If one parent has sickle cell anemia (SS) and the other parent is a carrier (AS), then there is a 50% chance of having a baby with sickle cell anemia (SS) and a 50% chance of having a baby who is a carrier (AS). If both parents are carriers (AS), then there is a 25% chance of having a baby with sickle cell anemia (SS).
Page 3 Carrying the sickle cell trait or having sickle cell anemia can confer resistance to the malaria parasite . Historically, this has provided a selective advantage in some parts of the world where malaria is prevalent, such as parts of Africa. Many people of African descent are sickle cell carriers. For example, it is estimated that 8% of African-Americans are sickle cell carriers. Therefore, development of new sickle cell anemia treatments and diagnostic testing are critical. Diagnosing sickle cell anemia and sickle cell carriers One common way of diagnosing sickle cell anemia is hemoglobin electrophoresis . In this technique, not conducted today, a patient’s hemoglobin protein is separated using electrophoresis. Normal hemoglobin migrates a different distance than sickle hemoglobin, allowing diagnosis. Today’s lab will illustrate diagnosis of sickle cell anemia on the DNA level. First, a patient’s beta-globin gene is amplified from his or her DNA using a technique called polymerase chain reaction or PCR . Then the amplified DNA is cut using an enzyme that specifically recognizes the DNA of the normal beta-globin gene not the sickle cell beta-globin gene. Enzyme-digested DNA is run on a gel, and a different size band appears for the normal gene and for the sickle cell form. We will run these DNA samples today. Southern blotting analysis Southern blotting analysis is a commonly used technique in cell and molecular biology. DNA is separated on a gel, transferred to a membrane, and then probed for a particular DNA sequence. Probes are usually radioactive or fluorescent; however, we will be simulating this step of the analysis by treating our membrane with a DNA dye. The stages of Southern blot analysis are highlighted in Figure 3 . Before DNA from a gel can be transferred to a membrane, the DNA is depurinated using HCL and denatured using NaOH. This means that purine bases are removed from the DNA, destabilizing phosphodiester bonds in the DNA, and introducing nicks in the DNA. NaOH disrupts hydrogen bonds between paired bases. Both of these treatments result in the formation of small fragments of DNA and conversion of the double-stranded DNA samples into a single stranded form. This process facilitates transfer of DNA to the membrane. .
Page 4 Figure 3: Southern blotting procedure (Molecular Biology of the Cell) Materials and Methods: Please follow the instructions for forming a group. Each group will have a gel loading team and a blot transfer/detection team. Also, select one individual to time all incubations.