Abstract
The report is on sickle cell genes and was carried out in the laboratory of hematology and transfusion science. It was aimed at enabling students to acquire a profound understanding of the effects of mutation in health and disease, particularly about sickle cell anemia. The report comprehensively describes the use of restriction enzymes in the detection of gene sequence mutations. In the experiment, agarose gel electrophoresis of genetic sequences is performed and its results interpreted.
The experiment is about testing the genes of three parties: patient B, her partner, and her unborn child. DNA samples from the white blood cells of each one of them are used alongside control samples which are sickle celled, normal, and one that has a sickle trait (carrier). The digesting enzyme used is in carrying out the test is Mst II.
The results show two DNA bands and a single band as well. The double bands are for the parents who have the sickle cell traits and the single band is for the fetus that is sickle celled. Sickle cell being a genetic disorder requires that both parents must have copies of mutant β global gene for the offspring to be sickle celled.
Introduction
Health and disease can be affected by changing a single nucleotide in an important gene’s DNA sequence. For instance, such a single change can lead to thalassemia (Knight, 2009, p. 30). A great number of genetic disorders are identified in situations where such diseases can be attributed to given changes in a single nucleotide (Campbell and Farell, 2007, p. 742).
In the recent past, cancer of the lungs, breasts, and the colon has been attributed to mutations in tumor suppressor genes and oncogenes (American Institute for Cancer Research, 1997, p. 178). Breast cancer can be diagnosed using gene mutations such as those occurring in BCRI and II genes. The normal adult hemoglobin A (Hb A) has its variant form as Haemoglobin S (Hb S).
In the former, there is an amino acid substitution in the B polypeptide. Valine (Val) is the one providing the amino acid substitution for glutamic acid (Glu) normal Hb A. Vernon Ingram reported this vital finding in 1957 when he used peptide mapping analysis to determine this structural change (Lichtman and Spivak, 2000, p. 46).
These procedures are not only difficult but also tedious. However, recombinant DNA technology is predated by this. A to T is the single base mutation in the triplet codon of amino acid number 6 from the end of the amino acid’s beta chain. Through this chain, an amino acid with a polar side chain valine is introduced rather than the acidic (negative) residue.
It also changes the hemoglobin molecule’s property. The electrophoretic mobility of Hb S is changed by this substitution as compared to Hb A. At a slightly basic PH, Hb S will be more positive than Hb A and will thus travel gradually towards the positive electrode (Bain et al, 2011, p. 20).
The change in mobility forms a diagnostic test for the presence of Hb S. Given the era of biotechnology, it is possible to accurately analyze parental or fetal DNA from cells acquired from amniocentesis (Rodeck and Whittle, 1999, p. 496; Yashon and Cummings, 2008, p. 46). Sufficient DNA can be obtained from the DNA of a few cells. This can be utilized to amplify using Polymerase Chain Reaction. Growth of cells in culture for about nine to twelve days can also be used to come up with sufficient DNA for analysis (Bruns, Ashwood, and Burtis, 2007, p. 34; Modrich, 2006, p. 229).
Recognition by restriction enzymes of specific palindromic sequences in DNA forms the basis of this test. CCT-GAG-GAG is the sequence of nucleotides that specify 5, 6, and 7 (Pro – Glu – Glu) in the normal β globin gene. Codon 6 has the point mutation which converts A to G hence changing the sequence to CCT – GTG – GTG.
CCTNAGG is the palindrome recognition site of the restriction enzyme Mst II where N represents any of the four nucleotides. Examining the sequence closely indicates that the normal β globin CCT – GAG – G will be recognized by Mst II where N is a G though not in its mutated form found in the sickle cell anemia gene. This leads to the ability of the enzyme to cut DNA’s normal sequence but not DNA in its mutated version. Electrophoresis can then be used to separate the varying lengths of DNA produced after exposing both the normal and β globin genes (mutated) to the Mst II enzyme.
This separation is possible because smaller DNA molecules will pass faster through the agarose’s molecular sieve as compared to larger pieces (Tomashefski, 2008, p. 59; Patrinos and Ansorge, 2005, p. 69; Mahesh and Vedamurthy, 2003, p. 26). Produced bands show the kind of genes that were present in any given DNA sample.
The Scenario
Based on last Practical’s results, DNA obtained from patient B’s white blood cells, a test is done to find out the genes that she has alongside the DNA from her partner’s white blood cells who is a male from Ghana. The patient is carrying a baby and there is also a sample of DNA from amniocentesis of the baby.
Samples from all these three will be analyzed together with normal control samples, who have sickle cell anemia and have a sickle trait. Before the practical, the DNA samples had already been digested using the Mst II enzyme. The practical is aimed at establishing which genes are carried by patient B, the baby she is carrying, and her male partner.
Methods
Preparing the gel bed
In preparing the gel bed, the open end of clean and dry gel ends was closed using tape. A tape with a width of ¾ inch was extended on the sides and the bed’s bottom edge. Contact points were pressed firm to come up with a good seal. A well former template was then put at the bed’s end to enhance the stability of the bed.
In casting Agarose gels, a 250 ml flask was used in the preparation of the solution. The following components were added to the flask: agarose of mass 0.8g, 2ml of a buffer that’s concentrated, and distilled water of about 98ml. This brought the total volume to 100ml. Using a marker pen, this volume was marked in a flask. The mixture was then swirled to ensure that clumps of agarose powder had been dispersed. The level of the solution’s volume was then marked with a marking pen.
The mixture was heated for the dissolution of the agarose powder to ensure that the final solution was clear. The flask was covered using plastic wrap to reduce evaporation. The mixture was then heated on high for one minute. It was heated again while swirling for a period of twenty-five seconds to fully dissolve agarose.
The solution was cooled to 55 degrees Celsius while swirling carefully to enhance even heat distribution. Distilled water was added if noticeable evaporation had taken place, bringing the solution level to the original volume as indicated on the flask. After the gel had cooled slightly, the gel bed’s interface was sealed with tape to ensure that the agarose solution did not leak. A small amount of agarose was transferred to both internal ends of the bed using a pipette. One minute was given to let the aragose solidify.
The cooled agarose solution was then poured into the bed, ensuring that the bed rested on a surface that’s level. The gel was allowed to solidify for twenty minutes after which it became cool to the touch.
Preparing the gel for electrophoresis
After total solidification of the gel, the tape was gradually and cautiously removed from the gel bed. By gradually pulling straight up, the comb was removed in an even and careful manner to prevent tearing of the sample contents. While still on the bed, the gel was carefully put in the electrophoresis chamber. 50X buffer was diluted in distilled water to produce 1 liter of 1X buffer. The electrophoresis apparatus chamber was filled with 1X buffer ensuring that the gel was covered with the buffer. The samples were then loaded and electrophoresis was conducted.
Loading of samples
During loading of samples, sample volumes were checked to ensure that the whole volume of the sample was at the bottom of the tubes before loading the gel. The DNA samples were loaded in tubes A to F into the wells consecutively. The amount of sample to be used in loading was 35μl. Tube A had a sample of sickle cell gene, B contained sickle cell carrier sample, C had a sample of the normal gene, D contained the DNA sample of patient B, E had the DNA sample of the unborn baby, and F had the DNA sample of the father.
Running the Gel
After loading the DNA samples, the cover was carefully snapped down onto the electrodes ensuring that there’s proper orientation of the positive and negative color codes. The black wire was then plugged into the negative input while the red wire was plugged into the positive input of the power source. The power source was set at a given voltage and electrophoresis conducted at a duration determined by the tutor. The two electrodes were checked for bubbles as a way of confirming that current was flowing properly.
After completion of electrophoresis, power was turned off and the power source unplugged. Leads were also disconnected and the power removed. The gel was then removed from the bed for staining with Methylene blue.
Staining of the Gel
The gel was placed on a flat surface after electrophoresis. Using a number of drops from electrophoresis buffer, the gel was moistened. The Methylene blue card’s blue side was placed on the well moistened gel using gloves. For several times, fingers were run on the whole surface of the card. The Methylene blue was allowed to be in contact with the gel for 15 minutes after which the card was removed and the gel was transferred to a big weigh board. Distilled water at 37°C was used in destaining.
During the first destaining, the gel was submerged in about 50-100 ml of distilled water at 37°C for 10 minutes. This was done with intermittent agitation. For the second destaining, the previous step was repeated for another 10 minutes. The gel was then carefully removed from the destain solution and visualized using a Visible Light Gel Visualization System. Both the staining and destaining procedures were to be repeated if the gel was too light making the bands not to be properly visible.
Results
At the end of the first destain, the larger DNA bands initially appeared as dark blue bands with a lighter blue background. When destained for the second and third time, the dark blue DNA bands became clearer, the smaller bands were visible, and the entire background turned light blue in colour. This is shown in figure 2 below. Two bands were observed in tubes D and F while tube E had only one band as shown in figure 1 below.
Discussion
In the homozygous normal individual β globin, there are two bands while in the homozygous sickle cell anemia β globin gene has only one band. An individual who has sickle cell anemia has one DNA band identified by the probe (lane A) as shown in figure 1 above. An individual that has a sickle cell trait has three DNA bands signifying a mutant allele. On the other hand, two DNA bands are present in an individual who is normal which points to heterozygous for mutant and normal alleles (Russell, 2007, p. 381-382; Malacinki, 2005, p. 378).
This is summarized in figure 1 above. The results indicate that patient B (mother) is a sickle cell carrier, the father to the unborn child is also a sickle cell carrier. This means that the unborn child is sickle celled as shown from the results. Hence none of the three individuals is normal. “Sickle cell anemia is a genetic recessive disease so that two copies of the mutant β – globin gene are necessary for sickle cell anemia to appear (Dudek, 2006, p. 201).”
The results obtained from this experiment were expected and correspond to what other people have found after performing the same experiment. In patients with sickle cell anemia, a target site recognized by the restriction enzyme Mst II is eliminated by a base substitution mutation within the β – globin gene (GAG – GTG). While the Mst II enzyme digests the DNA from the three individuals, gel electrophoresis separates it. The expected results for an individual suffering from sickle cell and heterozygous individuals are as obtained in this experiment.
One of the limitations of this method over others is that the procedures may cause possible risks to both the mother and the fetus. This may include fetal injury, infection, spontaneous abortion, and hemorrhage. As far as amniocentesis is concerned, the probability of the risk of miscarriage is between 0.5% and 1% (Cummings, 2008, p. 343).
A difficulty in interpreting the results of this experiment may arise due to the bands being invisible before staining and destaining. It’s after destaining that the bands can be visible. The alternative method that can be applied to minimize this difficulty is the use of PCR which amplifies small DNA samples to analyzable concentrations (Russell, 2007, p. 379).
Conclusion
The experiment has given clear results that indicate the effect of mutation on health and disease. After testing the DNA samples from the white blood cells of the three parties using the digestive enzyme Mst II, it can be concluded that two DNA bands will show that the individual is normal, three will show that the individual’s genes have sickle cell traits and one band indicates that the individual’s genes are sickle celled. The genes of both parents have to contain sickle cell traits for the child to be sickle cell anemic.
Reference List
American Institute for Cancer Research, 1997. Dietary fat and cancer: genetic and molecular interactions. Washington DC: Springer. Web.
Bain et al. 2011. Variant Haemoglobins: A guide to Identification. West Sussex, UK: John Wiley and Sons. Web.
Bruns, Ashwood and Burtis, 2007. Fundamentals of Molecular Diagnostics. London: Elsevier Health Sciences. Web.
Campbell, M. and Farell, S. 2007. Biochemistry. Belmont, USA: Thomson Higher Education. Web.
Cummings, M.R. 2008. Human Heredity: Principles and Issues, page 2. Belmont: Cengage Learning. Web.
Dudek, R.W. 2006. High Yield Cell and Molecular Biology. Baltimore: Lippincott Williams & Wilkins. Web.
Knight, J.C. 2009. Human Genetic Diversity: Functional Consequences for Health and Disease. Oxford: Oxford University Press. Web.
Lichtman, M. and Spivak, J. 2000. Hematology: landmark papers of the twentieth century. London: Academic Press. Web.
Mahesh, S. and Vedamurthy, A. Biotechnology-4: including recombinant DNA technology, environmental biotechnology, and animal cell culture. New Delhi: New Age International. Web.
Malacinki, G.M. 2005. Essentials of Molecular Biology. Sudbury: Jones & Bartlett Learning. Web.
Modrich, P. 2006. DNA Repair, Volume 408. London: Academic press. Web.
Patrinos, G. and Ansorge, W. 2005. Molecular Diagnostics. London: Academic Press. Web.
Rodeck, C.H. and Whittle, M.J. 1999. Fetal Medicine: Basic Science and Clinical Practice. London: Elsevier Health sciences. Web.
Russell, P. 2007. Biology: the Dynamic Science. Belmont, US: Thomson Higher Education. Web.
Tomashefski, J.F. 2008. Dail and Hammar’s Pulmonary Pathology: Neoplastic lung disease. NY: Springer. Web.
Yashon, R. and Cummings, M. 2008. Human Genetics and Society. Belmont, US: Cengage Learning. Web.