What is RFLP analysis?

The Procedure

Restriction fragment length polymorphism (RFLP) analysis is a method for distinguishing individuals and analyzing relatedness, based on genetic differences. RFLP analysis relies on small DNA sequence differences that lead to the loss or gain of restriction enzyme sites in a chromosome or to the change in size of a DNA fragment bracketed by restriction enzyme sites. These sequence differences lead to a different pattern of bands on a gel (reminiscent of a bar code) that varies from individual to individual.

RFLP analysis starts with the isolation of DNA. Typically, DNA isolation
requires the use of detergents, protein denaturants, RNA degrading enzymes, and alcohol precipitation to separate the DNA from the other cellular components. This DNA could be isolated from a blood sample provided by an individual, from evidence left at the scene of a crime, or from other sources of cells or tissues.

The purified DNA is then digested with a molecular “scissors” called a restriction enzyme. Restriction enzymes recognize and cut precise sequences, typically six base pairs in length. If one base pair is changed in that recognition sequence, the enzyme will not cut the DNA at that point. However, if a sequence that is not recognized by a restriction enzyme is altered by mutation, so that it now is recognized, the DNA will be cleaved at that point. In other cases, the DNA sequences recognized by the restriction enzymes themselves are not changed, but the length of DNA between two restriction enzyme sites differs between individuals. These types of mutations occur with enough regularity that often even two closely related individuals will have some detectable differences in the sizes of DNA fragments produced from restriction enzyme digestion.

Once the DNA has been digested with a restriction enzyme, it is separated by size in an agarose gel. At this point, the DNA appears, to the eye, to be a smear of molecules of all sizes, and it is not generally possible to differentiate the DNAs from different individuals at this stage. The size-fractioned DNA is next transferred to a nylon membrane in a process called Southern blotting. The result of the transfer is that the location and arrangement of the DNA fragments in the gel is maintained on the membrane, but the DNA is now single-stranded (critical for the next step in the process) and much easier to handle.

The final step in the process is to detect specific DNA fragments on the membrane. This is done by using a DNA fragment that is labeled to act as a probe, to home in on and identify similar DNA sequences on the membrane. Before use, the probe is made single-stranded, so it can bind to the single-stranded DNA on the membrane. The probe DNA can be labeled with radioactivity, in which case it is detected using X-ray film. The probe DNA can also be labeled with molecules that are bound by proteins, and the proteins can then be detected either directly or indirectly.

In a case in which a restriction enzyme site has been added or removed, the probe is normally a DNA fragment that is found in only one location in the genome. In cases in which one is looking at the size of fragments bracketed by restriction enzyme sites, the probe DNA is normally a DNA molecule that is found in several sites in the genome, and the DNA fragments that are identified in this analysis are ones that tend to vary between individuals. In many cases, the probe DNA binds to regions of DNA that consist of variable number tandem repeats (VNTRs). The number of VNTRs tends to vary between different individuals and, consequently, these sequences are useful for identification.

Applications

One of the earliest uses of this technique in clinical medicine was in the prenatal diagnosis of sickle-cell disease. Previous work had shown that many individuals with the disease had a mutation in their DNA that eliminated a restriction enzyme site in a gene encoding a hemoglobin protein. This information was used to develop a diagnostic RFLP procedure. A section of the hemoglobin gene is used as a probe. The size of restriction enzyme fragments identified is different in individuals who have sickle-cell disease (and therefore have two mutant alleles) compared with individuals who carry either one mutant allele or have two unmutated hemoglobin alleles. This method allowed for the identification of affected fetuses using DNA from cells isolated from amniotic fluid (a much simpler and
safer procedure than the previous method of diagnosis, which required isolating fetal red blood cells).

Another widely reported use of RFLP analysis has been in forensic science. RFLP methods have been critical in helping to identify criminals, and these methods have also helped exonerate innocent people. The first application of RFLP in forensic analysis was in the case of the murders of two young girls in England, in 1983 and 1986. Initially, a seventeen-year-old boy confessed to the murders. RFLP analysis, using DNA from the crime scene, indicated that he was not the murderer. After extensive investigation, including RFLP analysis of DNA from more than forty-five hundred men, a suspect was identified. Confronted with the evidence, the suspect pleaded guilty to both murders and was jailed for life. Since then, RFLP analysis has been used in thousands of criminal cases. Other forensic applications of RFLP include its use as evidence in court cases involving paternity determinations and its role in identifying the bodies of missing persons who otherwise could not be identified.

In addition to the clinical and forensic applications described above, RFLP analysis has been used in many subdisciplines of biology since the early 1980’s. The applications of RFLP analysis range from the conservation of endangered species to the identification of strains of bacteria associated with disease outbreaks to basic research involving the classification of organisms.

Although RFLP analysis has been widely used since its inception, it is increasingly being displaced by polymerase chain reaction (PCR) methods, which typically are much faster and require much less DNA. RFLP analysis was, however, an important step in the introduction of modern DNA analysis into the biology laboratory and the courtroom. The guiding principle behind RFLP analysis—identifying individuals, strains, and species, based on DNA sequence differences—remains a part of newer techniques.

Key terms



gel electrophoresis

:

a method for separating DNA molecules by size by applying electric current to force DNA through a matrix of agarose, which inhibits the migration of larger DNA fragments more than small DNA fragments




restriction enzymes

:

proteins that recognize specific DNA sequences and then cut the DNA, normally at the same sequence recognized by the enzymes




Southern blotting

:

a method for transferring DNA molecules from an agarose gel to a nylon membrane; once the DNA is on the membrane, it is incubated with a DNA containing an identifiable label and is then used to detect similar or identical DNA sequences on the membrane





Bibliography

Allison, Lizabeth A. “Recombinant DNA Technology and Molecular Cloning.” In Fundamental Molecular Biology. Malden, Mass.: Blackwell, 2007. Includes information about RFLP.

Chang, J. C., and Y. W. Kan. “A Sensitive New Prenatal Test for Sickle-Cell Anemia.” New England Journal of Medicine 307, no. 1 (July 1, 1982): 30-32. A short and readable scientific paper describing one of the first applications of RFLP analysis to clinical medicine. The same issue of the journal also has another, somewhat more detailed, article on the same topic by S. H. Orkin et al.

Guilfoile, P. A Photographic Atlas for the Molecular Biology Laboratory. Englewood, Colo.: Morton, 2000. An illustrated guide to molecular biology techniques, including a substantial illustrated section on RFLP analysis.

Hartl, D. L., and Elizabeth W. Jones. “Types of DNA Markers Present in Genomic DNA.” In Genetics: Analysis of Genes and Genomes. 7th ed. Sudbury, Mass.: Jones and Bartlett, 2009. This excellent introductory genetics textbook devotes a section of chapter 2 to a discussion of RFLP within the broader context of DNA structure and genetic variation.

Jeffreys, A., V. Wilson, and S. L. Thein. “Individual-Specific Fingerprints of Human DNA.” Nature 316, no. 6023 (July 4-10, 1985): 76-79. A technical article that describes some of the background information that led to the use of RFLP analysis in forensic science.

Karp, Gerald. “Genetic Analysis in Molecular Biology.” In Cell and Molecular Biology: Concepts and Experiments. 5th ed. Chichester, England: John Wiley and Sons, 2008. Includes information about RFLP.

Orkin, S. H., et al. “Improved Detection of the Sickle Mutation by DNA Analysis: Application to Prenatal Diagnosis.” New England Journal of Medicine 307, no. 1 (July 1, 1982): 32-36. Appears in the same issue as another article on the topic by J. C. Chang and Y. W. Kan.