Gel Electrophoresis In Analysis Of Sickle Cell Genes

Gel Electrophoresis In Analysis Of Sickle Cell Genes This experiment was carried out in order to outline a scheme for the investigation of Sickle Cell Gene Analysis and to understand and carry out the agarose gel electrophoresis. Besides that, the importance of restriction enzyme and the mechanism of the enzyme in separating the specific amino acid sequence were completely understood. The fragment bands observed on the gel electrophoresis from the experiment results was interpreted and understood. The controls (tube A-C) and 3 DNA samples [tube D (Mother), E (Unborn Child), F (Father)] which was amplified and digested with the MST 11 enzyme was analysed for the sickle cell genes using gel electrophoresis. Once the electrophoresis apparatus and the gel were prepared, the controls and the samples were loaded consecutively into the respective wells. The power source was set at 125V and left to run for 30 minutes. Following that, the gel was then stained using the Ethidium Bromide staining card. The gel was left to stain for 15 to 20 minut es and was later observed under a UV Transilluminator. Tube B (control), D (Mother) and F (Father) was heterozygous for Sickle Cell Disease, Hb AS whereas tube E (Unborn child) was homozygous for Sickle Cell Disease, Hb SS. Sickle Cell gene analysis is essential for reasons such as preparation of financial source to carry out treatment for the child if the child appears to be Hb SS. Treatments such as bone marrow transplantation and blood transfusion can be done to manage and treat Sickle Cell Disease children. Introduction Sickle Cell Disease (SCD) is an inherited blood disorder, characterized by the presence of sickle shaped red blood cells (Figure 1) (Wun and Hassell, 2009) (Oniyangi and Omari, 2006) (Stuart and Nagel, 2004). http://www.biologycorner.com/anatomy/blood/sickle_cell_anemia2.jpg Normal healthy individuals carries 3 main haemoglobins; Haemoglobin A (Hb A), Haemoglobin A2 (Hb A2) and Haemoglobin F (Hb F) (Wood et al, 1976). In Hb A, the glutamic acid (GAG) is situated on the 6th position of the ÃŽ ²- globin chain. Mutation of a single nucleotide (GAG to GTG) (Pan et al, 2007) (Stuart and Nagel, 2004) which takes place in the 6th position of the ÃŽ ²- globin chain (Figure 2) (Wun and Hassell, 2009) (Cleon et al, 2009) of Hb A results in the formation of Haemoglobin S (Hb S) which causes SCD (Pan et al, 2007) (Cleon et al, 2009) (Stuart and Nagel, 2004). The acidic (negative charged) glutamic acid is replaced by the polar (neutral) valine (Frenette and Atweh, 2007) (Cleon et al, 2009) (Stuart and Nagel, 2004). Sickle cell genes (autosomal recessive) are inherited from the parents (Figure 3) (Frenette and Atweh, 2007) (Wun and Hassell, 2009). One copy of gene are passed down from each parent to the fetus, therefore, a fetus will have 2 copies of genes to make the ÃŽ ²- globin. Heterozygous SCD patients (carriers) (Hb AS) had inherited 1 copy of normal gene and 1 copy of defective gene whereas homozygous SCD patients (Hb SS) had inherited 2 copies of defective genes (Frenette and Atweh, 2007) (Makani et al, 2010) (Oniyangi and Omari, 2006). The amount of defective genes in heterozygous patients is insufficient to cause symptoms because they have a proportion of normal genes as well. (Makani et al, 2010). Therefore, most SCD carriers are unaware of the presence of Hb S in them. Homozygous SCD patients has insufficient normal genes; therefore symptoms of SCD are clearly demonstrated by them (Makani et al, 2010) (Oniyangi and Omari, 2006). The image shows how sickle cell genes are inherited. A person inherits two copies of the hemoglobin gene-one from each parent. A normal gene will make normal hemoglobin (A). An abnormal (sickle cell) gene will make abnormal hemoglobin (S). When each parent has a normal gene and an abnormal gene, each child has: a 25 percent chance of inheriting two normal genes; a 50 percent chance of inheriting one normal gene and one abnormal gene; and a 25 percent chance of inheriting two abnormal genes. If both parents are carriers for SCD, the child has a 50% chance of being carriers, 25% chance of being homozygous for SCD and 25% chance of having 2 copies of normal genes (National Blood, Lung and Heart Institute). Sickle Cell gene analysis is an important screening test for a married couple. The fetus should be screened for SCD if one parent is a carrier for SCD (Tshilolo et al, 2008) (Streetly et al, 2009). It is important to ensure whether the child is heterozygous or homozygous for SCD for reasons such as financial preparation for treatment of the child (if child is homozygous for SCD) (Streetly et al, 2009) (Tshilolo et al, 2008). Sickle Cell gene analysis can be done using gel electrophoresis. The Deoxyribonucleic Acid (DNA) sample can be obtained from white blood cells (in adults) and from the amniocentesis fluid (in fetus). The samples can then be amplified with Polymerase Chain Reaction (PCR) (Rahimi et al, 2008) (Bruzzone et al, 2008). PCR produces copies of DNA which had been obtained from a small sample, to ensure the amount of sample is sufficient for the SCD gene analysis (Bruzzone et al, 2008) (VanGuilder et al, 2008). The multiplied DNA sample will then be digested with a restriction enzyme (Sasnauskas et al, 2007) (Rahimi et al, 2008), for instance MST 11. This enzyme recognizes the CCT-GAG-G sequence of the amino acid on the ÃŽ ²- globin and will bind to cleave (cut) that DNA strand which it recognizes (Figure 4). Due to the substitution of amino acid in SCD (CCT-GTG-G), the MST 11 enzyme cannot recognize that mutated sequence so therefore, will not cut that mutated DNA strand (Sasnauskas et al, 2007). As a result, the normal genes will be cut by the enzyme and will become short fragments whereas the sickle cell genes will be an uncleaved long fragment. The DNA from a normal individual (Hb AA), SCD carrier (Hb AS), and SCD homozygous individuals (Hb SS) produces fragments of different sizes and lengths (Rahimi et al, 2008) after digested with the restriction enzyme. Figure 4: The picture on the (left) shows the sites recognized and would be cleaved by the restriction enzyme in a normal ÃŽ ²- globin allele, whereas the enzyme would not cut the altered ÃŽ ²- globin allele (SCD). The picture on the (right) shows the different fragments bands of different sizes and lengths demonstrated in a gel electrophoresis (www.bio.miami.edu) These fragments are then separated by gel electrophoresis (Figure 4). Gel electrophoresis is used to separate molecules such as DNA by using an electric field applied to a gel matrix. Smaller DNA fragments are able to squeeze through the pores of the gel, compared to the larger fragments (Rahimi et al, 2008). The sickle cell genes (long fragments) will be slowest to move compared to the normal genes which had been cut (short fragments). The electrophoresis gel will finally be stained and observed under an Ultra Voilet Transilluminator to view the bands produced. This experiment was carried out in order to outline a scheme for the investigation of Sickle Cell Gene Analysis and to understand and carry out the agarose gel electrophoresis. Besides that, the importance of restriction enzyme and the mechanism of the enzyme in separating the specific amino acid sequence were completely understood. The fragment bands observed on the gel electrophoresis from the experiment results was interpreted and understood. Method Gel Bed Preparation The open ends of a clean and dry gel bed (casting tray) was closed by using a tape. The  ¾ inches wide tape was extended over the sides, and the bottom edges of the bed. The extended edges of the tape were then folded back onto the sides and bottom. The contact points were pressed firmly to form a good seal. Next, the well former template (comb) was placed in the first set of notches at the end of the bed. The comb was placed firmly and evenly across the bed. Casting Agarose Gels Agarose gel (0.8g), concentrated buffer (2ml of 50X), and distilled water (98ml) were added into a 250 ml flask to prepare the gel solution. The volume in the flask which gave the total volume of 100 ml was marked with a marker pen. Next, the mixture was swirled to disperse the agarose powder clumps. The level of the solution on the outside of the flask was indicated with a marker pen. The mixture was then heated to dissolve the agarose powder. The flask was covered with a plastic wrap to minimize the evaporation rate. The mixture was then heated in high temperature for a minute. After that, the mixture was swirled every 25 seconds while it was heated in high temperature, until all the agarose was completely dissolved. The final solution produced was clear (like water), without any undissolved particles in it. The agarose solution was then cooled to 550 C by swirling it carefully, so even dissipation of heat was promoted. Distilled water was added to bring the solution up to the orig inal volume as marked on the flask when evaporation was detected. The interface of the gel bed sealed with the tape, was checked once the gel has cooled slightly to avoid leakage of the agarose solution. A transfer pipette was used to deposit a small amount of cooled agarose solution to both ends of the bed and left for a minute for the agarose to solidify. Finally, the bed was placed on an even surface and the cooled agarose solution was poured onto the bed. The gel was allowed to completely solidify. The gel appeared firm and cool after approximately 20 minutes. Gel Preparation for Electrophoresis The tape was carefully and slowly removed from the gel bed once the gel was completely solidified. The comb was slowly pulled straight up to be removed. This was done carefully and evenly to prevent the sample wells from tearing. The gel (on the bed) was placed into the electrophoresis chamber, orientated properly and centred on a level and even platform. The 50X buffer was then diluted in distilled water to prepare a 500 ml of 1X buffer. Once the buffer was prepared, it was poured into the electrophoresis apparatus chamber. The gel was completely covered with the buffer. The samples were then loaded to conduct the electrophoresis, as discussed below. Loading of Samples The sample volumes were checked to ensure small amounts of samples were not clanged onto the walls of the tubes. The entire volume of the samples was ensured to be at the bottom of the tubes, before the gel was loaded with the samples. 25 µl of DNA samples from tubes A to F was loaded into the wells in a consecutive order (Table 1). Running the Gel The cover was snapped down carefully onto the electrode terminals after the DNA samples were loaded. The negative and positive colour coded indicators on the cover and the apparatus chamber was properly orientated. The plug of the black wire was inserted into the black input of the power source (negative input) and the plug of the red wire was inserted into the red input of the power source (positive input). The power source was set at 125V and the electrophoresis was conducted for 30 minutes. Bubbles observed on the two platinum electrodes indicated that the current was flowing properly. Once the electrophoresis was completed, the power was turned off and the plug was unplugged from the power source. The leads were disconnected and the cover was removed. The gel was removed from the bed to be stained with Ethidium Bromide. Staining the Gel After electrophoresis, the gel was placed on a flat surface and was moistened with several drops of the electrophoresis buffer. The Ethidium Bromide staining card adhesive was removed and placed onto the surface of the gel (where the wells were). Fingers were firmly run over the entire surface of the card several times. Once that has been done, an empty beaker or the casting tray was kept on top of the gel with the staining card to maintain good contact between the gel surface and the card. The gel was left to stain for about 15 to 20 minutes. The card was then removed from the gel surface and the surface of the gel was rinsed with the buffer. The gel was then examined on a Visible Light Gel Visualization System or an Ultra Violet Transilluminator. The fragment bands formed on the surface of gel was observed, and interpreted. Discussion Based on the results obtained, Sample A (Control for Normal Gene) had only 2 bands on the gel when observed under the UV Transilluminator. This is because, the restriction enzyme, MST 11 only cuts the amino acid sequence which it recognises. The MST 11 had cut the CCT- GAG- G sequence from the DNA strand of this normal Hb A gene. Therefore, two bands are formed instead of 1 band. The long fragment had now been cut to two short fragments. Since the MST 11 only recognises the CCT- GAG- G sequence which is on the beginning of the strand, this band appears shorter than the remaining band after it has been cut. Short fragments are able to squeeze through the gel pores easily compared to the longer fragments. Since glutamic acid (GAG) is negatively charged, and is therefore shorter than the remaining fragment, this fragment travels faster and further from the well towards the anode (positive terminal) compared to the remaining fragment. Sample D (Mothers DNA) had the same results as Sample F (Fathers DNA) and Sample B (Control for Sickle Cell Trait Gene). There were 3 bands observed on the gel under the UV Transilluminator. Since these samples are heterozygous for SCD (Hb AS), they have inherited 1 copy of normal gene and 1 copy of defective gene. The restriction enzyme did not recognize the sickle cell gene (CCT- GTG- G). Therefore, the MST 11 enzyme did not cut that sequence. As a result, that fragment appeared to be 1 long fragment and could not easily squeeze through the pores of the agarose gel. As a result, this fragment was not able to move fast and further from the well. However, the MST 11 restriction enzyme recognised and had cut the normal gene which was present. Therefore, when the DNA strand was cut, two shorter fragments were formed which easily travelled through the pores and were able to travel away from the wells. The A gene with the shorter fragment travelled further compared to the remaining fragm ent. Sample E (Unborn Childs DNA) had the same results as Sample A (Control for Sickle Cell Anaemia Gene). Only 1 band was observed on the gel under the UV Transilluminator. Since the sample is homozygous for sickle cell (Hb SS), the baby had inherited 2 copies of defective gene from the parents. The restriction enzyme did not recognize the Sickle cell gene (CCT- GTG- G) and did not cut the DNA strand. As a result, the fragment remained as 1 long fragment and was not able to squeeze though the pores of agarose gel easily. This fragment moved extremely slow and could not move very far from the wells. The inheritance pattern which had caused the child to be Homozygous for SCD had been discussed above (Figure 3). SCD can lead to complications such as anaemia, jaundice and stroke if left untreated (Pan et al, 2007) (Nagababu et al, 2008). This is because, when substitution of amino acid occurs due to the mutation, a new longitudinal polymer which has valine in the 6th position of the beta chain instead of glutamic acid is polymerized. Haemoglobin S is formed due to this. During hypoxia (less oxygen supply to tissues), aggregation of haemoglobin takes place due to the presence of the hydrophobic valine instead of the polar glutamic acid in the 6th position of the ÃŽ ² -globin chain (Cleon et al, 2009) (Stuart and Nagel, 2004). As a result, red blood cells with sickle shape which are fragile and not flexible are formed (Nagababu et al, 2008) (Pan et al, 2007) (Manchikanti et al, 2007). C:UserscompaqDesktoprd_sicklecell.jpg Figure 5: The complications of Untreated Sickle Cell Disease (ICAGEN Ion Channel Advances, 2010) Children with SCD will normally be closely observed by a paediatrician in order to ensure they are healthy. These children will be subscribed with folic acid to be taken daily to stay healthy. Besides that, penicillin is also required in order to prevent them from getting illnesses due to their weak and immature immune system (Ndefo et al, 2009). Complications due to Sickle Cell Disease Treatment Streptococcus Pneumoniae sepsis Newborn 5 years (Penicillin VK) 2years and above (23- valent Streptococcus Pneumoniae polysaccharide vaccine (PPV23) Bone marrow Aplasia and Megaloblastic erythropoiesis Folic acid Stroke Blood transfusions Pain Episodes Hydroxyurea (complete blood count monitored every 2 weeks, Hb F monitored every 6 -8 weeks, and serum chemistries monitored every 2- 4 weeks) Other than that, hydroxyurea are also given to children with SCD. Hydroxyurea works by activating the fetal haemoglobin production to replace the production of Haemoglobin S which results in SCD (Ndefo et al, 2008). Children with SCD who has a high risk for developing stroke, will be given blood transfusion frequently (Vichinsky et al, 2007) (Kirkham, 2007). A blood transfusion replaces the sickle cells with normal red blood cells which leads to a good supply of oxygen to the brain, which will reduce the chances of stroke in children (Kirkham, 2007) (Nagababu et al, 2008). Unfortunately, blood transfusions causes iron overload in the body because the body lacks mechanism to excrete iron (Vichinsky et al, 2007). An overload of iron results in iron deposition in various organs which can be fatal if untreated (Vichinsky et al, 2007). Therefore, children with regular blood transfusions will be given chelating agents (Vichinsky et al, 2007) such as desferrioxamine to excrete iron from the body through the urine and faeces. Bone marrow transplantation is the only cure for SCD (Ndefo et al, 2008). The affected bone marrow will be replaced with a closely matched Human Lymphocyte Antigen (HLA) bone marrow from a healthy individual (Ndefo et al, 2008). Bone marrow transplants enables, new and normal healthy red blood cells to be produced. The disadvantages of bone marrow transplantations are the occurrence of Graft Versus- Host Disease (GVHD). Based on the study by Ndefo et al (2008), it was suggested that there will be an 85% chance of survival free from SCD, 7% chance of developing bone marrow transplant complications and a 9% chance of GVHD failure rate with a bone marrow transplant procedure. Conclusion As a conclusion, gel electrophoresis is found to be an excellent way to aid in the analysis of sickle cell genes. Using gel electrophoresis, many samples can be analysed at the same time, and its results are specific and accurate. The disadvantages of this method are that analytical errors might occur due to the large amount of sample handling and it is costly. Although it is costly, this method can increase the awareness of the carriers of SCD about the presence of Hb S in them, which can aid in their future family planning. Children with homozygous SCD, can be prevented from suffering from SCD complicatio

Essay --

In the field of medicine, diagnosis and management are initiated by the presence of symptoms. Nevertheless, a number of technological advancements allow for an individuals disease susceptibility to be identified, through DNA-based diagnostic testing. Although seen as the â€Å"holy grail† in disease prevention and management, DNA testing raises issues that can lead to widespread disapproval amongst the public. Hereditary Haemochromatosis (HH), an autosomal recessive disorder, is a disease that can not only be treated effectively, but if identified early it can also be prevented. HH is a metabolic disorder caused by a mutation in the hemochromatosis (HFE) protein, leading to an increase in intestinal iron absorption despite adequate or even excessive iron stores that lead to liberal iron accumulation in the body, thus causing irreversible organ damage. (Fowler, 2008) HH is commonly known as adult onset-HH, as the disorder is often asymptomatic, with excessive iron accumulation observed after the age of 40 predominantly in the liver, but also in the pancreas, pituitary, heart, joints, and skin. This can lead to clinical complications such as cirrhosis, diabetes mellitus, arthritis and liver fibrosis, in addition to skin hyperpigmentation. (Papanikolaou and Pantopoulos, 2004) In the bloodstream, iron binds to transferrin, establishing an iron-transferrin complex (diferric transferrin). Iron is released from transferrin when the compound binds with the transferrin receptor at the hepatocyte surface. Binding of the HFE protein to the transferrin receptor reduces the affinity of the transferrin receptor for the iron-transferrin complex; which in turn reduces the amount of iron being released by the complex. (Vora, 2012) The HFE protein is ... ...d be tested for, in order to minimise the adverse effects it may have on any one individual or family. These individuals adopted a deontological approach, where they believe the well-being of each individual in a population should be accounted for by the health sector. Conclusively, genetic screening for HH can be approached from many interest groups, each with their own contrasting view, based on their rational. As a young scientist, I believe that the well-being of each individual in the population should be cared for when implementing any genetic screening. Even though haemochromatosis has a low penetrance rate, and is rarely seen in non Caucasian ethnicities, the continuous admixture of ethnicities, its 1 in 300 prevalence rate and its preventability, indicates that any test for it cannot be dismissed, regardless of its ethnic discrimination or other issues.

A Poetry Analysis on Modern Love

Modern Love George Meredith â€Å"Modern Love. † The term brings to mind the changing dynamic of today's society. This change has been present for decades and continues on to this day. In George Meredith's poem he illiterates the negative impact of this change in a case that could encompass so many couples; the pain of a loveless marriage. Through his use of diction, and metaphor Meredith show the pain and heartache of two people being so close, yet so emotionally distant. Similar essay: How to be Old Poem AnalysisThe first line of the poem begins the dark theme (By this he knew she wept with waking eyes), showing how the husband has seen his wife's suffering; as well as painting a memorable picture through the use of alteration. The alteration serves another purpose as well. It's smooth deliverance shows just how used to the situation the husband is to his wife's tears. In line 2 we see just how helpless the husband is to help, his hand â€Å"quivers† out of nervousness, and in line 3 we see the extent of the wife's sobs (Shook their common bed).The dark selection of diction continues as metaphors are employed in lines 5&6 (And strangled mute, like little gaping snakes, dreadfully venomous to him). The truly telling word in these lines is â€Å"Strangled,† this extremely active verb implies force. This describes the situation of any willfully married wife during the time the poem was written. They had little choice in not only their husb and, but also in the lifestyle handed to the by that husband, not to mention that divorce during this time period was early unheard of.The next lines hint at the wife's feeling of death, (â€Å"Stone-still†) showing her complete hopelessness at the situation imposed on her. Lines 8-12 have the same dark imagery (â€Å"Pale drug of silence†, â€Å"Sleep's heavy measure†, â€Å"move-less†, â€Å"Dead black years†), but those same images fit into another, larger image. The author uses them to describe her â€Å"Giant heart of memories and tears. † Meredith clearly shows the long lasting nature of the wife's pain, as well as the multitude of sleepless nights that all weigh against her heart.The only relief from the torture is presented in â€Å"Sleep's Heavy Measure. † The choice of the word â€Å"effigies† could possibly be the most significant of the entire work. Effigies are stone representations of a person, normally used only after death. The image of death is repeated in the 15th and 16th lines as well, (Upon their marriage-tomb, the sword between; Each wishing for the sword that severs all) these lines are used to show the reader several things. First, the emotional death of the parties involved, second the death of the marital bond, and third the longing for literal death.Meredith's choice of words and formulation of metaphors steer the reader away from the bright connotations of love and into the darker feelings that society in the Victorian era was happy to ignore. His work was very nearly satirical in nature, it challenged the social conventions of marriage at the time and began to show how both parties in a marriage were both influence by, and responsible for the health of a marriage. The true feeling and innate sadness in the poem comes from not only the lack of love and affection, but also the torture of conforming to the standers of the day.

Effects Of Child Death Rates On Children - 1211 Words

As a child, everyone remembers having to get shots. My first memorable experience with shots came at age four. I didn’t understand why I needed shots. All I knew was this sharp thing was going to be stuck in my arm and it was going to hurt. Before I got my four year old shots I received my first set of vaccines, vaccines that people are now claiming are dangerous. Research shows that child death rates have been reduced by more than half since 1990 due to vaccinations (Asia News Monitor). The vaccinations are given though out infancy and childhood to prepare the immune system against any possible outbreak of these potentially fatal diseases. Vaccinations involve an injection of, in most cases, an attenuated version†¦show more content†¦Both have reasons to believe what they do. It has been known that ever since the vaccines for diseases like diphtheria and measles were introduced in the twentieth century, the number of deaths related to these diseases decreased by more than 500 percent. On the other hand, the mid-twentieth century was also the time when such indicators as nutrition, sanitation and healthcare, and other important factors of spreading vaccine-preventable diseases have been greatly improved (McMahon-Pratt, 2005). It is rather difficult to objectively determine what influenced the decrease in the number of deaths – the invention of antibiotics in the 1940’s, the improvement of sanitation and healthcare standards, or the introduction of certain vaccines. Those opposing mandatory vaccination choose to believe that it was the first two factors rather than the third one. There are facts to support both versions. For example, death rates from the pertussis (whooping cough) declined from 12,000 deaths per year in 1905 to less than 1,000 deaths per year in 1950 – 12 times less. But as the pertussis vaccine was not widespread until late 1940’s, we can logically assume it obviously was not the vaccine that influenc ed the mentioned decrease. At the same time, since the vaccine was introduced and