RFLP and Genetic Screening PCR Analysis of the Cystic Fibrosis Gene Linked DNA Markers Study Questions Molecular Markers WWW Links Genetic Topics

RFLP and Genetic Screening Restriction fragment length polymorphisms (RFLP) have become key elements in systems to diagnose diseases. Let's look at how these experiments are performed. First, as with any genetic comparison we need variable phenotypes and some way to detect them. Human diseases can be detected clinically. Sickle cell anemia patients can be detected by the shape of their blood cells, and hemophiliacs can be identified by the inability of their blood to coagulate properly. But we now have the capability to detect in utero indivi duals that will express these traits. Two tools are needed to perform these DNA type diagnoses. First we need a restriction enzyme that will cut DNA in a manner that is informative for the disease in question, and second we need a nucleic acid probe that will detect that region. Not all restriction enzyme sites will reside in the same position in all individuals. Thus if we cut DNA with a restriction enzyme we may not obtain the same size DNA fragments. For example: Sample A: | 1 kb | 4 kb | _________________________________ Probe: XXXXXXXXXXXXX Sample A: | 5 kb | _________________________________ Probe: XXXXXXXXXXXXX Note: The symbol "|" denotes the restriction enzyme cleavage site. The procedure that is used to analyze the DNA is Southern hybridization. The two DNA samples are cut with the appropriate enzyme and are hybridized with the correct probe. The probe hybridized to the 1 and 4 kb fragments in sample A and the 5 kb fragment in sample B. This is an example were a restriction site has been gained in sample A (or lost in sample B) by mutation. Sample A Sample B ------------------- 5.0 kb ___ 4.0 kb ___ 1.0 kb ___ ------------------- Now let's look at a second example. Sample A: | 3 kb | 3 kb | _________________________________ Probe XXXXXXX Sample A: | 2 kb | 4 kb | _________________________________ Probe XXXXXXX For this example, the Southern Hybridization would look like this. Sample A Sample B ------------------- 3.0 kb ___ 2.0 kb ___ ------------------- In this example a 3 kb fragment is recognized in sample A and a 2 kb fragment is recognized in sample B. These two examples demonstrate a powerful technique that is becoming widely used and accepted: restriction fragment length polymorphism (or RFLP). These two examples demonstrate how this name is derived. All the samples we are looking at are examples of restriction enzyme fragments. In each example we see a polymorphism between the size of the fragments that are recognized by the probe. It is important to remember that any RFLP is unique to a specific restriction enzyme and nucleic acid probe. RFLP Screening for Human Diseases Let's look at specific examples. The first RFLP example will detect an individual with sickle cell anemia. Remember that sickle cell is the result of a change in the #6 amino acid of the ß- globin chain of hemoglobin. Specifically glutamic acid is converted to valine. This results from a change in the nucleotide A to T. This change eliminates a site recognized by the restriction enzyme DdeI. Restriction enzyme: DdeI (recognition sequence: 5'-GTNAG-3') Probe: fragment of ß-globin coding sequence Site Eliminated Normal ß-Globin | 175 bp | 201 bp | Allele ____________________________ Probe XXXXXXXXXXXXXXXXXXX Sickled ß-Globin | 376 bp | Allele ____________________________ Probe XXXXXXXXXXXXXXXXXXX Normal Sickled ß-Globin ß-Globin ------------------ 376 bp ___ 201 bp ___ 175 bp ___ ------------------ Each of the two marker patterns (376 bp vs. 201 + 175 bp) can be considered alleles. These markers can also be used to describe a biochemical phenotype. The second example is for a DNA markers for human X-linked disease hemophilia A. This disease affects 1/10,000 males. The affected gene produces Factor VIII a component of the blood coagulation pathway. This his gene is mutated in affected patients. For Hemophilia A two alleles exist: 1.2 kb fragment = hemophilia allele 0.9 kb fragment = normal allele Restriction enzyme: BclI Probe: Fragment from Factor VIII coding region Normal Allele | 0.9 kb | 0.3 kb | _________________________ Probe XXXXXXXXXXXXX Hemophilia Allele | 1.2 kb | _________________________ Probe XXXXXXXXXXXXX Representation of an autoradiogram for hemophilia individuals Normal Normal Carrier Affected Affected Male Female Female Female Male ---------------------------------------- 1.2 kb ___ ___2x ___ 0.9 kb ___ ___2x ___ ---------------------------------------- 2x means the intensity of the band is twice that of the other bands. Why is the female band twice as intense? Because they contain two X chromosomes, therefore twice the amount of DNA. Copyright © 1997. Phillip McClean