 change per kilobase. Highly polymorphic regions are usually located between genes, where small variations in sequence do not affect gene expression. The polymorphisms can be identified by the digestion of genomic DNA with a restriction enzyme (endonuclease). Differences in sequences that result in the gain or loss of a restriction site cause variations in the lengths of the fragments produced. Polymorphisms can also result from the insertion or deletion of stretches of DNA between two restriction sites.
To visualize an RFLP, Southern blotting techniques are used to identify fragments of various lengths based on the location of the sites of a particular restriction enzyme. Probes of known sequence that highlight restriction fragments that often vary in length among different individuals are used to generate clearly discernible patterns. A probe specific for a portion of a particular chromosome will reveal differences within that region of the genome from one individual to another.
Because restriction polymorphisms should occur near any particular target gene, RFLPs can be identified that show tight linkage with a mutant (or disease) phenotype. Comparison of restriction maps of patients suffering from a particular disease with those of unaffected individuals can reveal specific bands that are always present or absent in affected individuals.
After the identification of an RFLP that is tightly linked with a disease, it may be used as a molecular procedure to detect the disease, either in a prenatal screen or after birth. For instance, an RFLP has been identified that is associated with the genetic disease sickle cell anemia. Sickle cell anemia is caused by a mutation in the alpha-globin gene and results in an abnormal form of hemoglobin. Digestion of DNA with the restriction enzyme Hpa1 and Southern analysis using a probe specific for the alpha-globin gene results in the production of a 7-kb fragment in normal individuals. In contrast, patients with sickle cell anemia display a fragment of 13-kb. Carriers of the disease, those who do not have sickle-cell anemia, but who have inherited one copy of the mutant gene from a parent, can also be identified by RFLP analysis. They will produce both the 7- and 13-kb fragments. It is important to note that the change in DNA sequence that causes this RFLP is not the same change that causes sickle cell anemia itself. Instead, it is tightly linked to the alpha-globin gene. Other human genetic diseases that can be detected via RFLP analysis include Huntington's Chorea, phenylketonuria, and cystic fibrosis.
RFLP analysis is particularly useful for diagnosis of disease because it assays directly for a genotype (DNA sequence) and does not depend on expression of a gene or even phenotypic expression of the disease itself. Thus, a disease can be identified in an individual before symptoms of the disease are apparent. Additionally, a fetus can be monitored for diseases before birth.
Additionally, RFLPs provide a beginning point for the isolation of the gene responsible for a disease. If the mutation that causes an RFLP in fact lies within the gene responsible, an RFLP at this gene must occur in all cases of the disease. Therefore, it is difficult to prove that a defect in a particular gene is in fact responsible for causing a disease. However, mapping and RFLP analysis can exclude certain genes as candidates. They may also provide a point from which researchers may proceed along the DNA to identify the causal gene itself.
The technique termed DNA fingerprinting utilizes RFLPs and other polymorphic markers to identify individuals based on their particular patterns. Through this technique, hair, blood or other bodily fluids found at a crime scene and blood obtained from a suspect can be used to compare DNA fingerprint patterns. A match between the two patterns provides strong evidence against the suspect. RFLP patterns are also used to establish parent-child relationships by comparison of the map of a suitable region of the chromosome between potential parents and the child.
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