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Using RFLPs for mapping genetic diseases and for DNA fingerprinting

I. Definition:

Restriction Length Fragment Polymorphisms, RFLP's, are DNA differences that are inherited and can be used as genetic markers for diseases such as sickle cell anemia and phenylketonuria (Heller et al, 2001). RFLP's can also be used to construct a family pedigree and determine paternity.

These fragments are generated by cutting genomic DNA with a restriction endonuclease at a particular nucleotide sequence and separating the resulting fragments on a gel by performing a Southern blot (Campbell, 2001). The resulting RFLP's can be compared to RFLP's from corresponding DNA sequences from family members or unrelated people. By definition, this method detects DNA differences and therefore can only be used to distinguish polymorphic alleles from each other.

II. Making a RFLP:

The first step in producing a RFLP is to obtain a sample of blood, hair root, or other biological sample. The DNA from this sample is then cut with a restriction enzyme and multiple fragments are produced based on the DNA sequence as is shown in figure 1:

Figure 1: Cutting a Nucleotide Sequence at Particular Restriction Sites. A particular restriction enzyme cuts genomic DNA of person 1 and 2 at the GCGC nucleotide sequence. This image was obtained pending permission from Dr. Simon Lewis of Deakin University and the original reference is found here.

In this figure, the blue arrows show that the restriction enzyme cuts the nucleotide sequence between the first G and the first C. Person 1 will therefore have this DNA sequence cut into three fragments while Person 2 will have this DNA sequence cut into two fragments (Lewis, 2001). These fragments are then run on an agarose gel in separate lanes and the fragments will migrate towards the positive electrode to different degrees based on the molecular weight of each fragment. (The smaller fragments will move farther on the gel than the larger fragments.) The fragments then need to be visualized. This is commonly done by transferring the bands to a nitrocellulose gel and probing for the various DNA sequences contained in the fragments. Ideally, this probe is 6-10 bp, but in the simplified example above, the probe could be GCG, which would bind to the red CGC sequence contained by all fragments. The probe needs to be able to be visualized, and this can be done by exposing the radioactive probe to x-ray film. At this point, the fragments of various lengths can be visualized and the sequence differences between these two people can be visualized. Person one will show 3 DNA fragments and Person 2 will show 2 DNA fragments.


III. The Use of RFLPs in Mapping Genetic Diseases

In the following illustration, DNA from the hemoglobin gene from each family member is subjected to a particular restriction endonuclease. Since the hemoglobin gene is polymorphic, there is more than one DNA sequence encoding for this gene. Hb A is the wild type allele, and Hb S is the allele that codes for the sickling of red blood cells (Huskey, 2001). RFLP's are produced using this polymorphic DNA sequence and the resulting fragments are separated by gel electrophoresis and as shown in Figure 2:

Figure 2: RFLP's produced from fragments of the hemoglobin gene. Hb A corresponds to the wild type hemoglobin gene and Hb S corresponds to the diseased hemoglobin gene. This image was reproduced with permission by Dr. Robert J Huskey from the University of Virgina and can be found in its original version here.


The wild-type hemoglobin gene, Hb A, appears at 1.15 kb, while the sickled hemoglobin gene, Hb S, appears at 1.35 kb. A person homozygous for sickle cell anemia (S/S) shows only one RFLP at 1.35 kb, while people heterozygous for this disease (A/S) have RFLP's at 1.35 kb and 1.15 kb. People who have not inherited this gene (A/A) show one RFLP at 1.15 kb. (Figure 1 does not show a MW marker, but this marker is neccessary in order to determine the MW of the fragments.)

Can we positively conclude from this one pedigree as to which family members are homozygous and heterozygous for sickle cell anemia?

No. We must be absolutely sure that the genetic disease and the DNA sequence that produces these RFLP's are linked. It could be that these family members have inherited mutations on the hemoglobin gene, and that these mutations have absolutely nothing to do with the disease. Because of this, RFLP's need to be produced using various restriction enzymes and various DNA sequences to be sure that the polymorphic DNA sequence is directly related to the disease.


IV. The Use of RFLPs in DNA Fingerprinting

DNA fingerprinting is often used in criminal cases to determine a suspect's guilt. DNA from a blood, hair root, or semen sample found at the scene of a crime can prove guilt or innocence with high precision. The DNA sample is cleaved with a restriction enzyme and the resulting fragments are separated using Southern blotting techniques. DNA from the crime scene is analyzed on the same gel as DNA from the potential criminals, and therefore, DNA collected from the scene can be compared with that from various suspects and the RFLP's produced from the DNA of the guilty suspect will match with the RFLP's produced from DNA collected from the crime scene.

In the following example, a woman was raped while she and her fiance were sleeping in their car. They were found the next morning in the woods next to a recreation area and both had died of gunwounds. One man was later found driving the stolen vehicle and he told authorities of the friend that was with him the night of the murders. In order to determine which suspect was guilty of raping the woman, DNA fingerprinting (RFLP analysis) was used. DNA from a semen sample retrieved from the body was compared with DNA from blood samples from both suspects. These DNA samples were cut with a particular restriction enzyme and fragments were separated by gel electrophoresis. Figure 3 shows the RFLP's from the semen sample in yellow and the RFLP's from both suspects in purple and blue. It is shown in this figure that suspect 2 is the man that raped this woman. It is possible that an innocent person could show the exact same restriction fragments, but the chance of this is 1 in 9,390,000,000, which is twice the human population of the world! Suspect 2 was conviced of rape and murder and received a double death sentence. This happened to be the first case in the world in which the conviction of the death sentence was based on DNA fingerprinting (The Dolan DNA Learning Center, 2000).




Figure 3: RFLP analysis of the semen sample collected from the raped woman versus blood samples of two suspects. The pink and blue fragments were produced from genomic DNA from the suspects and the yellow fragments were produced from genomic DNA from the victim. This figure was reproduced pending permission by the Dolan DNA Learning Center.


Campbell MA. 2001. Southern Blot Method. Davidson College, Davidson. <http://bio.davidson.edu/Courses/genomics/method/Southernblot.html>. Accessed 2003, February 14.

Heller HC, Orians GH, Purves WK, Sadava D. 2001. Life: the Science of Biology, sixth edition. Sunderland, Massachusetts: Sinauer Associates, Inc., 337.

Huskey RJ. 1996. Genotype Determination USing RFLP's and a Gene Probe. University of Virginia. <http://www.people.virginia.edu/~rjh9u/hbsrflp.html>. Accessed 2003 February 14.

Lewis SW. 2001 June 5. RFLP DNA Typing. Deakin University. <http://www.deakin.edu.au/forensic/Chemical%20Detective/RFLP%20DNA%20Typing.htm>. Accessed 2003 February 17.

The Dolan DNA Learning Center. 2000. DNA Detective. <http://www.dnalc.org/resources/dnadetective.html>. Accessed 2003, February 14.


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