Cybertory™ Virtual Molecular Biology Laboratory
RFLP and Electrophoresis
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In this exercise, we will use gel electrophoresis and restriction fragment length polymorphism (RFLP) analysis to determine which samples of human hemoglobin DNA have the mutation that causes sickle cell anemia. This will let us identify who has the disease, who is homozygous for normal genes, and who is a heterozygous carrier of the mutation.
- Be able to follow a protocol to successfully carry out enzymatic reactions in the Virtual Lab.
- Learn to use gel electrophoresis to separate DNA fragments by size, and interpret the resulting bands.
- Understand the implications of a diploid genome for genetic diagnosis.
- Be able to recognize partial digestion, and list several conditions that might cause it.
Sickle cell anemia was the first human disease to be attributed to a specific genetic mutation. It is caused by a single nucleotide difference from the normal hemoglobin B gene, which causes a single amino acid difference in the protein. The mutant protein clumps together into long strands, especially in the deoxygenated state; this causes red blood cells to deform, and leads to circulatory problems and anemia.
We will be analyzing PCR products amplified from the genomes of people with and without the mutation. Because the mutation is a substitution, rather than an insertion or deletion, the size of the PCR product is not changed in the mutant; both are 771 base pairs long.
The restriction enzyme Bsu36I cuts at the sequence 'CCTNAGG' (where 'N' indicates that the nucleotide in the middle position doesn't matter). Our PCR products from normal genes is cut by this enzyme at base positions 228, 429, and 517, resulting in four fragments. The nucleotide substitution in the sickle cell mutation happens to eliminate the site at base 228 by changing 'CCTGAGG' to 'CCTGTGG'. This means that we can detect the mutation by digesting the gene with this enzyme, since the mutant gene leads to only three fragments.
Read the user interface section of the FAQ for clues on how to use the Virtual Lab.
Below is a list of the various pieces of furniture and equipment used in this experiment. Each item links to its entry in the Simzymes catalog, where you may find further information about that item and how to use it.
You may want to print this protocol so you don't have to keep flipping back and forth between windows.
- Be sure the micropipette has a clean tip.
- Set up a restriction digestion for each sample. For each reaction, use:
- 5 µl DNA
- 2 µl 10x buffer
- 1 µl Bsu36I restriction enzyme
- 12 µl H2O (to 20 µl total)
- Place the reaction tubes in the waterbath. It should be set to 37°C, and the display should be green, meaning it has equilibrated to the set temperature. Incubate for one virtual hour (if you fast-forward the clock it only takes a few seconds!) Set the clock back to normal speed when you are done.
- Load 15 µl from each sample into its own well on the gel.
- Load 15 µl marker into the next well after your samples (from the tube labeled 'MWM_100', the 100 base pair DNA ladder).
- Turn on the power to the gel by clicking 'Run' on the power supply. This will take about half a virtual hour, so you should fast-forward the clock. Be careful not to let your bands run off the end of the gel!
- Open the door to the second cabinet under the bench. Drag out the camera hood, and drop it onto the gel to take a picture. Experiment with the different file type buttons on the camera. Save an image of your results.
- Compare the band patterns from the patients to the normal and sickle cell controls to determine which patient(s) are normal, which have the disease, and which are heterozygous.
- Which patients have sickle cell anemia? Which are heterozygous?
- Can you think of a way to eliminate any of the pipetting steps from the protocol?
- Click on the Camera Hood link in the Equipment section to read about that piece of equipment in the Simzymes catalog. Why might we want to save gel images in different formats?
- For the 771 bp PCR product from normal hemoglobin B, with Bsu36I cuts sites at positions 228, 429, and 517, what are the expected sizes of the restriction fragments?
- What are the expected sizes of the Bsu36I restriction fragments for the PCR product from mutant hemoglobin B, where the restriction site at position 228 has been eliminated?
- The protocol calls for one (virtual) hour of incubation with one microliter of restriction enzyme per 20 microliter reaction. What happens if you use a shorter incubation, or if you dilute the enzyme? Can you find conditions where the enzyme cuts only some of the DNA molecules (partial digest)?
- The fourth drawer contains one tube of 1M HCl, and a tube of 1M NaOH. What happens if you add these to a restriction digestion? Why might pH affect enzyme activity? Over what range of pH can you achieve complete digestion?
- Experimentally determine the minimum amount of enzyme required to achieve complete digestion in 30 minutes. Repeat the determination with enzyme that has been pre-incubated iin the 37 degree wateerbath for 12 (virtual) hours. What is the difference, and why?
- What aspects of the virtual lab are different from what you would expect in a real lab?
- Sickle-cell disease. (2009, September 24). In Wikipedia, The Free Encyclopedia. [Link]
- Pauling L, Itano H, Singer SJ, and Wells IC. "Sickle Cell Anemia, a Molecular Disease." Science 110: 543-548, 1949. [Link]
- Husain SM, Kalavathi P, Anandaraj MP. Analysis of sickle cell gene using polymerase chain reaction & restriction enzyme Bsu 361. Indian J Med Res. 101:273-6, 1995.
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