Genetic Diagnosis. electrophoresis. During the lab, genetic testing was done for the cystic fibrosis gene in a young
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1 Meyers 1 Keya Meyers Genetic Diagnosis Abstract: In this lab two processes were observed: restriction fragment polymorphism and gel electrophoresis. During the lab, genetic testing was done for the cystic fibrosis gene in a young child, Jeff, that was diagnosed. Two restriction enzymes were used to cut positive control, negative control, and the Jeff s DNA; Eco RI and BAM HI. The positive control was DNA collected from a cystic fibrosis individual and the negative control was DNA collected from a normal individual. When Jeff s DNA was compared to that of positive and negative control, it resembled that of the positive control. Therefore, it was concluded that Jeff was positive for the cystic fibrosis mutation gene. Introduction: One way to test for genetic diseases is through restriction fragment polymorphism (RFLP). This analysis is a powerful technique used in identification of genetic anomalies associated with disease and also involves the electrophoretic analysis of DNA fragment sizes generated by restriction enzymes [De Cicco, 2012]. In general, restriction enzymes help to cleave DNA at specific recognition sites; they cleave the phosphate bonds between both strands. These proteins also known as endonucleases and are obtained from bacteria. They require Mg+2 for activity and generate a 5 prime (5') phosphate and a 3 prime (3') hydroxyl group at the point of cleavage [De Cicco, 2012]. They are usually named after the bacteria they originated from and are assigned a number that tells the order of when it was found. Strains found in a species are responsible for producing specific types of restriction enzymes. A Roman numeral is always used to designate one out of possibly several different restriction enzymes produced by the same organism [De Cicco, 2012]. With regard to the cleavage specificity, the types have the following characteristics: type I enzymes bind to the recognition
2 Meyers 2 sequence but cleave DNA randomly; type II enzymes cut DNA at defined positions close to or within their recognition sequence; type III enzymes require two recognition sequences in opposite orientations within the same DNA molecule to accomplish cleavage and cleave outside these sequences [Griesenbach, 2005]. Each enzyme will have recognize sites with about 4-8 base pair sequences. Recognition sites can have the same DNA sequence when read 5 to 3 which is known as palindromes [De Cicco, 2012]. The cleavage pattern of Eco RI can be used as an example: Eco RI restriction site: 5'-GAATTC-3' 5'-G AATTC-3' 3'-CTTAAG-5' 3'-CTTAA G-5' Each enzyme will have recognize sites with about 4-8 base pair sequences and cut at specific site and create sticky or blunt ends. Sticky ends occur when the cleavage pattern causes the cuts to be complementary to each other; such as the example of Eco RI. Other enzymes cut blunt ends in which the sequences are opposite of each other. Also, with the recognition of palindromes is that fact that restriction enzymes will only possibly cut a certain amount of times; depending on how many times a palindrome occurs in the DNA sequence. Furthermore, based on the cuts made, the size of the DNA fragments will be determined and mainly depends on the distance between recognition sites [De Cicco, 2012]. The larger the DNA sequence is the greater number of cuts is expected. Testing for genetic diseases is one of ways in which RFLP can be utilized to track genetic mutations. A genetic disease is usually caused by a mutation in the DNA sequence of an individual organism that can occur through heredity or independently. This mutation will be responsible for the dysfunction of a protein that has a special cellular purpose. A single nucleotide mutation can result in a change in a single amino acid and therefore changing the amino acid sequence of a protein can affect the structure and therefore the function of the protein [De Cicco, 2012]. Mutations can contribute to the
3 Meyers 3 altering or removal of bases at a recognition site which would affect the restriction enzyme, what it cuts, and how it cuts. In this experiment, gel electrophoresis was used to separate the DNA fragments obtained from RFLP analysis and through visualization of the gel the effect of a genetic mutation on the DNA sequence will be observed. The case of a young child, Jeff, who is sick with different symptoms was presented. It was suggested that the child suffers from cystic fibrosis (CF) which is a mutation is a deletion of three base pairs resulting in the removal of a single phenylalanine amino acid at position 508 in the cystic fibrosis transmembrane conductance regulator protein [De Cicco, 2012]. This protein is responsible for the transport of ions and if defected will cause an impairment of ion transfer. The low volume hypothesis postulates that due to absent chloride transport and increased sodium absorption the height of the ASL is reduced, leading to impaired mucociliary clearance; reduced mucociliary clearance, which is explained by both theories, leads to formation of thickened dehydrated mucus, which provides an ideal environment for bacterial infection, leading to chronic inflammation and ultimately organ failure in the CF lung [Ivancic-Jelecki, 2006]. Through electrophoresis of the DNA fragments, the presence of the CF mutation gene, F508, can be observed. If the DNA from the child is obtained then ran through gel electrophoresis and compared to that of positive and negative control patients, then the presence of the CF mutation will confirm the diagnosis that the child does indeed carry the CF mutation gene. Methods: The samples of negative control DNA and Jeff s DNA were obtained along with four microcentrifuge tubes and labeled Jeff 1, Jeff 2, neg 1 and neg µl of reaction buffer were then added to each of the four tubes. 15 µl of Jeff s was transferred to tube Jeff 1 and 15 µl was added to tube Jeff µl of negative control DNA was transferred to tube neg 1and 15 µl of negative control DNA was transferred to tube neg µl of enzyme 1 (Eco RI) was added to tubes Jeff 1 and neg µl of enzyme 2 (BAM HI) was added to tubes Jeff 2 and neg 2. The tubes were then incubated for 30 minutes in 37 degrees Celsius water bath. A 0.8% agarose gel was then prepared as follows: (1) a gel holder was placed into a preparation rack and a comb was placed near one end of the gel space. (2) 0.4 grams of
4 Meyers 4 agarose was weighed out on a balance. (3) The agarose was then transferred to a glass flask and 50 ml of 1x TAE buffer was added to the flask. The solution was swirled in order for it to mix properly and then placed in the microwave for 1 minute (two thirty second intervals). (4) Once cooled, Ethidium Bromide was added to the solution then the solution was poured into the gel rack. The gel was allowed to cool and solidify. After the gel solidified, using gloves, the comb was removed from the geld holder. The gel holder was then removed from the preparation rack and was placed into the electrophoresis chamber. TAE buffer was added until the top of the gel was covered by liquid. The tubes were then recovered containing the restriction digests. 5 µl of loading dye was then added to each tube and were then placed on ice until gel was ready to be loaded. A tube of marker DNA, a tube of positive control DNA cut with enzymes 1 and a positive control DNA cut with enzyme 2 were all obtained. The tubes were arranged in the order intended to load in the gel. Each sample was loaded into separate wells in this precise order from left to right: marker, positive control 1, negative control 1, Jeff 1, positive control 2, negative control 2, Jeff 2. Once wells were loaded, the apparatus was covered and the gel ran at 120 V until the faster of the two dyes was about half-way through the gel, about minutes. The power supply was turned off and the leads were disconnected from the apparatus. It was important to wear gloves when removing the gel from the apparatus. The gel was carried to a UV transilluminator to be viewed under UV light. An image was taken and the measurements of the marker and the samples migrated were determined. Results: Based on the symptoms presented we know that Jeff could have cystic fibrosis. In order to test this diagnosis, the gel was specifically loaded with DNA from a patient that was positive with cystic fibrosis and a patient that was negative with cystic fibrosis. The negative and positive DNA were purposely loaded adjacent to each other so that the number of cuts and band sizes could be compared. If compared, it is evident that enzyme 1, Eco RI made two cuts at 2150 and 4300 base pairs and enzyme 2, BAM H I, made two cuts as well but at 1500 and 7200 base pairs. The cystic fibrosis mutation gene altered the first recognition site of enzyme 2 and therefore removed the first cut. Because the CF gene had no effect on the recognition sites for enzyme one, the positive control, negative control and Jeff s DNA
5 Meyers 5 had the same amount of bands which were appeared at the same amount of base pairs. Based on the cuts made in Jeff s DNA by enzyme 2, he does suffer from cystic fibrosis. Figure 1: UV Image of Gel M P1 N1 J1 P2 N2 J2 Figure 2: DNA Standard Curve
6 Meyers 6 Table 1: Band Sizes of Each Well Well First Band Second Band Third Band Positive bp 4300 bp Negative bp 4300 bp Jeff bp 4300 bp Positive bp 7200 bp Negative bp 1800 bp 7200 bp Jeff bp 7200 bp Discussion: It was concluded from the results that Jeff did in fact have the CF mutation gene and suffers from the disease. This is known because if his DNA fragments are compared to that of the positive control cut by enzyme 2, they are identical both in the number of bands and their sizes. The controls are necessary for diagnostic experiments because they provide specific patterns in which the sample being tested can be compared to. In this experiment there were two controls that helped to test Jeffs DNA. The positive control DNA of a patient with cystic fibrosis and the negative control DNA of a patient without cystic fibrosis were used. Also, it is a good way to make sure to get rid of any other other variable could explain the results. The loss of a single amino acid in a protein can cause devastating diseases such as cystic fibrosis because the basis of protein synthesis is through translation of genetic material into amino acids. If there is an alteration in a DNA sequence such as a mutation, during translation, the amino acid needed to allow the cystic fibrosis transmembrane conductance regulator protein to become functional will not be produced. Therefore, the missing amino acid or substituent amino acid produced would then be incorporated into the polypeptide that will soon become the protein would not be functional and can no longer be used by the cell for ion transport.
7 Meyers 7 References: De Cicco- Skinner, Katie. Case Study: Genetic Diagnosis of Disease Lab protocol. Biology Department: Cell Biology Griesenbach, U. (2005). Pre-clinical and clinical endpoint assays for cystic fibrosis gene therapy. Journal of Cystic Fibrosis, 4(2), Ivancic-Jelecki, J. (2006). Restriction enzyme cleavage of fluorescently labeled DNA fragments Analysis of the method and its usage in examination of digestion completeness. Analytical Biochemistry, 349(2),
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