An Inquiry Into Hamlets Madness Essays - Characters In Hamlet

An Inquiry Into Hamlet's Madness In the event of examining the nature of Hamlet's madness,we will need to probe into Hamlet's state of mind at different periods and circumstances in the play. Hamlet can be seen to be and not to be mad by different people at different stages. From one perspective, Hamlet can be seen to be mad when Ophelia goes to her father and gives a description of Hamlet's disposition when he goes to see her, also when he goes to see his mother in her closet as can be seen in his tone of voice and his murder of Polonius and his lack of repentance for his death. also, his psychological trauma and emotional depression at the begining of the play may have plunged him into emotional insanity, and lastly his encounter with Leartes in Ophelia's grave. Reversly, evidence is also shown to prove Hamlet's sanitysuch as, when he initially tell Horatio about his intended change of disposition, also, when he tells both Rosencrantz and Guildestern that he is not mad. Also the things which he claimed to have done on the ship bound for England goes to show his sanity, and lastly his encounter with Leartes in Ophelia's grave. Upon the revelation of the ghost who is supposedly Hamlet's father's spirit, we witness a marked change in Hamlet's disposition both in words and in deeds, one of such can be seen when (in Act 2 scene 1) Ophelia goes to see her father, apparently scared gives him a brief but vivid description of Hamlet's disposition when he came to see her. she describes him as having a look so pietous in purport as if he had been loosed out of hell. This shows us a marked change in Hamlet's disposition, the statement As if he had been loosed out of hell raises a lot of questions such as, what happened to Hamlet?. Possibly,some spirit or demon may have taken over him thus his appearance as being hellish in nature or it could be that he had lost his wits to hell and thus is not aware of his appearance and we are made to believe that that he appears thus throughout most of the play. Secondly, to further back up the point that hamlet was indeed mad is or can be seen with the encounter he had with his mother in her closet, where he lashes out at her to the extent that he is rude and also armed with such venomous words that frighten his mother. Possibly, he does this out of mere outrage at finding Claudius' guilt and unable to take revenge but has to see his mother and thus speaks daggers to her heart and seizes her arm possibly in a fit of madness rather than outrage as it should be noted, the act was not premeditated but rather spontaneous and Getrude in shock screams for help and Polonious who is behind the arras(curtains) screams the same and Hamlet hearing him draws his sword and kills him. And when he finally realizes whom he had killed he shows no remorse whatsoever but rather sees his actions as being justified as he says Thou wretched, rash, intrudig fool, farewell. This action and statement show a completely different personality as in most periods in the book we see Hamlet in a suicidal melancholy but never in a murderous mood as we see him here so thus it would be safe to say that he was probably momentarily taken over by a fit of madness. Also from the begining of the book, we see the tragic hero as being psychologically disturbed by the death of his father and the overhasty marriage of his mother to his uncle Claudius, and to further compound matters his love is rejected by Ophelia on the advice of her father over her true feelings and Hamlet's feelings, thus driving him into a state of emotional depression as well as psychological instability as Hamlet now saw himself as loosing both parents as well as a confidant, thus leaving him with no womanly affection whatsoever as he could no longer enjoy the sole monopoly of his mother's affection which had now gone to his uncle (Hamlet is said by some critics

Atonement Ian Mc Ewan Essay Example

Atonement Ian Mc Ewan Essay Example Atonement Ian Mc Ewan Essay Atonement Ian Mc Ewan Essay Essay Topic: A Woman Killed With Kindness Twelfth Night A radical revolt, questioning the notion of a scent. What we have In our discourse is not simply that language reflects a particular truth; the sign doesnt refer to a fixed kind of object. Language Is composed of a variety of signs which continually refer to other signs. To differ substitution of signs. The infinite differing of meaning = difference. If the truth is questioned, the concept of humanism is also questioned; it reflects the values off particular historical point. This is called discursively constructive = SST that is created, but does not pre-exist. Shift from universal to particular values: everything Is constructed and Is not pre- existing. There is ethical concern is Atonement. Its a post modernist novel. We move from fiction to metrification always systematically. Metrification is dealing with how fiction is produced. Something textual its not an essence, constructed and divided. The distinction between fiction and metrification is blurred. The fictionally of the world is questioned. Intellectuality becomes also Important allusions to other authors. The language citations are used and show how constructed and how artificial English literature Is. It Is called In the post-modernist era (Linda Hutchison), a parody = referring to the sat with a difference. History Is seen as very Toll Ana prominent moving away Trot revolutionize tendencies. They want to reemphasize the importance of historical detail. Novels are often a way of questioning the process in which we can write about history. Its not a historical novel in the conventional sense, but its a novel in order to prompt ourselves to ask the question about the perspective of history. In Atonement is showed how difficult it is to reconstruct the totality of the war. Questioning of writing history, historiographer writing history is some kind of story writing. This novel explicitly shows that its constructed from reality. It shows the different phases of writing a book. Emphasis on the ex-centric: emphasis on ethnic minorities, women After the war, many writers start to write about groups that start to be ignored. There is no notion of a centre and the centre cannot speak from everybody. Modernism privileges things that are fluid and unfixed. Its often difficult to distinguish between the two literary movements. We focus on how fiction is close to reality. Blurring of science and fiction. Ms Anew He was born in 1948 and studied at the University of East Anglia in Norwich which is till famous for the course of creative writing. This is a course for apprentice writers. The novelists found at this course are Malcolm Bradbury novels about campus life. A great number of writers in the UK have graduated from his creative writing course, including Ian Ms Anew. In the early period, there was a focus on the bizarre: The Comfort of Strangers: Story of young honeymooners in Venice. They face a danger of meeting an older couple until finally they are trapped in the house who are sexual perverts and who eventually killed them. Later, he became concerned with the notion of good and evil. Black Dogs (1992) deals with the world after the collapse of the Berlin Wall. Black Dogs is a symbol that fascism is still with us; they are the Nazi dogs who used to torture the prisoners. Atonement 2 Saturday (2005) one day in the life of a neurosurgeon, who is attacked during the day and on the very same day, he has to perform an operation on the man who attacked him -> moral dilemma. There allusions of the post 9/1 1 and terrorism. How do we have to react towards such situations? The Gothic note of the novel is adapted to the contemporary period. The Gothic was a mind of style that developed in the 18th century which reintroduced the irrational in contrast to the philosophical enlightenment of romanticism. It is prominent in the novels by Jane Austin (Northerner Abbey, Emma), Bronze (Withering Heights). Atonement is in the same tradition. At the beginning of Atonement, there is a quotation from Northerner Abbey, Ian Ms Anew questions the rationality and the social structure of the British society. Atonement In modernism, there is a preoccupation with shifting points of view and with morality. This preoccupation with ethics is typically British. There is always this difficulty of seeing, because the weather is too bright. Historiographer metrification of the second World War. Story analysis. The first chapter is about a play written by Bryony. The beginning is ir onic because the play reflects the wider plot and shows what is going to happen to Bryony herself. Bryony is interested in gothic prose, she is like Mrs. Mooreland, unable to distinguish what is gothic and what is not. The end of Britons play is characterized with a happy ending and its what is certainly not going to happen in Atonement. Bryony studies ere mothers face because she wants her to approve of her play > study of detail. Bryony wants to control people, Just like Jane Statutes Emma. She is so meticulous; she is Just like the general of an army unlike her sister who is closed in her room among books. Bryony is a contradiction because she likes order and she likes secrets. What she enjoys are very insignificant things. There is a great emphasis placed on her sense of order. She did not have it in her to be cruel ironic passage of the book, she doesnt realize that shes cruel. You can be cruel without even knowing it; its something shes ongoing to discover at the end of the book. Bryony is an artist. The book is about being a writer. The young Bryony is the centre of the attention but we can sense an omniscient narrator telling the story. l en trials AT Ordeal Bryony play Is centralize as a melodrama, exactly want is going to happen to her. 1945- very conservative social background. Thinking of divorce isnt popular. The story of the sister is of a failed marriage like an echo. P. 13 there is already a conflict between the characters about the leading roles of the play. Lola wants to have the leading role and Bryony eventually decides that she is ongoing to be the director of the play. She is very authoritarian. The difficulties of putting out a play PROBLEM OF MEDICATION. Chapter 2 We are introduced to Cecilia. We have an omniscient narrator who explores what goes in the mind of the character. Cilias estate is a kind of paradise. Being expelled from the Tallish estate is like being expelled from paradise. Emphasis on the notion of gaze, look > looking doesnt always mean to understand. P. 21 Another literary allusion Claries by Richardson. This story is about love. The world of Atonement is the world of books. The first chapters take place in the library, something unusual in post modernism. The novel is about social values, transgressing the rules : Cecilia would like to have her independence. Cecilia is quite bold, she has much more freedom; she is wild and in that sense she is opposed to Bryony. Cecilia has feelings of attachment for Robbie but she wants to remain independent. Memories of the war are already present in the first chapters. They have a symbolic value. The vase is symbolic of the war, its something shes always been very attached to. Everything in this novel is associated with the war. . 27 He is in a very difficult position, he is in between two classes in a very structured society which is rather tight. He is somewhat different from everybody else. Roadie wants to study malice, Just Like In ten l orals AT Areola, winner ten woman falls in love with a medical doctor. There is a omniscient perspective but we are in Cilias mind. There is some kind of tension between them and she doesnt want to acknowledge the fact that she is in love with him. Restatement of different positions in the society. She is in a very difficult position herself, because she went to Cambridge. She didnt have good grades ( different from Robbie) and she is in a position of inferiority. The tension of the vase is in between them, because its going to break. The book is full of echoes because the vase is going to break again a second time at another part of the novel. It is a symbol of kindness and all sort of things. One can read in this breaking vase a proliferation of Robbers and Cilias relationship. Emphasis on light. Because its supposed to be written by Bryony who is trying to imitate Virginia Wolfs style. Its almost a pastiche. There is a reminiscence of the stream of consciousness in Virginia Wolf, we know hats going on in Cilias mind. When Bryony receives the rejection letter from the horizon, its quite similar. P. 30 The sense of being not willing to surrender. She is in a furry. As she climbed into the water in her underwear insignificant detail, like in Virginia Wolfs Solid Objects. Little details are going to cause a disaster. The Trials of Rubella, the trials of the heroin who later has to atone what she has done. Rubella is an opera by Richard Strauss and it is extremely refined, its about how husband and wife are finally reconciled and enjoyed a peaceful life of domesticity. Richard Strauss was a major composer of the 20th century and he was the last on to be a romantic. His latest compositions are about death and transfiguration but they are in an extremely romantic style. 1949 is very late indeed ; because there was a lot of experimental music by other composers. Medieval castle gothic allusions. Emphasis on nature. Like in Northerner Abbey, there is a mistake of perception. Its a gothic and post modern perspective. Do we have the right to make such mistakes? I Nils novel Is written Day ten 010 Bryony Ana It could not De peduncles ruling near lifetime. Metrification. P. 8 stream of consciousness, limited view. Robbie is present as a rapist. Bryony likes t o rearrange reality, typical of writers. But do writers have the right to do p. 39 gazing gazing doesnt give the ability to understand. Unambiguous sunlight emphasis on light but light doesnt seem to help. Irony because she things shes getting it wrong when shes not getting it wrong. She is initiated to adult life and its the beginning of her career as a writer. P. 40 hidden observer like herself sense of perspective. Is she not becoming the villain? Can we say that a point of view is always legitimate? After all, its not all relative, its not a matter of playful reference, its also about ethics. There did not have to be a moral thats what she thought at the time. However, the fact that the Atonement novel is made of divisions questions this. She is obviously making a mistake as not to commit herself. She writes in the style of V. Wolf, its not completely neutral, but she doesnt assume that all you need to do is to write about the same thing described from different perspectives but dismissing the concept of values this is her mistake as a writer. The voice of the late Bryony is aware that she was conceited. Its the wrong genre like if drama wasnt appropriate to what she has written. Metrification again. Paul Marshall is introduced to her. From the start, he is depicted as someone very old-baring, someone who is aware of his importance. The villain of the gothic tale. Paul Marshall is interested in Lola. There is a conversation and hes talking about himself all the time. He is talking also about the Rainbow ammo (=love) bar. Shes not mistaken about the fact that Marshall is stupid. She learns that Robbie has been invited by her brother to have diner with the family and shes upset about it. Chapter 5 Lola is again a possible literary allusion. Lola Nabob, Elliot, represents sexuality. Ian Ms Anew wants to show that. There is a conversation between Paul Marshall, the children and Lola for the first time. A literary allusion to Hamlet wanly suggests Tanat you neednt read to much because you start to think different things and that you begin to behave like a snob, like Paul Marshal Chapter 6 Perspective of the mother, reminiscent of Mrs. Dally, p. 68 shes a little bit like Mrs. Dally, she can no longer procreate. Shes missing the comfort of having a child to take care of. The crisis of middle age and the regret of no a nger having children, consciously an imitation of Virginia Wolf. Chapter 7 Bryony is persuaded that she has her great talent developing. P. 2 pastoral ideal emphasis on the nature, rather than on the rational- typical of gothic. Emphasis on perspective. There is an attempt of Bryony to imitate the expressionistic style of Virginia Wolf. P. 76 come back like a leitmotiv used in different contexts used between Cecilia and Robbie and Cecilia and her sister, like come back from a nightmare. So she was not able to come back! Chapter 8 Focuses on Robbie. He is in his study, where youre going to find medical and literary kooks. P. 82 Addends poems major poet of the 20th century. It is important because it has to do with the destruction of the values of Europe. All these magazines really existed. T. S Eliot is another poet. ( on the waste land? ). This is an echo when Bryony receives a rejection from another magazine. He lives in a world that is full of books. Its a novel about fiction, something that the movie couldnt reproduce because its another media. Allusion to Shakespearean comedies Twelfth Night a very dark comedy in which the main character is fooled by everybody and he is unhappily in love. At the end, we eave an Impression Tanat everyone Is nappy except t ml. This applies to Robbie. Its textual. Other authors: Freud p. 6 Robbie is unease but he is free of any sense of inappropriateness. He is very spontaneous and doesnt feel awkward of being in between two situations. Relationship of kindness between mother and son. Very intense feelings are expressed very economically. when I look for my face in my spoon, I see only you. P. 92 he thought of himself in 1962 Robbie wont live in 1962. Science religion-literature = three differ ent ways to see the world. Something you cant detect in the movie. It took Bryony a second to ruin an entire life. P. 103 Bryony feels different from the rest of the family, shes less of a conformist. They are going to have a diner and face a heat wave. Heat wave // sexual. Bryony hands in the letter to Cecilia who asks her if she has read it has! Bryony is an illustration of the role of the artist in the society. Chapter Ten of course she p. 113 Feeling guilty is a major theme in the novel. The writing of this book is a form of atonement. At the end of the book, Bryony is getting more and more sick = physical atonement. Atonement isnt going to resurrect people. Not only is she mistaken of what has happened, she is also thinking of how profitable its going to be for her writing. She is cruel without even knowing. With the letter some cruelness has been introduced. She thinks that theres a monster in the house echo of Northerner Abbey, gothic. Order is not always a good thing because its imposing SST wrong on the society. P seen could never Torture Roadie Nils Lusting mina I nerve Is good Ana evil and she is determined that the source of evil is Robbie himself. She tells Lola about it. Lola enjoys the power of being the centre of attention. Shes very selfish. There is a degree of fascination. the mans a maniac! they are thinking of committing something that will destroy Robbers and Cilias relationship. They are very naive. The fountain episode is very important and shes creating stories about this fountain. There is no doubt that the climax has to take place in the library. P. 122 the atmosphere is an imitation of the gothic novel. what she saw must have been shaped in part by what she already knew, or believed she knew. She saw them emphasis on gaze. Books are put in parallel with over-anxious imagination. Bryony misinterprets the situation, she is reshaping the world according to her imagination; then she leaves the room. Chapter 11. Diner p. 127 has England even been hotter? Robbie compares the hot weather to sexuality. There is a lot of gazing in the whole novel, especially at the diner; however, there is a feeling of discomfort. P. 128 Marshall speaking. All the rules change allusion to sexual heat but its also and equivalent of the transgressions of the rules of the whole family. P. 131 We are in the mind of Robbie who is sure of his relationship with Cecilia; direct quota tion from Shakespeare but Robbie is wrong about it. . 137 Another perspective of the library scene quite sensuous. The supposed attacked was in fact a moment of pleasure. Ironic parallels : Bryony also thinks shes reaching another stage in her life. Importance AT ten Moving moment of togetherness; the only moment of togetherness that theyre going to have. The notion of witnessing Robbie has the impression that God is watching them like a marriage contract. Religious aspect. P. 142 The twins decide to leave the diner. The leave a letter and they are escaping. P. 144 importance of the number of words in common with seeing. Two mistakes : Interpretation Bryony Teaching Robbie : giving the letter to Bryony and to participate in the search. We are in the mind of the wife, a typical Victorian woman. She doesnt agree to give money to her husband to be educated. She is aware that there is going to be a war, that there will be a massacre. There is a great number of echoes, including characterization. Robbie is a hybrid because he has been educated, he has been to Cambridge. He has humble origins but doesnt have the accent of lower people. He doesnt really fit among other soldiers. Emily doesnt like Jacks attitude because she thinks hes too generous. Robbie was manipulated by the British society. There is a rigidity of classes. Chapter 13. Focus on Bryony. P. 157 She is excited of this situation in terms of fiction writing. She is exhibiting the selfishness of the artist. Steam of is it good or not good to be a writer? Misconception of reality. She feels that she is the next on Robbers raping list. P. 158 Bryony is misinterpreting reality. She is mistaken. She has a crush on Robbie as a very young girl. He taught her how to swim. How could someone so benevolent become a villain in the wide imagination of a child? nothing of that sort would happen in England Northerner Abbey it did happen.

Ethic theory on the Workplace Case Study Example | Topics and Well Written Essays - 1500 words

Ethic theory on the Workplace - Case Study Example First, there are two kinds of ethical theories, consequentialist and non-consequentialist. There are two consequentialist theories that include egoism and utilitarianism (Shaw et al., 2009). Ethical egoism is the first theory that can be used to give an answer as to what Kehal should do. According to the theory, Kehal should give the gifts that the influential individuals are demanding. In this way, he will be acting in his own interest as prescribed by the theory. This is because it is assured that the airline will be granted the landing rights once the influential individuals receive what they demanded. Once the company has obtained the landing rights, Kehal will receive the promotion as well as a bonus. This will help him to cater for the medical expenses of his ailing parents and sort out all his commitments. Egoism is largely viewed as a consequentialist theory since it focuses on the consequence of an action for the agent instead of the final outcome. In other words, the theory is based on self interest and its major strength is that it evades the possibility of a conflict between self interest and morality. It would be rational for Kemal to offer the gifts to the influential individuals since by pursuing his interest morality is equated to rationality.   The second consequentialist theory that can help in cracking the ethical dilemma is utilitarianism. Based on this theory Kemal should not hand the gifts demanded by the local manager to be given to the influential individuals.

America and the end of the Cold War Research Paper

America and the end of the Cold War - Research Paper Example The â€Å"Cold War† can be defined as â€Å"a state of political tension and military rivalry which stops short of full-scale war, especially that which existed between the United States and Russia after World War 2† (www.freedictionary.com) The United States was in favor of capitalism, while the Soviet Union favored Communism. Some countries in Europe and Asia aligned themselves with the United States or the USSR. â€Å"During the Cold War, the Soviet Union and United States dominated international politics as opposing superpowers.† (â€Å"Notions of Security: Shifting Concepts and Perspectives†12) There were persistent concerns over Soviets infringing on the national security of these nations. The Americans and the Soviets had nuclear weapons. This resulted in the nuclear arms race between the two governments. There were fears of nuclear war but it never transpired.1 Both nations also wanted to be the first in space. This as well as Communist rule left t he USSR with an inactive economy for many years. When Mikhail Gorbachev was appointed as president in 1985 his goal was to renew the nation’s economy. He and President Ronald Reagan set out to resolve the policy and arms disagreements between their nations. These issues were resolved peacefully between them. In 1990 Boris Yeltsin was elected as president of Russia. In 1991 the Soviet Union officially came to an end subsequently leading to the fall of Communism. The American public was cautiously optimistic about the end of the Cold War because no one was certain that the new form of government in Russia would last.2 â€Å"Communism went out with a whimper, not a bang, hobbling the victory dance.† (Allen & Schweikart 768) The United States and Russia no longer felt threatened by each other. â€Å"The expectation of violence between the two major strategic powers has been drastically reduced.† (Reisman860) Immediately after the Cold War ended President George H.W. Bush began the process of reducing military forces. Unfortunately this resulted in economic problems. Aerospace and shipbuilding companies were nearly bankrupt. There were fewer defense contractors. Soldiers, airmen and sailors were laid off.3 The Recession of the early 1990’s followed not long after this happened. Bush felt that the world had changed for the better as a result of the fall of Communism and felt that Russia and other former Communist nations had to cooperate. In 1991, Congress provided financial help to Russia and other nations so that they could destroy their nuclear weapons. 4 Russia had a seat at the United Nations Security Council that the Soviet Union held previously. The United Nations was able to forge ahead with a new focus once the Cold War was over. Russia (Allen & Schweikart) During the Cold War the primary focus of the United States Intelligence Agency had been the military threat the Soviet Union and its allies imposed upon the nation. When the C old War ended it â€Å"called into question the continued efficacy of U.S intelligence activities in the post Cold War world.† (DeConcini1) The agency knew that there may be potential for a threat but many officials felt that U.S. intelligence needed to help American international firms to compete for business in other countries. They felt that this would protect national security. During the Bush and Clinton administrations private businesses were not supported by intelligence agencies. Former Director of Central Intelligence Robert

Write a management report Essay Example | Topics and Well Written Essays - 1000 words

Write a management report - Essay Example The memo therefore proposes that management should consider identifying the cost drivers and monitor its activities to control their costs. In assessing whether to accept special orders, management needs to accurately compute contribution margin by only considering the variable costs. Fixed costs should only help in determining the break-even point for special orders. Performance evaluation should as well focus on factors that are within employees control rather than incorporating non-controllable costs elements to evaluate the employees working in the SMU2 sector. The major concern of the SMU2 department is the use of inappropriate cost allocation model for the MP product where material costs, labour costs and variable costs are allocated to the product based on estimates. The allocation of material and labour costs to the MP product is acceptable since these are direct costs incurred in the product manufacturing. Allocation of factor overheads especially remaining factory costs should however be reviewed. These overheads should not be allocated to MP on a fixed percentage basis since MP special order sales only accounts for 2% of the revenue. Allocating the overheads on a fixed proportion therefore inflates the costs and reduces the contribution margin. In addition, allocation of media and promotion costs for SMU1 and SMU2 further fails to consider the cost drivers of the activities. Marketing costs should not be allocated depending on the weight of the products but the revenue derived from the effectiveness of such advertisement. This was, management will have to identify the increase in sales resulting from the incremental costs on advertisement. Allocation of management costs to the various products and departments has also failed to take into account the volume allocation and the level of activities performed by such shared staff on the various products. Activity based costing should be used to monitor the activities of the management to the

Advances in DNA Sequencing Technologies

Advances in DNA Sequencing Technologies Abstract Recent advances in DNA sequencing technologies have led to efficient methods for determining the sequence of DNA. DNA sequencing was born in 1977 when Sanger et al proposed the chain termination method and Maxam and Gilbert proposed their own method in the same year. Sangers method was proven to be the most favourable out of the two. Since the birth of DNA sequencing, efficient DNA sequencing technologies was being produced, as Sangers method was laborious, time consuming and expensive; Hood et al proposed automated sequencers involving dye-labelled terminators. Due to the lack of available computational power prior to 1995, sequencing an entire bacterial genome was considered out of reach. This became a reality when Venter and Smith proposed shotgun sequencing in 1995. Pyrosequencing was introduced by Ronagi in 1996 and this method produce the sequence in real-time and is applied by 454 Life Sciences. An indirect method of sequencing DNA was proposed by Drmanac in 1987 called sequen cing by hybridisation and this method lead to the DNA array used by Affymetrix. Nanopore sequencing is a single-molecule sequencing technique and involves single-stranded DNA passing through lipid bilayer via an ion channel, and the ion conductance is measured. Synthetic Nanopores are being produced in order to substitute the lipid bilayer. Illumina sequencing is one of the latest sequencing technologies to be developed involving DNA clustering on flow cells and four dye-labelled terminators performing reverse termination. DNA sequencing has not only been applied to sequence DNA but applied to the real world. DNA sequencing has been involved in the Human genome project and DNA fingerprinting. Introduction Reliable DNA sequencing became a reality in 1977 when Frederick Sanger who perfected the chain termination method to sequence the genome of bacteriophage ?X174 [1][2]. Before Sangers proposal of the chain termination method, there was the plus and minus method, also presented by Sanger along with Coulson [2]. The plus and minus method depended on the use of DNA polymerase in transcribing the specific sequence DNA under controlled conditions. This method was considered efficient and simple, however it was not accurate [2]. As well as the proposal of the chain termination sequencing by Sanger, another method of DNA sequencing was introduced by Maxam and Gilbert involving restriction enzymes, which was also reported in 1977, the same year as Sangers method. The Maxamm and Gilbert method shall be discussed in more detail later on in this essay. Since the proposal of these two methods, spurred many DNA sequencing methods and as the technology developed, so did DNA sequencing. In this lite rature review, the various DNA sequencing technologies shall be looked into as well their applications in the real world and the tools that have aided sequencing DNA e.g. PCR. This review shall begin with the discussion of the chain termination method by Sanger. The Chain Termination Method Sanger discovered that the inhibitory activity of 23-didoxythymidine triphosphate (ddTTP) on the DNA polymerase I was dependent on its incorporation with the growing oligonucleotide chain in the place of thymidylic acid (dT) [2]. In the structure of ddT, there is no 3-hydroxyl group, by there is a hydrogen group in place. With the hydrogen in place of the hydroxyl group, the chain cannot be extended any further, so a termination occurs at the position where dT is positioned. Figure 1 shows the structure of dNTP and ddNTP. Sanger discovered that the inhibitory activity of 23-didoxythymidine triphosphate (ddTTP) on the DNA polymerase I was dependent on its incorporation with the growing oligonucleotide chain in the place of thymidylic acid (dT) [2]. In the structure of ddT, there is no 3-hydroxyl group, by there is a hydrogen group in place. With the hydrogen in place of the hydroxyl group, the chain cannot be extended any further, so a termination occurs at the position where dT is positioned. Figure 1 shows the structure of dNTP and ddNTP. In order to remove the 3-hydroxyl group and replace it with a proton, the triphosphate has to undergo a chemical procedure [1]. There is a different procedure employed for each of the triphosphate groups. Preparation of ddATP was produced from the starting material of 3-O-tosyl-2-deoxyadenosine which was treated with sodium methoxide in dimethylformamide to produce 2,3-dideoxy-2,3-didehydroadenosine, which is an unsaturated compound [4]. The double bond between carbon 2 and 3 of the cyclic ether was then hydrogenated with a palladium-on-carbon catalyst to give 2,3-dideoxyadenosine (ddA). The ddA (ddA) was then phosphorylated in order add the triphosphate group. Purification then took place on DEAE-Sephadex column using a gradient of triethylamine carbonate at pH 8.4. Figure 2 is schematic representation to produce ddA prior to phosphorylation. In the preparation of ddTTP (Figure 3), thymidine was tritylated (+C(Ph3)) at the 5-position and a methanesulphonyl (+CH3SO2) group was introduced at the 3-OH group[5]. The methanesulphonyl group was substituted with iodine by refluxing the compound in 1,2-dimethoxythane in the presence of NaI. After chromatography on a silica column the 5-trityl-3-iodothymidine was hydrogenated in 80% acetic acid to remove the trityl group. The resultant 3-iodothymidine was hydrogenated to produce 23-dideoxythymidine which subsequently was phosphorylated. Once phosphorylated, ddTTP was then purified on a DEAE-sephadex column with triethylammonium-hydrogen carbonate gradient. Figure 3 is a schematic representation to produce ddT prior phosphorylation. When preparing ddGTP, the starting material was N-isobutyryl-5-O-monomethoxytrityldepxyguanosine [1]. After the tosylation of the 3-OH group the compound was then converted to the 23-didehydro derivative with sodium methoxide. Then the isobutyryl group was partly removed during this treatment of sodium methoxide and was removed completely by incubation in the presence of NH3 overnight at 45oC. During the overnight incubation period, the didehydro derivative was reduced to the dideoxy derivative and then converted to the triphosphate. The triphosphate was purified by the fractionation on a DEAE-Sephadex column using a triethylamine carbonate gradient. Figure 4 is a schematic representation to produce ddG prior phosphorylation. Preparing the ddCTP was similar to ddGTP, but was prepared from N-anisoyl-5-O-monomethoxytrityldeoxycytidine. However the purification process was omitted for ddCTP, as it produced a very low yield, therefore the solution was used directly in the experiment described in the paper [2]. Figure 5 is a schematic representation to produce ddC prior phosphorylation. With the four dideoxy samples now prepared, the sequencing procedure can now commence. The dideoxy samples are in separate tubes, along with restriction enzymes obtained from ?X174 replicative form and the four dNTPs [2]. The restriction enzymes and the dNTPs begin strand synthesis and the ddNTP is incorporated to the growing polynucleotide and terminates further strand synthesis. This is due to the lack of the hydroxyl group at the 3 position of ddNTP which prevents the next nucleotide to attach onto the strand. The four tubes are separate by gel-electrophoresis on acrylamide gels (see Gel-Electrophoresis). Figure 6 shows the sequencing procedure. Reading the sequence is straightforward [1]. The first band that moved the furthest is located, this represents the smallest piece of DNA and is the strand terminated by incorporation of the dideoxynucleotide at the first position in the template. The track in which this band occurs is noted. For example (shown in Figure 6), the band that moved the furthest is in track A, so the first nucleotide in the sequence is A. To find out what the next nucleotide, the next most mobile band corresponding to DNA molecule which is one nucleotide longer than the first, and in this example, the band is on track T. Therefore the second nucleotide is T, and the overall sequence so far is AT. The processed is carried on along the autoradiograph until the individual bands start to close in and become inseparable, therefore becoming hard to read. In general it is possible to read upto 400 nucleotides from one autoradiograph with this method. Figure 7 is a schematic representation of an autoradiograph. E ver since Sanger perfected the method of DNA sequencing, there have been advances methods of sequencing along with the achievements. Certain achievements such as the Human genome project and shall be discussed later on in this review. Gel-Electrophoresis Gel-Electrophoresis is defined as the movement of charged molecules in an electric field [1][8]. DNA molecules, like many other biological compounds carry an electric charge. With the case of DNA, this charge is negative. Therefore when DNA is placed in an electric field, they migrate towards the positive pole (as shown in figure 8). There are three factors which affect the rate of migration, which are shape, electrical charge and size. The polyacrylamide gel comprises a complex network of pores through which the molecules must travel to reach the anode. Maxam and Gilbert Method The Maxam and Gilbert method was proposed before Sanger Method in the same year. While the Sangers method involves enzymatic radiolabelled fragments from unlabelled DNA strands [2]. The Maxam-Gilbert method involves chemical cleavage of prelabelled DNA strands in four different ways to form the four different collections of labelled fragments [6][7]. Both methods use gel-electrophoresis to separate the DNA target molecules [8]. However Sangers Chain Termination method has been proven to be simpler and easier to use than the Maxam and Gilbert method [9]. As a matter of fact, looking through the literature text books, Sangers method of DNA sequencing have been explained rather than Maxam and Gilberts [1][3][9][10]. With Maxam and Gilberts method there are two chemical cleavage reactions that take place [6][7]. One of the chemical reaction take places with guanine and the adenine, which are the two purines and the other cleaves the DNA at the cytosine and thymin e, the pyrimidines. For the cleavage reaction, specific reagents are used for each of the reaction. The purine specific reagent is dimethyl sulphate and the pyrimidine specific reagent is hydrazine. Each of these reactions are done in a different way, as each of the four bases have different chemical properties. The cleavage reaction for the guanine/adenine involves using dimethyl sulphate to add a methyl group to the guanines at the N7 position and at the N3 position at the adenines [7]. The glycosidic bond of a methylated adenines is unstable and breaks easily on heating at neutral pH, leaving the sugar free. Treatment with 0.1M alkali at 90oC then will cleave the sugar from the neighbouring phosphate groups. When the resulting end-labelled fragments are resolved on a polyacrylamide gel, the autoradiograph contains a pattern a dark and light bands. The dark bands arise from the breakage at the guanines, which methylate at a rate which is 5-fold faster than adenines. From this reac tion the guanine appear stronger than the adenosine, this can lead to a misinterpretation. Therefore an Adenine-Enhanced cleavage reaction takes place. Figure 9 shows the structural changes of guanine when undergoing the structural modifications involved in Maxam-Gilbert sequencing. With an Adenine-Enhanced cleavage, the glycosidic bond of methylated adenosine is less stable than that of methylated guanosine, thus gentle treatment with dilute acid at the methylation step releases the adenine, allowing darker bands to appear on the autoradiograph [7]. The chemical cleavage for the cytosine and thymine residues involves hydrazine instead of dimethyl sulphate. The hydrazine cleaves the base and leaving ribosylurea [7]. After partial hydrazinolysis in 15-18M aqueous hydrazine at 20oC, the DNA is cleaved with 0.5M piperidine. The piperidine (a cyclic secondary amine), as the free base, displaces all the products of the hydrazine reaction from the sugars and catalyzses the b-elimination of the phosphates. The final pattern contains bands of the similar intensity from the cleavages at the cytosines and thymines. As for cleavage for the cytosine, the presence of 2M NaCl preferentially suppresses the reaction of thymine with hydrazine. Once the cleavage reaction has taken place each original strand is broken into a labelled fragment and an unlabelled fragment [7]. All the labelled fragments start at the 5 end of the strand and terminate at the base that precedes the site of a nucleotide along the original strand. Only the labelled fragmen ts are recorded on the gel electrophoresis. Dye-labelled terminators For many years DNA sequencing has been done by hand, which is both laborious and expensive[3]. Before automated sequencing, about 4 x 106 bases of DNA had been sequenced after the introduction of the Sangers method and Maxam Gilbert methods [11]. In both methods, four sets of reactions and a subsequent electrophoresis step in adjacent lanes of a high-resolution polyacrylamide gel. With the new automated sequencing procedures, four different fluorophores are used, one in each of the base-specific reactions. The reaction products are combined and co-electrophoresed, and the DNA fragments generated in each reaction are detected near the bottom of the gel and identified by their colour. As for choosing which DNA sequencing method to be used, Sangers Method was chosen. This is because Sangers method has been proven to be the most durable and efficient method of DNA sequencing and was the choice of most investigators in large scale sequencing [12]. Figure 10 shows a typical sequence is ge nerated using an automated sequencer. The selection of the dyes was the central development of automated DNA sequencing [11]. The fluorophores that were selected, had to meet several criteria. For instance the absorption and emission maxima had to be in the visible region of the spectrum [11] which is between 380 nm and 780 nm [10], each dye had to be easily distinguishable from one another [11]. Also the dyes should not impair the hybridisation of the oligonucleotide primer, as this would decrease the reliability of synthesis in the sequencing reactions. Figure 11 shows the structures of the dyes which are used in a typical automated sequencing procedure, where X is the moiety where the dye will be bound to. Table 1 shows which dye is covalently attached to which nucleotide in a typical automated DNA sequencing procedure Dye Nucleotide Attached Flourescein Adenosine NBD Thymine Tetramethylrhodamine Guanine Texas Red Cytosine In designing the instrumentation of the florescence detection apparatus, the primary consideration was sensitivity. As the concentration of each band on the co-electrophoresis gel is around 10 M, the instrument needs to be capable of detecting dye concentration of that order. This level of detection can readily be achieved by commercial spectrofluorimeter systems. Unfortunately detection from a gel leads to a much higher background scatter which in turn leads to a decrease in sensitivity. This is solved by using a laser excitation source in order to obtain maximum sensitivity [11]. Figure 12 is schematic diagram of the instrument with the explanation of the instrumentation employed. When analyzing data, Hood had found some complications [11]. Firstly the emission spectra of the different dyes overlapped, in order to overcome this, multicomponent analysis was employed to determine the different amounts of the four dyes present in the gel at any given time. Secondly, the different dye molecules impart non-identical electrophoretic mobilities to the DNA fragments. This meant that the oligonucleotides were not equal base lengths. The third major complication was in analyzing the data comes from the imperfections of the enzymatic methods, for instance there are often regions of the autoradiograph that are difficult to sequence. These complications were overcome in five steps [11] High frequency noise is removed by using a low-pass Fourier filter. A time delay (1.5-4.5 s) between measurements at different wavelength is partially corrected for by linear interpolation between successive measurements. A multicomponent analysis is performed on each set of four data points; this computation yields the amount of each of the four dyes present in the detector as a function of time. The peaks present in the data are located The mobility shift introduced by the dyes is corrected for using empirical determined correction factors. Since the publication of Hoods proposal of the fluorescence detection in automated DNA sequence analysis. Research has been made on focussed on developing which are better in terms of sensitivity [12]. Bacterial and Viral Genome Sequencing (Shotgun Sequencing) Prior to 1995, many viral genomes have been sequenced using Sangers chain termination technique [13], but no bacterial genome has been sequenced. The viral genomes that been sequenced are the 229 kb genome of cytomegalovirus [14], and the 192 kb genome of vaccinia [15], the 187 kb mitochondrial and 121 kb cholorophast genomes of Marchantia polymorpha have been sequenced [16]. Viral genome sequencing has been based upon the sequencing of clones usually derived from extensively mapped restriction fragments, or ? or cosmid clones [17]. Despite advances in DNA sequencing technology, the sequencing of genomes has not progressed beyond clones on the order of the size of the ~ 250kb, which is due to the lack of computational approaches that would enable the efficient assembly of a large number of fragments into an ordered single assembly [13][17]. Upon this, Venter and Smith in 1995 proposed Shotgun Sequencing and enabled Haemophilus influenzae (H. influenzae) to become the first bacterial genome to be sequenced [13][17]. H. influenzae was chosen as it has a similar base composition as a human does with 38 % of sequence made of G + C. Table 2 shows the procedure of the Shotgun Sequencing [17]. When constructing the library ultrasonic waves were used to randomly fragment the genomic DNA into fairly small pieces of about the size of a gene [13]. The fragments were purified and then attached to plasmid vectors[13][17]. The plasmid vectors were then inserted into an E. coli host cell to produce a library of plasmid clones. The E. coli host cell strains had no restriction enzymes which prevented any deletions, rearrangements and loss of the clones [17]. The fragments are randomly sequenced using automated sequencers (Dye-Labelled terminators), with the use of T7 and SP6 primers to sequence the ends of the inserts to enable the coverage of fragments by a factor of 6 [17]. Table 2 (Reference 17) Stage Description Random small insert and large insert library construction Shear genomic DNA randomly to ~2 kb and 15 to 20 kb respectively Library plating Verify random nature of library and maximize random selection of small insert and large insert clones for template production High-throughput DNA sequencing Sequence sufficient number of sequences fragments from both ends for 6x coverage Assembly Assemble random sequence fragments and identity repeat regions Gap Closure Physical gaps Order all contigs (fingerprints, peptide links, ÃŽ », clones, PCR) and provide templates for closure Sequence gaps Complete the genome sequence by primer walking Editing Inspect the sequence visually and resolve sequence ambiguities, including frameshifts Annotation Identify and describe all predicted coding regions (putative identifications, starts and stops, role assignments, operons, regulatory regions) Once the sequencing reaction has been completed, the fragments need to be assembled, and this process is done by using the software TIGR Assembler (The Institute of Genomic Research) [17]. The TIGR Assembler simultaneously clusters and assembles fragments of the genome. In order to obtain the speed necessary to assemble more than 104 fragments [17], an algorithm is used to build up the table of all 10-bp oligonucleotide subsequences to generate a list of potential sequence fragment overlaps. The algorithm begins with the initial contig (single fragment); to extend the contig, a candidate fragment is based on the overlap oligonucleotide content. The initial contig and candidate fragment are aligned by a modified version of the Smith-Waterman [18] algorithm, which allows optional gapped alignments. The contig is extended by the fragment only if strict criteria of overlap content match. The algorithm automatically lowers these criteria in regions of minimal coverage and raises them in r egions with a possible repetitive element [17]. TIGR assembler is designed to take advantage of huge clone sizes [17]. It also enforces a constraint that sequence from two ends of the same template point toward one another in the contig and are located within a certain range of the base pair [17]. Therefore the TIGR assembler provides the computational power to assemble the fragments. Once the fragments have been aligned, the TIGR Editor is used to proofread the sequence and check for any ambiguities in the data [17]. With this technique it does required precautionary care, for instance the small insert in the library should be constructed and end-sequenced concurrently [17]. It is essential that the sequence fragments are of the highest quality and should be rigorously check for any contamination [17]. Pyrosequencing Most of the DNA sequencing required gel-electrophoresis, however in 1996 at the Royal Institute of Technology, Stockholm, Ronaghi proposed Pyrosequencing [19][20]. This is an example of sequencing-by-synthesis, where DNA molecules are clonally amplified on a template, and this template then goes under sequencing [25]. This approach relies on the detection of DNA polymerase activity by enzymatic luminometric inorganic pyrophosphate (PPi) that is released during DNA synthesis and goes under detection assay and offers the advantage of real-time detection [19]. Ronaghi used Nyren [21] description of an enzymatic system consisting of DNA polymerase, ATP sulphurylase and lucifinerase to couple the release of PPi obtained when a nucleotide is incorporated by the polymerase with light emission that can be easily detected by a luminometer or photodiode [20]. When PPi is released, it is immediately converted to adenosine triphosphate (ATP) by ATP sulphurylase, and the level of generated ATP is sensed by luciferase-producing photons [19][20][21]. The unused ATP and deoxynucleotide are degraded by the enzyme apyrase. The presence or absence of PPi, and therefore the incorporation or nonincorporation of each nucleotide added, is ultimately assessed on the basis of whether or not the photons are detected. There is minimal time lapse between these events, and the conditions of the reaction are such that iterative addition of the nucleotides and PPi detection are possible. The release of PPi via the nucleotide incorporation, it is detected by ELIDA (Enzymatic Luminometric Inorganic pyrophosphate Detection Assay) [19][21]. It is within the ELIDA, the PPi is converted to ATP, with the help of ATP sulfurylase and the ATP reacts with the luciferin to generate the light at more than 6 x 109 photons at a wavelength of 560 nm which can be detected by a photodiode, photomultiplier tube, or charge-coupled device (CCD) camera [19][20]. As mentioned before, the DNA molecules need to be amplified by polymerase chain reaction (PCR which is discussed later Ronaghi observed that dATP interfered with the detection system [19]. This interference is a major problem when the method is used to detect a single-base incorporation event. This problem was rectified by replacing the dATP with dATPaS (deoxyadenosine a–thiotrisulphate). It is noticed that adding a small amount of the dATP (0.1 nmol) induces an instantaneous increase in the light emission followed by a slow decrease until it reached a steady-state level (as Figure 11 shows). This makes it impossible to start a sequencing reaction by adding dATP; the reaction must instead be started by addition of DNA polymerase. The signal-to-noise ratio also became higher for dATP compared to the other nucleotides. On the other hand, addition of 8 nmol dATPaS (80-fold higher than the amount of dATP) had only a minor effect on luciferase (as Figure 14 shows). However dATPaS is less than 0.05% as effective as dATP as a substrate for luciferase [19]. Pyrosequencing is adapted by 454 Life Sciences for sequencing by synthesis [22] and is known as the Genome Sequencer (GS) FLX [23][24]. The 454 system consist of random ssDNA (single-stranded) fragments, and each random fragment is bound to the bead under conditions that allow only one fragment to a bead [22]. Once the fragment is attached to the bead, clonal amplification occurs via emulsion. The emulsified beads are purified and placed in microfabricated picolitre wells and then goes under pyrosequencing. A lens array in the detection of the instrument focuses luminescene from each well onto the chip of a CCD camera. The CCD camera images the plate every second in order to detect progression of the pyrosequencing [20][22]. The pyrosequencing machine generates raw data in real time in form of bioluminescence generated from the reactions, and data is presented on a pyrogram [20] Sequencing by Hybridisation As discussed earlier with chain-termination, Maxamm and Gilbert and pyrosequencing, these are all direct methods of sequencing DNA, where each base position is determined individually [26]. There are also indirect methods of sequencing DNA in which the DNA sequence is assembled based on experimental determination of oligonucleotide content of the chain. One promising method of indirect DNA sequencing is called Sequencing by Hybridisation in which sets of oligonucleotide probes are hybridised under conditions that allow the detection of complementary sequences in the target nucleic acid [26]. Sequencing by Hybridisation (SBH) was proposed by Drmanac et al in 1987 [27] and is based on Dotys observation that when DNA is heated in solution, the double-strand melts to form single stranded chains, which then re-nature spontaneously when the solution is cooled [28]. This results the possibility of one piece of DNA recognize another. And hence lead to Drmanac proposal of oligonucleotides pro bes being hybridised under these conditions allowing the complementary sequence in the DNA target to be detected [26][27]. In SBH, an oligonucleotide probe (n-mer probe where n is the length of the probe) is a substring of a DNA sample. This process is similar to doing a keyword search in a page full of text [29]. The set of positively expressed probes is known as the spectrum of DNA sample. For example, the single strand DNA 5GGTCTCG 3 will be sequenced using 4-mer probes and 5 probes will hybridise onto the sequence successfully. The remaining probes will form hybrids with a mismatch at the end base and will be denatured during selective washing. The five probes that are of good match at the end base will result in fully matched hybrids, which will be retained and detected. Each positively expressed serves as a platform to decipher the next base as is seen in Figure 16. For the probes that have successfully hybridised onto the sequence need to be detected. This is achieved by labelling the probes with dyes such as Cyanine3 (Cy3) and Cyanine5 (Cy5) so that the degree of hybridisation can be detected by imaging devices [29]. SBH methods are ideally suited to microarray technology due to their inherent potential for parallel sample processing [29]. An important advantage of using of using a DNA array rather than a multiple probe array is that all the resulting probe-DNA hybrids in any single probe hybridisation are of identical sequence [29]. One of main type of DNA hybridisation array formats is oligonucleotide array which is currently patented by Affymetrix [30]. The commercial uses of this shall be discussed under application of the DNA Array (Affymetrix). Due to the small size of the hybridisation array and the small amount of the target present, it is a challenge to acquire the signals from a DNA Array [29]. These signals must first be amplified b efore they can be detected by the imaging devices. Signals can be boosted by the two means; namely target amplification and signal amplification. In target amplification such as PCR, the amount of target is increased to enhance signal strength while in signal amplification; the amount of signal per unit is increased. Nanopore Sequencing Nanopore sequencing was proposed in 1996 by Branton et al, and shows that individual polynucleotide molecules can be characterised using a membrane channel [31]. Nanopore sequencing is an example of single-molecule sequencing, in which the concept of sequencing-by-synthesis is followed, but without the prior amplification step [24]. This is achieved by the measurement of ionic conductance of a nucleotide passing through a single ion channels in biological membranes or planar lipid bilayer. The measurement of ionic conductance is routine neurobiology and biophysics [31], as well as pharmacology (Ca+ and K+ channel)[32] and biochemistry[9]. Most channels undergo voltage-dependant or ligand dependant gating, there are several large ion channels (i.e. Staphylococcus aureus a-hemolysin) which can remain open extended periods, thereby allowing continuous ionic current to flow across a lipid bilayer [31]. If a transmembrane voltage applied across an open channel of appropriate size should d raw DNA molecules through the channel as extended linear chains whose presence would detect reduce ionic flow. It was assumed, that the reduction in the ionic flow would lead to single channel recordings to characterise the length and hence lead to other characteristics of the polynucleotide. In the proposal by Branton, a-hemolysin was used to form a single channel across a lipid bilayer separating two buffer-filled compartment [31]. a-Hemolysin is a monomeric, 33kD, 293 residue protein that is secreted by the human pathogen Staphylococcus aureus [33]. The nanopore are produced when a-hemolysin subsunits are introduced into a buffered solution that separates lipid bilayer into two compartments (known as cis and trans): the head of t Advances in DNA Sequencing Technologies Advances in DNA Sequencing Technologies Abstract Recent advances in DNA sequencing technologies have led to efficient methods for determining the sequence of DNA. DNA sequencing was born in 1977 when Sanger et al proposed the chain termination method and Maxam and Gilbert proposed their own method in the same year. Sangers method was proven to be the most favourable out of the two. Since the birth of DNA sequencing, efficient DNA sequencing technologies was being produced, as Sangers method was laborious, time consuming and expensive; Hood et al proposed automated sequencers involving dye-labelled terminators. Due to the lack of available computational power prior to 1995, sequencing an entire bacterial genome was considered out of reach. This became a reality when Venter and Smith proposed shotgun sequencing in 1995. Pyrosequencing was introduced by Ronagi in 1996 and this method produce the sequence in real-time and is applied by 454 Life Sciences. An indirect method of sequencing DNA was proposed by Drmanac in 1987 called sequen cing by hybridisation and this method lead to the DNA array used by Affymetrix. Nanopore sequencing is a single-molecule sequencing technique and involves single-stranded DNA passing through lipid bilayer via an ion channel, and the ion conductance is measured. Synthetic Nanopores are being produced in order to substitute the lipid bilayer. Illumina sequencing is one of the latest sequencing technologies to be developed involving DNA clustering on flow cells and four dye-labelled terminators performing reverse termination. DNA sequencing has not only been applied to sequence DNA but applied to the real world. DNA sequencing has been involved in the Human genome project and DNA fingerprinting. Introduction Reliable DNA sequencing became a reality in 1977 when Frederick Sanger who perfected the chain termination method to sequence the genome of bacteriophage ?X174 [1][2]. Before Sangers proposal of the chain termination method, there was the plus and minus method, also presented by Sanger along with Coulson [2]. The plus and minus method depended on the use of DNA polymerase in transcribing the specific sequence DNA under controlled conditions. This method was considered efficient and simple, however it was not accurate [2]. As well as the proposal of the chain termination sequencing by Sanger, another method of DNA sequencing was introduced by Maxam and Gilbert involving restriction enzymes, which was also reported in 1977, the same year as Sangers method. The Maxamm and Gilbert method shall be discussed in more detail later on in this essay. Since the proposal of these two methods, spurred many DNA sequencing methods and as the technology developed, so did DNA sequencing. In this lite rature review, the various DNA sequencing technologies shall be looked into as well their applications in the real world and the tools that have aided sequencing DNA e.g. PCR. This review shall begin with the discussion of the chain termination method by Sanger. The Chain Termination Method Sanger discovered that the inhibitory activity of 23-didoxythymidine triphosphate (ddTTP) on the DNA polymerase I was dependent on its incorporation with the growing oligonucleotide chain in the place of thymidylic acid (dT) [2]. In the structure of ddT, there is no 3-hydroxyl group, by there is a hydrogen group in place. With the hydrogen in place of the hydroxyl group, the chain cannot be extended any further, so a termination occurs at the position where dT is positioned. Figure 1 shows the structure of dNTP and ddNTP. Sanger discovered that the inhibitory activity of 23-didoxythymidine triphosphate (ddTTP) on the DNA polymerase I was dependent on its incorporation with the growing oligonucleotide chain in the place of thymidylic acid (dT) [2]. In the structure of ddT, there is no 3-hydroxyl group, by there is a hydrogen group in place. With the hydrogen in place of the hydroxyl group, the chain cannot be extended any further, so a termination occurs at the position where dT is positioned. Figure 1 shows the structure of dNTP and ddNTP. In order to remove the 3-hydroxyl group and replace it with a proton, the triphosphate has to undergo a chemical procedure [1]. There is a different procedure employed for each of the triphosphate groups. Preparation of ddATP was produced from the starting material of 3-O-tosyl-2-deoxyadenosine which was treated with sodium methoxide in dimethylformamide to produce 2,3-dideoxy-2,3-didehydroadenosine, which is an unsaturated compound [4]. The double bond between carbon 2 and 3 of the cyclic ether was then hydrogenated with a palladium-on-carbon catalyst to give 2,3-dideoxyadenosine (ddA). The ddA (ddA) was then phosphorylated in order add the triphosphate group. Purification then took place on DEAE-Sephadex column using a gradient of triethylamine carbonate at pH 8.4. Figure 2 is schematic representation to produce ddA prior to phosphorylation. In the preparation of ddTTP (Figure 3), thymidine was tritylated (+C(Ph3)) at the 5-position and a methanesulphonyl (+CH3SO2) group was introduced at the 3-OH group[5]. The methanesulphonyl group was substituted with iodine by refluxing the compound in 1,2-dimethoxythane in the presence of NaI. After chromatography on a silica column the 5-trityl-3-iodothymidine was hydrogenated in 80% acetic acid to remove the trityl group. The resultant 3-iodothymidine was hydrogenated to produce 23-dideoxythymidine which subsequently was phosphorylated. Once phosphorylated, ddTTP was then purified on a DEAE-sephadex column with triethylammonium-hydrogen carbonate gradient. Figure 3 is a schematic representation to produce ddT prior phosphorylation. When preparing ddGTP, the starting material was N-isobutyryl-5-O-monomethoxytrityldepxyguanosine [1]. After the tosylation of the 3-OH group the compound was then converted to the 23-didehydro derivative with sodium methoxide. Then the isobutyryl group was partly removed during this treatment of sodium methoxide and was removed completely by incubation in the presence of NH3 overnight at 45oC. During the overnight incubation period, the didehydro derivative was reduced to the dideoxy derivative and then converted to the triphosphate. The triphosphate was purified by the fractionation on a DEAE-Sephadex column using a triethylamine carbonate gradient. Figure 4 is a schematic representation to produce ddG prior phosphorylation. Preparing the ddCTP was similar to ddGTP, but was prepared from N-anisoyl-5-O-monomethoxytrityldeoxycytidine. However the purification process was omitted for ddCTP, as it produced a very low yield, therefore the solution was used directly in the experiment described in the paper [2]. Figure 5 is a schematic representation to produce ddC prior phosphorylation. With the four dideoxy samples now prepared, the sequencing procedure can now commence. The dideoxy samples are in separate tubes, along with restriction enzymes obtained from ?X174 replicative form and the four dNTPs [2]. The restriction enzymes and the dNTPs begin strand synthesis and the ddNTP is incorporated to the growing polynucleotide and terminates further strand synthesis. This is due to the lack of the hydroxyl group at the 3 position of ddNTP which prevents the next nucleotide to attach onto the strand. The four tubes are separate by gel-electrophoresis on acrylamide gels (see Gel-Electrophoresis). Figure 6 shows the sequencing procedure. Reading the sequence is straightforward [1]. The first band that moved the furthest is located, this represents the smallest piece of DNA and is the strand terminated by incorporation of the dideoxynucleotide at the first position in the template. The track in which this band occurs is noted. For example (shown in Figure 6), the band that moved the furthest is in track A, so the first nucleotide in the sequence is A. To find out what the next nucleotide, the next most mobile band corresponding to DNA molecule which is one nucleotide longer than the first, and in this example, the band is on track T. Therefore the second nucleotide is T, and the overall sequence so far is AT. The processed is carried on along the autoradiograph until the individual bands start to close in and become inseparable, therefore becoming hard to read. In general it is possible to read upto 400 nucleotides from one autoradiograph with this method. Figure 7 is a schematic representation of an autoradiograph. E ver since Sanger perfected the method of DNA sequencing, there have been advances methods of sequencing along with the achievements. Certain achievements such as the Human genome project and shall be discussed later on in this review. Gel-Electrophoresis Gel-Electrophoresis is defined as the movement of charged molecules in an electric field [1][8]. DNA molecules, like many other biological compounds carry an electric charge. With the case of DNA, this charge is negative. Therefore when DNA is placed in an electric field, they migrate towards the positive pole (as shown in figure 8). There are three factors which affect the rate of migration, which are shape, electrical charge and size. The polyacrylamide gel comprises a complex network of pores through which the molecules must travel to reach the anode. Maxam and Gilbert Method The Maxam and Gilbert method was proposed before Sanger Method in the same year. While the Sangers method involves enzymatic radiolabelled fragments from unlabelled DNA strands [2]. The Maxam-Gilbert method involves chemical cleavage of prelabelled DNA strands in four different ways to form the four different collections of labelled fragments [6][7]. Both methods use gel-electrophoresis to separate the DNA target molecules [8]. However Sangers Chain Termination method has been proven to be simpler and easier to use than the Maxam and Gilbert method [9]. As a matter of fact, looking through the literature text books, Sangers method of DNA sequencing have been explained rather than Maxam and Gilberts [1][3][9][10]. With Maxam and Gilberts method there are two chemical cleavage reactions that take place [6][7]. One of the chemical reaction take places with guanine and the adenine, which are the two purines and the other cleaves the DNA at the cytosine and thymin e, the pyrimidines. For the cleavage reaction, specific reagents are used for each of the reaction. The purine specific reagent is dimethyl sulphate and the pyrimidine specific reagent is hydrazine. Each of these reactions are done in a different way, as each of the four bases have different chemical properties. The cleavage reaction for the guanine/adenine involves using dimethyl sulphate to add a methyl group to the guanines at the N7 position and at the N3 position at the adenines [7]. The glycosidic bond of a methylated adenines is unstable and breaks easily on heating at neutral pH, leaving the sugar free. Treatment with 0.1M alkali at 90oC then will cleave the sugar from the neighbouring phosphate groups. When the resulting end-labelled fragments are resolved on a polyacrylamide gel, the autoradiograph contains a pattern a dark and light bands. The dark bands arise from the breakage at the guanines, which methylate at a rate which is 5-fold faster than adenines. From this reac tion the guanine appear stronger than the adenosine, this can lead to a misinterpretation. Therefore an Adenine-Enhanced cleavage reaction takes place. Figure 9 shows the structural changes of guanine when undergoing the structural modifications involved in Maxam-Gilbert sequencing. With an Adenine-Enhanced cleavage, the glycosidic bond of methylated adenosine is less stable than that of methylated guanosine, thus gentle treatment with dilute acid at the methylation step releases the adenine, allowing darker bands to appear on the autoradiograph [7]. The chemical cleavage for the cytosine and thymine residues involves hydrazine instead of dimethyl sulphate. The hydrazine cleaves the base and leaving ribosylurea [7]. After partial hydrazinolysis in 15-18M aqueous hydrazine at 20oC, the DNA is cleaved with 0.5M piperidine. The piperidine (a cyclic secondary amine), as the free base, displaces all the products of the hydrazine reaction from the sugars and catalyzses the b-elimination of the phosphates. The final pattern contains bands of the similar intensity from the cleavages at the cytosines and thymines. As for cleavage for the cytosine, the presence of 2M NaCl preferentially suppresses the reaction of thymine with hydrazine. Once the cleavage reaction has taken place each original strand is broken into a labelled fragment and an unlabelled fragment [7]. All the labelled fragments start at the 5 end of the strand and terminate at the base that precedes the site of a nucleotide along the original strand. Only the labelled fragmen ts are recorded on the gel electrophoresis. Dye-labelled terminators For many years DNA sequencing has been done by hand, which is both laborious and expensive[3]. Before automated sequencing, about 4 x 106 bases of DNA had been sequenced after the introduction of the Sangers method and Maxam Gilbert methods [11]. In both methods, four sets of reactions and a subsequent electrophoresis step in adjacent lanes of a high-resolution polyacrylamide gel. With the new automated sequencing procedures, four different fluorophores are used, one in each of the base-specific reactions. The reaction products are combined and co-electrophoresed, and the DNA fragments generated in each reaction are detected near the bottom of the gel and identified by their colour. As for choosing which DNA sequencing method to be used, Sangers Method was chosen. This is because Sangers method has been proven to be the most durable and efficient method of DNA sequencing and was the choice of most investigators in large scale sequencing [12]. Figure 10 shows a typical sequence is ge nerated using an automated sequencer. The selection of the dyes was the central development of automated DNA sequencing [11]. The fluorophores that were selected, had to meet several criteria. For instance the absorption and emission maxima had to be in the visible region of the spectrum [11] which is between 380 nm and 780 nm [10], each dye had to be easily distinguishable from one another [11]. Also the dyes should not impair the hybridisation of the oligonucleotide primer, as this would decrease the reliability of synthesis in the sequencing reactions. Figure 11 shows the structures of the dyes which are used in a typical automated sequencing procedure, where X is the moiety where the dye will be bound to. Table 1 shows which dye is covalently attached to which nucleotide in a typical automated DNA sequencing procedure Dye Nucleotide Attached Flourescein Adenosine NBD Thymine Tetramethylrhodamine Guanine Texas Red Cytosine In designing the instrumentation of the florescence detection apparatus, the primary consideration was sensitivity. As the concentration of each band on the co-electrophoresis gel is around 10 M, the instrument needs to be capable of detecting dye concentration of that order. This level of detection can readily be achieved by commercial spectrofluorimeter systems. Unfortunately detection from a gel leads to a much higher background scatter which in turn leads to a decrease in sensitivity. This is solved by using a laser excitation source in order to obtain maximum sensitivity [11]. Figure 12 is schematic diagram of the instrument with the explanation of the instrumentation employed. When analyzing data, Hood had found some complications [11]. Firstly the emission spectra of the different dyes overlapped, in order to overcome this, multicomponent analysis was employed to determine the different amounts of the four dyes present in the gel at any given time. Secondly, the different dye molecules impart non-identical electrophoretic mobilities to the DNA fragments. This meant that the oligonucleotides were not equal base lengths. The third major complication was in analyzing the data comes from the imperfections of the enzymatic methods, for instance there are often regions of the autoradiograph that are difficult to sequence. These complications were overcome in five steps [11] High frequency noise is removed by using a low-pass Fourier filter. A time delay (1.5-4.5 s) between measurements at different wavelength is partially corrected for by linear interpolation between successive measurements. A multicomponent analysis is performed on each set of four data points; this computation yields the amount of each of the four dyes present in the detector as a function of time. The peaks present in the data are located The mobility shift introduced by the dyes is corrected for using empirical determined correction factors. Since the publication of Hoods proposal of the fluorescence detection in automated DNA sequence analysis. Research has been made on focussed on developing which are better in terms of sensitivity [12]. Bacterial and Viral Genome Sequencing (Shotgun Sequencing) Prior to 1995, many viral genomes have been sequenced using Sangers chain termination technique [13], but no bacterial genome has been sequenced. The viral genomes that been sequenced are the 229 kb genome of cytomegalovirus [14], and the 192 kb genome of vaccinia [15], the 187 kb mitochondrial and 121 kb cholorophast genomes of Marchantia polymorpha have been sequenced [16]. Viral genome sequencing has been based upon the sequencing of clones usually derived from extensively mapped restriction fragments, or ? or cosmid clones [17]. Despite advances in DNA sequencing technology, the sequencing of genomes has not progressed beyond clones on the order of the size of the ~ 250kb, which is due to the lack of computational approaches that would enable the efficient assembly of a large number of fragments into an ordered single assembly [13][17]. Upon this, Venter and Smith in 1995 proposed Shotgun Sequencing and enabled Haemophilus influenzae (H. influenzae) to become the first bacterial genome to be sequenced [13][17]. H. influenzae was chosen as it has a similar base composition as a human does with 38 % of sequence made of G + C. Table 2 shows the procedure of the Shotgun Sequencing [17]. When constructing the library ultrasonic waves were used to randomly fragment the genomic DNA into fairly small pieces of about the size of a gene [13]. The fragments were purified and then attached to plasmid vectors[13][17]. The plasmid vectors were then inserted into an E. coli host cell to produce a library of plasmid clones. The E. coli host cell strains had no restriction enzymes which prevented any deletions, rearrangements and loss of the clones [17]. The fragments are randomly sequenced using automated sequencers (Dye-Labelled terminators), with the use of T7 and SP6 primers to sequence the ends of the inserts to enable the coverage of fragments by a factor of 6 [17]. Table 2 (Reference 17) Stage Description Random small insert and large insert library construction Shear genomic DNA randomly to ~2 kb and 15 to 20 kb respectively Library plating Verify random nature of library and maximize random selection of small insert and large insert clones for template production High-throughput DNA sequencing Sequence sufficient number of sequences fragments from both ends for 6x coverage Assembly Assemble random sequence fragments and identity repeat regions Gap Closure Physical gaps Order all contigs (fingerprints, peptide links, ÃŽ », clones, PCR) and provide templates for closure Sequence gaps Complete the genome sequence by primer walking Editing Inspect the sequence visually and resolve sequence ambiguities, including frameshifts Annotation Identify and describe all predicted coding regions (putative identifications, starts and stops, role assignments, operons, regulatory regions) Once the sequencing reaction has been completed, the fragments need to be assembled, and this process is done by using the software TIGR Assembler (The Institute of Genomic Research) [17]. The TIGR Assembler simultaneously clusters and assembles fragments of the genome. In order to obtain the speed necessary to assemble more than 104 fragments [17], an algorithm is used to build up the table of all 10-bp oligonucleotide subsequences to generate a list of potential sequence fragment overlaps. The algorithm begins with the initial contig (single fragment); to extend the contig, a candidate fragment is based on the overlap oligonucleotide content. The initial contig and candidate fragment are aligned by a modified version of the Smith-Waterman [18] algorithm, which allows optional gapped alignments. The contig is extended by the fragment only if strict criteria of overlap content match. The algorithm automatically lowers these criteria in regions of minimal coverage and raises them in r egions with a possible repetitive element [17]. TIGR assembler is designed to take advantage of huge clone sizes [17]. It also enforces a constraint that sequence from two ends of the same template point toward one another in the contig and are located within a certain range of the base pair [17]. Therefore the TIGR assembler provides the computational power to assemble the fragments. Once the fragments have been aligned, the TIGR Editor is used to proofread the sequence and check for any ambiguities in the data [17]. With this technique it does required precautionary care, for instance the small insert in the library should be constructed and end-sequenced concurrently [17]. It is essential that the sequence fragments are of the highest quality and should be rigorously check for any contamination [17]. Pyrosequencing Most of the DNA sequencing required gel-electrophoresis, however in 1996 at the Royal Institute of Technology, Stockholm, Ronaghi proposed Pyrosequencing [19][20]. This is an example of sequencing-by-synthesis, where DNA molecules are clonally amplified on a template, and this template then goes under sequencing [25]. This approach relies on the detection of DNA polymerase activity by enzymatic luminometric inorganic pyrophosphate (PPi) that is released during DNA synthesis and goes under detection assay and offers the advantage of real-time detection [19]. Ronaghi used Nyren [21] description of an enzymatic system consisting of DNA polymerase, ATP sulphurylase and lucifinerase to couple the release of PPi obtained when a nucleotide is incorporated by the polymerase with light emission that can be easily detected by a luminometer or photodiode [20]. When PPi is released, it is immediately converted to adenosine triphosphate (ATP) by ATP sulphurylase, and the level of generated ATP is sensed by luciferase-producing photons [19][20][21]. The unused ATP and deoxynucleotide are degraded by the enzyme apyrase. The presence or absence of PPi, and therefore the incorporation or nonincorporation of each nucleotide added, is ultimately assessed on the basis of whether or not the photons are detected. There is minimal time lapse between these events, and the conditions of the reaction are such that iterative addition of the nucleotides and PPi detection are possible. The release of PPi via the nucleotide incorporation, it is detected by ELIDA (Enzymatic Luminometric Inorganic pyrophosphate Detection Assay) [19][21]. It is within the ELIDA, the PPi is converted to ATP, with the help of ATP sulfurylase and the ATP reacts with the luciferin to generate the light at more than 6 x 109 photons at a wavelength of 560 nm which can be detected by a photodiode, photomultiplier tube, or charge-coupled device (CCD) camera [19][20]. As mentioned before, the DNA molecules need to be amplified by polymerase chain reaction (PCR which is discussed later Ronaghi observed that dATP interfered with the detection system [19]. This interference is a major problem when the method is used to detect a single-base incorporation event. This problem was rectified by replacing the dATP with dATPaS (deoxyadenosine a–thiotrisulphate). It is noticed that adding a small amount of the dATP (0.1 nmol) induces an instantaneous increase in the light emission followed by a slow decrease until it reached a steady-state level (as Figure 11 shows). This makes it impossible to start a sequencing reaction by adding dATP; the reaction must instead be started by addition of DNA polymerase. The signal-to-noise ratio also became higher for dATP compared to the other nucleotides. On the other hand, addition of 8 nmol dATPaS (80-fold higher than the amount of dATP) had only a minor effect on luciferase (as Figure 14 shows). However dATPaS is less than 0.05% as effective as dATP as a substrate for luciferase [19]. Pyrosequencing is adapted by 454 Life Sciences for sequencing by synthesis [22] and is known as the Genome Sequencer (GS) FLX [23][24]. The 454 system consist of random ssDNA (single-stranded) fragments, and each random fragment is bound to the bead under conditions that allow only one fragment to a bead [22]. Once the fragment is attached to the bead, clonal amplification occurs via emulsion. The emulsified beads are purified and placed in microfabricated picolitre wells and then goes under pyrosequencing. A lens array in the detection of the instrument focuses luminescene from each well onto the chip of a CCD camera. The CCD camera images the plate every second in order to detect progression of the pyrosequencing [20][22]. The pyrosequencing machine generates raw data in real time in form of bioluminescence generated from the reactions, and data is presented on a pyrogram [20] Sequencing by Hybridisation As discussed earlier with chain-termination, Maxamm and Gilbert and pyrosequencing, these are all direct methods of sequencing DNA, where each base position is determined individually [26]. There are also indirect methods of sequencing DNA in which the DNA sequence is assembled based on experimental determination of oligonucleotide content of the chain. One promising method of indirect DNA sequencing is called Sequencing by Hybridisation in which sets of oligonucleotide probes are hybridised under conditions that allow the detection of complementary sequences in the target nucleic acid [26]. Sequencing by Hybridisation (SBH) was proposed by Drmanac et al in 1987 [27] and is based on Dotys observation that when DNA is heated in solution, the double-strand melts to form single stranded chains, which then re-nature spontaneously when the solution is cooled [28]. This results the possibility of one piece of DNA recognize another. And hence lead to Drmanac proposal of oligonucleotides pro bes being hybridised under these conditions allowing the complementary sequence in the DNA target to be detected [26][27]. In SBH, an oligonucleotide probe (n-mer probe where n is the length of the probe) is a substring of a DNA sample. This process is similar to doing a keyword search in a page full of text [29]. The set of positively expressed probes is known as the spectrum of DNA sample. For example, the single strand DNA 5GGTCTCG 3 will be sequenced using 4-mer probes and 5 probes will hybridise onto the sequence successfully. The remaining probes will form hybrids with a mismatch at the end base and will be denatured during selective washing. The five probes that are of good match at the end base will result in fully matched hybrids, which will be retained and detected. Each positively expressed serves as a platform to decipher the next base as is seen in Figure 16. For the probes that have successfully hybridised onto the sequence need to be detected. This is achieved by labelling the probes with dyes such as Cyanine3 (Cy3) and Cyanine5 (Cy5) so that the degree of hybridisation can be detected by imaging devices [29]. SBH methods are ideally suited to microarray technology due to their inherent potential for parallel sample processing [29]. An important advantage of using of using a DNA array rather than a multiple probe array is that all the resulting probe-DNA hybrids in any single probe hybridisation are of identical sequence [29]. One of main type of DNA hybridisation array formats is oligonucleotide array which is currently patented by Affymetrix [30]. The commercial uses of this shall be discussed under application of the DNA Array (Affymetrix). Due to the small size of the hybridisation array and the small amount of the target present, it is a challenge to acquire the signals from a DNA Array [29]. These signals must first be amplified b efore they can be detected by the imaging devices. Signals can be boosted by the two means; namely target amplification and signal amplification. In target amplification such as PCR, the amount of target is increased to enhance signal strength while in signal amplification; the amount of signal per unit is increased. Nanopore Sequencing Nanopore sequencing was proposed in 1996 by Branton et al, and shows that individual polynucleotide molecules can be characterised using a membrane channel [31]. Nanopore sequencing is an example of single-molecule sequencing, in which the concept of sequencing-by-synthesis is followed, but without the prior amplification step [24]. This is achieved by the measurement of ionic conductance of a nucleotide passing through a single ion channels in biological membranes or planar lipid bilayer. The measurement of ionic conductance is routine neurobiology and biophysics [31], as well as pharmacology (Ca+ and K+ channel)[32] and biochemistry[9]. Most channels undergo voltage-dependant or ligand dependant gating, there are several large ion channels (i.e. Staphylococcus aureus a-hemolysin) which can remain open extended periods, thereby allowing continuous ionic current to flow across a lipid bilayer [31]. If a transmembrane voltage applied across an open channel of appropriate size should d raw DNA molecules through the channel as extended linear chains whose presence would detect reduce ionic flow. It was assumed, that the reduction in the ionic flow would lead to single channel recordings to characterise the length and hence lead to other characteristics of the polynucleotide. In the proposal by Branton, a-hemolysin was used to form a single channel across a lipid bilayer separating two buffer-filled compartment [31]. a-Hemolysin is a monomeric, 33kD, 293 residue protein that is secreted by the human pathogen Staphylococcus aureus [33]. The nanopore are produced when a-hemolysin subsunits are introduced into a buffered solution that separates lipid bilayer into two compartments (known as cis and trans): the head of t

Faust Essay -- essays research papers

The legend of Faust was a legend that occurred in the 1500’s in Europe. Over time, as the story was told and passed on through generations, many different ideas on what happened were brought up, but the main idea of the story is the same in most cases. One of the most interesting things about this legend is the fact that though this story is more than four hundred years old, it is still told in some contemporary films to this day. All though it is not always as direct as a deal with the actual devil, the same basis of the story can be seen in present day films. In one of the most successful movies of the year 2000, The Matrix, a Faustian theme is evident. The Matrix is a science fiction movie directed by the Wachowski brothers. The old legend of Faust is, in short, about a young scholar who made a deal with Mephistopheles, the devil. Faust was seeking ultimate knowledge and in the deal the devil said he would grant Faust ultimate knowledge in return for his soul. Faust agrees to the deal and after a certain time period of possessing ultimate knowledge Faust suddenly dies. There are many different versions of the story as to exactly how he died, and some versions of the story go into more detail than others. As time passed, Faustian legends were being told in many different stories, many different ways. To have a Faust story, four basic elements should be present: a Faust figure, a devil figure, some sort of temptation, and a price.   Ã‚  Ã‚  Ã‚  Ã‚  The Matrix is about a man, called Neo, who was living an average life, and was heavily into computer hacking. One day he receives messages appearing on his computer leading him towards a meeting with a powerful man named Morphius. Morphius alerts Neo that the reason that the reason that all of federal agents were chasing him and all of these other things were happening to him because he was â€Å"the one†. He was searches for a greater truth in the world than what was just there in his face, and Morphius says that he could show Neo that truth. Morphius then holds two pills in his hands, one pill would lead him to the truth, the other would just take him back to his regular life as if nothing ever happened. Neo wanted the truth about the world so Morphius explained it. He said that the perception is that our day-in, day-out world is real; in reality, that world is a hoax, an elaborate deception spun by al... ...ant to make Faust a hero of some sort.   Ã‚  Ã‚  Ã‚  Ã‚  If someone could go back in time and show this film to people that know of the Faust legend back in around the 1700’s, it would make absolutely no sense. It would seem like a whole lot of unrealistic garbage to those people, but in present times, though the Matrix is very futuristic, it almost seems as if it could be possible. The whole idea of the movie plot seems brilliant and makes people question themselves to whether or not this concept could actually be possible.   Ã‚  Ã‚  Ã‚  Ã‚  The links between the Matrix and the Faust legend can be seen throughout the entire film. Even though the storyline of the legend has gone through a number of changes over time, it is truly amazing how a story that probably is not even true still comes into use in society today. The Faust legend is used in other situations besides just films, stories, or plays; it is also seen in everyday situations for people. There are many ways in which a Faustian theme can come into play in everyday life. For example, any one who has ever sold themselves out for something, gambled, or done drugs has been in a Faustian situation.

E-Procurement And E-Logistics

our site – CUSTOM ESSAY WRITING – Business Management Dissertation Ideas ABSTRACT In this paper, we analyze the e-procurement and e-logistics of the Dell Inc. Company. This will include a brief overview of the company, an exploration of its Customer Relationship Management, the Supply Chain management and an analysis of the various softwares used by Dell Inc in promoting its relationship marketing. INTRODUCTION Today, many people have discovered the significance of E-commerce. E-commerce, also known as electronic commerce refers to business transactions and communication via computers especially over the internet and networks (Botha, Bothma and Geldenhuys, 2008: p.23). This involves buying and selling of services and goods, and transfer of funds among other commercial communications through the internet, mainly through the World-Wide Web (Botha, Bothma and Geldenhuys, 2008: p.23). E-commerce takes place in different situations such as between businesses and customers (B2C), between one business/company and another (B2B), and between customer and customer (C2C). It is mainly divided into two main parts, which are e-procurement and e-logistics. E-procurement is defined as an electronic method of conducting business transactions while e-logistics refers to the transfer of goods sold over the internet to customers (Botha, Bothma and Geldenhuys, 2008: p.24). A well implemented e-procurement system is highly effective in connecting businesses and other business processes with suppliers while running all interactions between them. According to Botha, Bothma and Geldenhuys (2008: p.23), the development and advancement of technology, many businesses now sell their products through computer technology, which is a brilliant way of making companies reduce overhead costs and reach a wide customer base. Thus, e-procurement benefits not only the business owners, but also customers since they can shop without leaving their homes. Also, customers can easily find the lowest price of products when buying their goods via the internet. In this paper, we analyze the e-procurement and e-logistics of the Dell Inc. Company. DELL INCORPORATED Dell Inc. is a computer company that was established by Michael S. Dell, in 1984 (Krauss 2003: p.7). It offers a wide range of technology product categories (Krauss (2003: p.8). These products range from personal computers to services such as storage solutions. Also, it gives a variety of services, which range from business services and configurable information technology including product-related support services, consulting and applications and infrastructure technology (Krauss, 2003: p.8). As stated by Levy (1999: p.20), Dell Inc. operates in four global business segments, which include public, Large Enterprise, Consumer, and small and Medium Business. The company designs its own products, manufactures and markets them, sells, as well as supports a range of products and services, which can be modified to individual requirements of customers (Perret and Jaffeux, 2007: p.4). Dell Inc. is considered among the companies that are most profitable. The company offers the most innovative customer service, as well as product custom configuration in the world (Perret and Jaffeux, 2007: p.5). For this reason, the company is faced with the challenge of satisfying the customers’ needs while maintaining a stable relationship with them. E-PROCUREMENT AT DELL Dell Inc. is widely known for selling its computers and others services through the internet to other business (B2B) and to individual customers (B2C) (Perret and Jaffeux, 2007: p.5). B2B refers to business transactions between one company and another such as business customers, suppliers and distributors. The B2C refers to business transactions between a company and consumers. At the beginning of the 1990’s, Dell Inc. attempted to distribute wares by retailing. However, the management found out later that this method was unprofitable for business (Gattorna, 2003: p.51). Hence, Dell Inc. decided to key on boosting its customer support and services by allowing customers to make orders directly (Gattorna, 2003: p.52). This was considered a unique strategy for Dell customization. Recently, Dell Inc. improved its sourcing and buying processes by implementing a leading e-procurement solution known as Ariba Buyer (Krauss, 2003: p.8). In order to ease the business processes between Dell Inc. and its supplier companies, Ariba Buyer which is an e-procurement solution is used. It is quite useful in automating and streamlining sourcing. (Li, 2007: p.20). In earlier years, making purchase orders at Dell was a highly laborious process since company workers filled out forms for each purchase process every time they ordered an item, which included collecting about ten approval signatures (Li, 2007: p.21). The buyers were then expected to re-enter the data into two different systems that included a home-grown Access database and the legacy purchasing system. This paper-based process was challenging for Dell to track its purchases by commodity, as well as analyze its purchasing patterns in terms of where, how much and from whom the supplies were bought, hence the change in its procurement process. Thus, Dell Inc. implemented an e-procurement solution known as Ariba Buyer. E-procurement enabled Dell to streamline its supplying base. This helped in the elimination of maverick spending, as well as standardization of the ordering processes for its suppliers. (Krauss, 2003: p.8). This was followed, by Dell’s move, to assess 3 e-procurement systems depending on five criteria. These criteria included a user-friendly boundary, cost-effectiveness, and integration with existing back-end system (Krauss, 2003: p.8). Others included e-commerce links to most of Dell’s supplying companies, and compatibility with the current IT policy of Dell servers (Li, 2007: p.20). According to Gattorna (2003: p.50), close to seven months were spent by the personnel that were responsible for implementing Ariba. This time was spent in developing twenty interfaces that would facilitate connection of Ariba buyer with Dell’s legacy systems. They created linkages for Ariba and Dell’s purchase order, catalog data, cost center, accounting code validation, and employee data among other systems (Gattorna, 2003: p.50). This was made to ascertain that all the processed orders had been validated. This resulted in a final product, which facilitates making purchases online. This product is known as Dell Internet Requisition Tool (DIREQT) (Gattorna, 2003: p.51). Currently, DIREQT has made it easy for Dell employees to complete purchasing orders online by loging into DIREQT Web site, as well as conducting searches for certain products, suppliers or services, which usually give accurate status reports (Levy, 1999: p.23). Immediately, Ariba Buyer forwards the catalog items and requisition straight to the right manager at the cost center who signs the order electronically. The system then automatically creates an approving chain before directing it to an employee network. (Gattorna, 2003: p.51). However, if the product ordered is not present in the catalogue, Ariba Buyer includes a Dell buyer to source the product and hands over the request for last signatures (Perret and Jaffeux, 2007: p.6). After the requisition has been approved, it is moved to the Ariba Commerce Services Network (ASCN). ASCN is a shared network infrastructure that helps to connect with buyers and marketplaces, on the Ariba Business to Business (B2B). Commerce stand (Perret and Jaffeux, 2007: p.6). Ariba uses ASCN to communicate its orders to suppliers, which includes shipping through e-mail, faxes, Extensive Markup Language (XML) and electronic data interchange (EDI) (Perret and Jaffeux, 2007: p.6). Moreover, Ariba Buyer also accelerates the payment process in Dell Inc. The receipts that Dell’s central receiving department prepares for wares are brought into the organization and matched automatically with the right invoice. This is then fed into the system by the account payable processors (Bothma and Geldenhuys, 2008: p.25). In addition, the purchasers create receipts of the service given to them, which is also matched in an automatic manner. Therefore, this practice helps to avoid the early routine of service invoices, which is time-consuming, when making purchases for approval. As stated by Botha, Bothma and Geldenhuys (2008: p.25), with the Ariba Buyer at Dell, the requisition cycle time is likely to be reduced by 62%, and lessen operation costs by 61%. However, Dell Inc. believes that it stands to benefit on a larger scale from the perception into the buying process attained through combining customers’ information. Moreover, through the use of Ariba, Dell is able to gather information necessary to evaluate its supply base and re-evaluate key business to market communications services, office products and consulting, among many more kinds of expenditures (Gattorna, 2003: p.50). CUSTOMER RELATIONSHIP MANAGEMENT According to Perret and Jaffeux (2007: p.7), Customer Relationship Management (CRM) is the creation and maintenance of relations with customers. The key aim of Dell is to offer its customers technologically reliable customer service requirements. Perret and Jaffeux (2007: p.7) argue that the software that help in facilitation facilitate Dell’s CRM include marketing automation software, a system that benefits the sales, and custom designed Web pages that contain purchase data. According to Ross (2010: p.88), today, one fifth of standard-based computers sold in the world is Dell’s product. The key concept of Dell Inc. is to sell computers directly to customers. This will increase their success in the computer business. (Ross, 2010: p.88). Before Dell Inc. invented the made-to-order concept, its customers used to buy its products from electronic shops and retail stores. In this case, customers interacted only with the salesperson of the store and not the manufacturer. Therefore, Dell introduced the concept of interacting directly with the customer via the internet so as to fulfil the demands of its clients and deliver quality services. E-LOGISTICS AT DELL INC. For Dell Inc., the E-logistics has entirely changed it way of distributing its products. Traditionally, Dell used to pick up components from the warehouses of suppliers then collect them in its central or regional distribution centres, and finally merge them in stock in order to deliver the final products to customers (Ross, 2010: p.88). Currently, through implementation of e-logistics, Dell Inc. can now pick up components from the ware houses of suppliers and then forward the merging of components made during the transit to the logistic-service providers through USP or Airborne Express (Li, 2007: p.36). This has resulted in less fixed costs spent in warehouse centers and distribution, no product technological obsolescence, and no stock-keeping units (SKU). SUPPLY CHAIN MANAGEMENT AT DELL INC. Supply Chain Management (SCM) is a system that Dell established to ensure the availability of precise computer components for its customers on demand and location. SCM describes how the company manages how raw materials are transformed into the end products and how products and services get to all its consumers (William, 2003: p.150). This has enabled the company to develop a tight bond with its supplier companies and consumers. In this regard, Mencarini (2003: p.19) states that Dell Inc. have one of the most effective SCM system in the world, and that it is focusing on creating the best SCM through the i2. This will improve the supply chain process through connecting its suppliers and planners in order to satisfy the requirements, as well as demands of their customers. SOFTWARE USED BY DELL IN PROMOTING RELATIONSHIP MARKETING Dell also uses a variety of software to promote relationship marketing such as Hotlink, Premier Pages and an enhanced CRM system, among others (Gattorna, 2003: p.57). Its database software is highly efficient and effective with customer relationship management, which stores tables of data used to check the information of customers and establish promotional campaigns. These databases mainly include the information of customers, their products and interests. According to Gattorna (2003: p.57), customer database helps to increase profits since it contains the information of clients, which determines the efficient and effective ways to target and divide the consumers. Hotlink is an automation software program, which facilitates tergeting and marketing communication, monitoring of customers and market development (Mencarini, 2003: p.21). This software gives Dell a free opportunity to advertise its products through the word of mouth. Also, it impacts its customer base to ensure that customers receive better services than before. Premier Pages are a transparent online system/software custom designed Web pages, which contain all the purchasing data (Gattorna, 2003: p.57). In addition, the software contains a paperless ordering process, which captures the technology configurations of customers. Mencarini (2003: p.21) argues that Dell created Premier Pages in order to gather less clientele details than they already have and develop a win-win situation that is more realistic. This starts when the clients places their orders for a computer and built later. Another system that Dell uses is an enhanced CRM system, helped by an information system company called the IS Partners (Moon, 2003: p.45). ProClarity offers a comprehensive analytical ability that highlights negative and positive areas of the business. Moreover, the company breaks down its sales by region where each team enables Dell to measure its own trend and success. ProClarity significantly benefits all the financial sections of the company. It also helps the Dell staff to easily access detailed demographic information about customers. The marketing department is able to follow product sales, customer activity and marketing mixes via this software. The management can follow activities in customer accounts, and act on lapsed quotes. Additionally, Dell installed the e-commerce software i2 Collaboration Planner, i2 Supply Chain Planner and i2 Factory Planner in order to meet its supply chain needs (Moon, 2003: p.45). This is applicable in the management of build-order procedures that exist between placing orders and customer support. The software enables Dell Inc. to classify customers and target them through their most preferred medium, obtain and analyze results (Moon, 2003: p.45). Moreover, Dell Inc. has signed an agreement with Part-Miner (Gattorna, 2003: p.51). Part-Miner is a vertical portal in electronic components industry, which provides information and helps to meet the demand and supply of the components. FUTURE PLANS OF DELL INC In future, Dell plans to update its processes of purchase such as the establishment of online auctions for products and services like printing, shipping, and paper (Li, 2007: p.20). The company also plans to make order status, payment information and receipts easily accessible to suppliers online. In coming years, Dell intends to expand its catalogue base and purchase choices by convincing its main suppliers to use the Ariba Business to Business Commerce Platform (Li, 2007: p.20). CONCLUSION CRM-SCM integration tries to satisfy clients through prompt delivery of products, ensuring its accessibility and maintain the manufacturer’s profits and returns. Thus, there are several lessons that can be drawn from Dell’s application of e-business. This trend can be emulated by other organizations in the industry. This will result in offering of better services to customers. It can be portrayed via the way Dell Inc. uses CRM to its advantage. Customer satisfaction will increase their trust in the organization, improving its reputation. In addition, custom-building a PC desired by the clients has formed a particularly strong relationship between Dell and its customers (Moon, 2003: p.50). In addition to this, implementing technology in a phased fashion has helped Dell to achieve a strong relationship with its clients. Dell set up simulated environments in order to support the i2 system in blotches without affecting the live form. Dell ensured that all stages of the comp leted process allowed future growth of the company before developing the whole system. Hence, this reduced risk and increasing efficiency. Another significant lesson from Dell would be to extend the link from the customer to the supplier, while maximizing its operation efficiency as well as customer satisfaction (Ross,2010: p.92). As a result, customers were able to spend less money on purchasing customized machines. This is because Dell approved the savings that resulted from managing its inventories efficiently. The company was, therefore, able to share information with suppliers about customer requirements and buying patterns in real-time. REFERENCES Botha, J., Bothma, C. & Geldenhuys, P. 2008. Managing E-commerce in Business, New York, Juta and Company Ltd. Gattorna, J. 2003. Gower handbook of supply chain management, Burlington, Gower Publishing Ltd. Krauss, M. 2003. Dell looks to Sears to extend buyer reach. Marketing News, April 28, 2003, Vol. 37, Issue 9. Li. L. 2007. Supply chain management: concepts, techniques and practices enhancing the value through collaboration, Tokyo,World Scientific. Moon, K. 2003. Dell Computers: A Leader in CRM. Retrieved February 20, 2010 Mencarini A. 2003. E-Business: Dell Case Study, UK, Strathclyde Business School. Perret, F. & Jaffeux, C. 2007. Essentials of logistics and management, London, EPFL Press. Levy, R. H. 1999. 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