Illusions Within the Great Gatsby

American Illusions in The Great Gatsby The American dream. Every American has his or her own ideals and preferences, but all share more or less the same dream. In The Great Gatsby, Fitzgerald explores what happens when this dream is taken too far. What is one to do when the dream begins to overshadow reality? What are the consequences when a successful man allows the dream to matter more than life itself? Fitzgerald tells all through the hopeless Gatsby, idealistic Nick, and ignorant Myrtle. Mansions, cars, jewels, and extravagant parties- what more could a person want?Gatsby had it all, yet he was still empty inside, craving more. All the riches Gatsby has mean nothing without his great love, Daisy. Gatsby strived to become successful for the sole purpose of capturing Daisy's heart. However, Gatsby's dream is an unattainable and hopeless dream for he can never win her love. Daisy and Gatsby live only miles apart, but their relationship is eons apart, as Daisy is already attached. Ga tsby is pursuing â€Å"a transcendent significance outside of society and beyond the notability of history† (Lynn 180).Gatsby is dreaming â€Å"the American dream† that anything is possible, but the tragic flaw within him is that he is living in the past and cannot see the destructive future that lies ahead. Gatsby says, â€Å"I'm going to fix everything just the way it was before,†¦ She'll see,† and he does not realize that he cannot make it the way it was before (Fitzgerald 114). When Gatsby does get the chance to prove himself to Daisy, it is already too late. According to Fitzgerald, â€Å"the whole caravansay had fallen in like a card house at the disapproval in her eyes,† (Fitzgerald 114).Gatsby's downfall is in the fact that he is unable to determine the fine line that divides reality and illusion in his life. The green light at the end of Daisy's dock burns bright for Gatsby, but Gatsby does not realize that he cannot ever capture the light. He continues to dream blindly. This is evident when Nick tells Gatsby that he cannot relive the past and Gatsby replies, â€Å"Why of course you can, old sport! † (Fitzgerald 116). Gatsby's dream of capturing Daisy's love is based on a fantasy of romance, but the truth is that Daisy is already taken and no amount of money or popularity can change hat. His obsession leads him to come out and profess his love for Daisy, but Daisy does not follow in suit and his dream is over. Nick sums up Gatsby by saying â€Å"He did not know that [his dream] was already behind him†¦ † Gatsby's â€Å"American dream† was a one-way street and, with unrequited love, Gatsby's dream can never come true. Gatsby's â€Å"American dream† leads him to protect Daisy and causes his subsequent downfall, death. Nick has a haughty and idealistic dream of America, which clouds his own judgment.Nick is more aware than Gatsby. He makes a clear distinction between fantasy and reality. N ick is able to separate romance from real life. He has a perfectionist vision of America, and he judges people against his ideal American society. Nick is the right person in the wrong city associating with the wrong crowd. Nick is raised in an idealist middle-west society â€Å"where dwellings are still called through decades by a family's name,† and where tradition is part of everyday life (Fitzgerald 184).Nick dreams the â€Å"American dream† that he can travel to the East and become wealthy and still have all the old-fashioned ways of his town. After spending a few months in the East, Nick comes to the realization that the East is corrupt, materialistic, and self-centered. Nick's view on life is based on Western morals and a â€Å"western† American dream; one to which the East cannot measure up. Nick follows his dream according to his western morals of hard work and righteousness, yet in the East he possesses â€Å"some deficiency† which made him â₠¬Å"subtly unadaptable to Eastern life† (Fitzgerald 184).Nick is portrayed as a young man who comes to the East to make his fortune, but finally goes back to the mid-west, horribly disillusioned. Nick is let down by his â€Å"American dream† and loses a part of his faith in society. Myrtle Wilson, like many other commoners among American society, has the â€Å"American dream† to move ahead and become successful. Myrtle is tired of her life as a â€Å"nobody from nowhere. † She wanted power, wealth, and fame. Myrtle's way out of the ruins of society and the valley of the ashes is Tom. Myrtle feels a sense of power and with Tom she is closer to her â€Å"American Dream. Myrtle, as many Americans do, dreams of moving up in the social ladder, up towards the extravagant parties of the rich- the ultimate â€Å"American dream. † Myrtle had no chance of moving up with George Wilson, and she ended up having an affair with Tom to get ahead. Myrtle does not re alize she is just a toy on the side for Tom, and her dream will not likely come true. Myrtle is too foolishly stuck in her optimistic and ideal world to realize that Tom will never take a chance at moving a step down the social ladder.The naive Myrtle dreams of moving ahead with Tom and towards the rich society, yet in the end it is carelessness of that same society which causes her death. Myrtle Wilson turns out to be the brutal victim of Gatsby's and Daisy's love affair, as she left in the street for death while Gatsby and Daisy drive off without any thought of what they had done. â€Å"They saw that her left breast was swinging loose like a flap and there was no need to listen for the heart beneath† (Fitzgerald 145).Myrtle just wanted to attain her â€Å"American dream† like many others, but her ambition and lust for power caused her to pay the ultimate price, death. The hopelessness of Gatsby, Nick's idealism, and Myrtle's dream all contribute to the illusion of th e American Dream. It is not simply American to dream, it is the duty of every person to dream. However, to dream too far and become too entranced and engrossed in one's dream leads to either destructive behavior, ruin, or to disappointment. The Great Gatsby is based on this idea of an â€Å"American dream. † However, dreams that are taken too far become harmful illusions.

Malaria Issue Report

The issue I am going to be talking about in my report is about how to prevent the transmission of malaria around the world. Malaria is a mosquito-borne, climate sensitive disease caused by the parasite Plasmodium.[1] Malaria is caused by one of four species of the protoctist Plasmodium[2]. The Plasmodium parasites multiply in red blood cells; this can cause symptoms such as fever and headache, but in severe cases the disease can lead to death. Transmission Malaria is transmitted through the bite of an infected female (Anopheles) mosquito; although it can be transmitted through contact of infected blood but this is very uncommon.[3] The cycle of malaria is an Anopheles mosquito bites an infected person; a small amount of infected blood is taken. The plasmodium parasite grows and matures in the mosquito's gut for approximately a week before it then travels to the mosquito's salivary glands. When the mosquito next bites someone, these parasites mix with the saliva, are then injected with the bite, and the transmission of malaria is complete.[4] Cycle in the Body Once in the blood, the parasites travel to the liver and enter liver cells, to grow and multiply. After some time, the parasites leave the liver cells and enter red blood cells. In the red blood cells the malaria parasites continues to grow and multiply. After they mature, the infected red blood cells rupture, freeing the parasites to attack and enter other red blood cells.[5] Symptoms Toxins released when the red cells burst, these are what cause the common symptoms of malaria, these are: 1. Fever 2. Chills 3. Flu like symptoms Statistics According to the World Health Organisation (WHO) malaria report 2011, there were 216 million cases of malaria and an estimated 655,000 deaths in 2010. Malaria mortality rates have fallen by more than 25% globally since 2000. Most deaths occur amongst children living in Africa where a child dies every minute of malaria and the disease accounts for approximately 22% of all childhood deaths.[6] Malaria is a disease that only occurs in certain climates, due to the fact that malaria is passed on through mosquito bites so only certain countries and regions have the conditions that mosquitos can survive in are affected by the disease; this includes sub-Saharan Africa, Asia and Latin America as shown in the picture below: [7] This picture shows that malaria affects countries mainly in the Southern Hemisphere, especially South America (but north of Argentina) Sub-Saharan Africa and Southern Asia. Malaria has been on the rise since the 1970s and constitutes a serious risk to health in many tropical countries.[8] The Problem with malaria is that it mainly affects people who live in LEDCs (Less Economically Developed Countries) the problem with this is that they cannot afford to get the healthcare to treat the disease unlike with MEDCs (More Economically Developed Countries) where they don’t get diseases of this kind commonly but are able to afford the healthcare. Malaria also causes a number of deaths and illnesses every year, especially in countries which suffer from a lot of poverty. This means that if parents catch the diseases then their children will be orphaned and left to fend for themselves. Solutions to the Problem There are several methods of treating malaria and these are some of the best methods to protect us from the disease: Nets A mosquito net protects against mosquitos, flies, and other airborne insects. Mosquito nets are the first line of defence against the deadly bite of mosquitos. The fine, see-through mesh stops insects from biting or affecting the person using the net. The mesh is fine enough to stop these insects entering the sleeping area of the person. Research has found that the latest Long Lasting Insecticidal Nets (LLINs) save lives; they have been proven to cut malaria cases in children by half as well as reducing child deaths by 20%[9]. The use of these nets can also help protect other people sleeping nearby who are not sleeping under a net. It’s estimated that when 80% of people sleep under a net, the entire community is protected.[10] Although it is vital that the people using the mosquito nets regularly check the nets for any holes big enough for insects to get through. Scientists in Senegal have been researching the success rate of mosquito nets after six million nets were distributed over five years. Within three weeks of the nets being introduced, the scientists found that the number of malaria attacks had started to fall – it had fell by 13 times since before the nets had come in. The researchers also collected specimens of Anopheles gambiae, which is the type of mosquito species responsible for transmitting malaria to humans in Africa. Between 2007 and 2010 the proportion of the insects with a genetic resistance rose from 8% to 48%. By 2010 the proportion of mosquitoes resistant to Deltamethrin was 37%.[11] Testing Malaria parasites in the blood can best be detected under a microscope, but, when this is not available malaria cases can also be quickly and accurately identified using a Rapid Diagnostic Test (RDT). RDTs are disposable blood testing kits which can be used anywhere and provide a quick result, they measure the amount of plasmodium in the blood; therefore showing if you have malaria or not. RDTs mean that a potentially life-saving diagnosis of malaria, which usually could only be possible in hospitals, can now be made available anywhere.[12] The reliability of RDTs is a successful diagnostic reading of 80% or in other words; 4 out of 5 people tested for malaria get a correct result back.[13] Implications of the solutions to the problem There are a number of solutions to malaria, although they do have economic and environmental implications. The solutions include: Nets The economic problem with nets is that malaria is mainly affecting LEDCs; therefore people are unable to afford the cost of buying drugs nets as they can be expensive. Environmental issues with mosquito nets are that mosquitos are possibly starting to get immune to the chemical Deltamethrin which is used in the bed nets; this means that they are no longer being useful by stopping the female mosquito from biting and killing harmless insects instead. The social implications of using nets are that nets prevent people being bitten by malaria carrying mosquitos, this means that less access to health services are needed; therefore less time is lost from work because of malaria. Medicines Artemisinin-based Combination Therapies (ACTs) are therapies made by combining an extract of the Artemisia plant and another medicine. They are the most effective and advance treatments for malaria, and are strongly recommended for the treatment of malaria in Africa.[14] The implication to this method is that they are highly expensive to produce and take around 15 years to research and develop. And seeing as malaria mainly affects LEDCs it wouldn’t be much use to them, as they will not be able to afford to buy them. Also the problem with using drugs is that a lot of testing takes place on animals which have ethical implications along with it. Benefits/Risks of solutions to the Problem Medicines In the table below there is a list of advantages and disadvantages for using different medicines for treating malaria: Chemoprophylaxis drug Presentation Advantages Disadvantages Proguanil Paludrine tablets 1. Low cost 2. Well tolerated 3. Suitable for pregnant or breastfeeding women 1. Increasing resistance of P. falciparum to chloroquine 2. Large number of tablets required if taken in combination Chloroquine Nivaquine syrup Avloclor tablets 1. Mefloquine Lariam tablets 1. Weekly dose 2. Effective for most areas of the world 3. Can be used in the last 2 trimesters of pregnancy 1. Needs to be commenced 2 to 3 weeks prior to departure 2. May cause neuro-psychiatric adverse events Doxycycline Capsule 1. Low cost 2. Generally well tolerated 3. Can be commenced close to departure date 1. May cause photosensitivity 2. May cause vaginal yeast infections in women 3. Unsuitable for children under 12 years Atovaquone/proguanil Malarone tablets 1. Well tolerated 2. Can be commenced close to departure date 3. Short course 1. Expensive [15] According to the table above the advantages outweigh the disadvantages in the five different medicines, although with Doxycycline the advantages and disadvantages are in the same proportion. This would suggest that Doxycycline may not as effective anti-malarial medicine as some of the others. Testing Another solution to the problem could be to use malaria testing kits such as the Rapid Diagnostic Tests (RDT). The benefit of this is that it can create a diagnosis in fraction of the time that a standard test takes. This means that people who have malaria can be treated a lot quicker than they usually could be by the standard method, which involves looking at a blood sample under a microscope to detect plasmodium. An issue with RDTs is that they can be expensive for people living in LEDCs as they can range from $1.20 – $13.50 per test.[16] The risks of RDT is that, although it is a quick test, it is not always effective at identify the plasmodium in the blood. The danger is that you may get inaccurate result, leading to you being quite unaware of having the disease, therefore getting the treatment early enough to take effect. Alternative Solutions to the Problem Quinine Another solution to the problem which was used up until the 1930s as the only cure for malaria is quinine. It is found in the Andes Mountain range of Peru and Ecuador[17]. The medicinal properties to quinine has been around since the 17th century, but back then people used to chew the bark off of the cinchona tree as it was believed to be a useful treatment for malaria. Although people didn’t have medicinal knowledge, due to the positive result, they thought it was an effective method of malaria treatment. The active ingredient is quinine which is found in the cinchona tree. Quinine works by lowering the body’s core temperature, thereby killing the plasmodium parasites that cause malaria in the red blood cells. Quinine can also be found in tonic water; some doctors/ GPs may recommend drinking tonic water when going to a high risk country. Vaccine A vaccination involves injecting a small harmless amount of infection into you. Finding a vaccine to protect people from malaria would be the best possible defence[18]. Around the world scientists are working on this important research.[19] However, malaria parasite, plasmodium has proven to be adapting to the different cures and vaccines that scientists have been creating. This means that they change their characteristics as antibodies are developed. This adaptability has made finding a vaccine especially challenging.[20] Currently there is no vaccine, but there are several being developed. Several potential malaria vaccines are already being tested in clinical trials; although no vaccine has been licensed for use.[21] It will be a long wait before a vaccine is available to prevent the spread of malaria.[22] Dr Sandy Douglas (Welcome Trust Clinical Research Training) at the University of Oxford said â€Å"We have found a way of making antibodies that kill all different strains of malaria parasites. This is still in an early research phase; the next step is to do clinical trials in people.†[23]

Baking as a Favourite Activity During the Holidays Essay

Baking as a Favourite Activity During the Holidays - Essay Example My brother became particularly interested in baking and, thus, he went on to join a culinary institute. I realized I am better with accounting and finances, therefore, I sought to gain the knowledge in business management and marketing as I horn my other skills from dedicated lecturers at a renowned culinary institution. Moreover, while in high school, my brother and I decided to have a bake sale to raise funds for a classmate that had lost her father to cancer and needed money to settle the medical bills and pay for funeral arrangements. To my surprise, the bake sale became such a huge success and people kept telling us how much they enjoyed them. This sparked my desire to own a bakery and my brother to come up with new innovative recipes. However, currently, I believe my raw skills and limited knowledge in business management skills are the only challenge to running this business. This is why I am fervent about joining an institution which is well known to ensure that my skills are better developed to ensure success in my chosen trade. I strongly feel that it would equip me with the necessary business management skills to set up a bakery with my brother and run it efficiently. In addition, I would like to interact with the different academicians at such an institution and seek their mentorship and guidance as I set out on my endeavor. Last year, my aunt was diagnosed with diabetes that made her less active and cheerful especially since she is well-advanced in age. This has further ignited my ambition as now my brother and I endeavor to create healthy recipes that would be enjoyed by all. This, coupled with the fact that we would like to use social media and new technologies to boost our business, has also contributed to my desire to join a university which will help us develop the necessary skills. This is because many universities, to my knowledge, always continuously aim to remain up to date in equipping their students with recent technologies and business trends that have helped to provide them with relevant skills while making them versatile. I look forward to participating in the extra-curricular activities offered at the university that I join, as I know they will enable me to further build my confidence and overall character. Therefore, I hope that at least one of the applications that I have made to various universities will come through as I am certain the learning environment will provide the ideal foundation as I set out to actualize my business idea. One of the greatest benefits that I have personally come to reap from being able to help the my aunt in baking is the confidence in my ability to do all the tasks that are assigned to me, most especially those concerned with the principles behind this practice. This new confidence in myself has enabled me to think of a greater role for myself in future than I had previously considered when I was still a child. Moreover, through the teamwork that developed between my brother and I, I have managed to see first hand how the skills displayed by bakers are necessary for dealing with prospective issues concerning health promotion. It has, indeed, been a privilege to work and share activities and experience with my aunt and brother, because they are individuals who were not only friendly, but have also supported me whenever I have experienced any problems. Moreover, all members of my family have

Women in the Second World War Case Study Example | Topics and Well Written Essays - 500 words

Women in the Second World War - Case Study Example While Germany, Italy, and other allies seized majority of Europe, President Roosevelt of the US accepted to back Britain. Concisely, due to the significant booming of the Harbor of Pearl, the US president declared a war through the congress. Consequently, with the declaration of war by President Roosevelt, Hitler responded by declaring war on the US; hence the commencement of WW II (Library of congress, 2011). The exhibition has featured numerous women chosen based on their strength, as well as, variety in the library collections. Significantly, similar to their friends, they concentrated on certain ways in their wartime assignments. This document has selected three from the eight women in order to explore a comparison, as well as, a contrast in order to reveal who was significant in the exhibition. For instance, all women in WW II employed photographs in their elaboration of what happened in the WW II. Accordingly, my document has chosen Therese Bonney, Esther Bubley, and Toni Frissell. Considerably the three women attracted about million viewers with their WW II images. However, they differ significantly because Bonney’s pictures portrayed images of numerours homeless nurses, soldiers in the front line, WACs, afro- American airmen, and numerous orphaned children. In contrast, Bubbley’s images portrayed wartime subjects around the capital of the nation. In addition, her ima ges portrayed the life of the city, especially the boarding houses for workers of the war. It is worth noting that, Bonney’s images reveal that, she could go forth alone and try to establish the truth and bring it back by trying to make the others intermingle with it and act on it. Therefore, she believed that, the emergency of the WW II had significantly threatened civilization in Europe. Consequently, Bonney wanted to reveal that, people would find measures I order to end the war. Another critical point of contrast was Frissell’s desire t

Changes in media and its effects on personal, social and work lives Essay

Changes in media and its effects on personal, social and work lives - Essay Example However, all this came to change with the fast development of technology. Before we knew it, we had television, and all this changed everything because we could not only hear the news but could also see who was presenting it as well as videos of the news itself. It has indeed been a privilege for me to live long enough to see the development of media from its infancy to where it is today. Question: What are your feelings concerning the development of the Internet and social networking? Granddad: it is quite fascinating how the technology for transmitting media has developed and how fast that development has been. The things that we used to think were impossible have now become reality and this is something quite extraordinary. The Internet is a technology that I did not become aware of until sometime in the mid nineties when it was first introduced to me. I found it to be most useful because I could use email to send information, which I would have otherwise sent by mail, and I could find information instantly online, without having to go to a library, as used to be the case. I have come to adapt to this new technology like a fish in water, because it has made my life all much easier, especially now that I do not move around as much as I used to. I am an avid user of social networking sites such as Facebook, where I have come to encounter plenty of information as well as made many friends. I have also been able to rekindle old relationships with people whom I knew when I was younger, but life put us in different paths. Furthermore, the different sources of news have ensured that any biased news can be detected. I personally prefer watching news online and during the Iraq War, recently, I used to watch Al Jazeera and BBC to get a balanced picture of the news. Question: Which media do you think has managed to survive way into the modern times? Granddad: In my opinion, I believe that television, and most especially cartoons, have not lost the charm that kept peopl e hooked to them. When I was young, I used to love the Disney cartoons and I followed them every chance I got. I still see the same fascination in cartoons among kids today and this, I believe, is continuity. Personally, I am still a huge fan of cartoons because they not only remind me of my younger days but also make me laugh, something that I have to admit, has become extremely rare for me in other forms of entertainment. Question: What was the relationship between politicians and media, and has this relationship changed? Granddad: In previous decades, it was extremely difficult to find politicians who were open with any form of media. In fact, media was always treated with suspicion because of the belief that their main purpose was to destroy political careers by creating scandals. This, I believe, has changed since many politicians have come to embrace the media to further their own political careers. President Obama in the United States is an example of this new trend among pol iticians. He has used the media so well that he has managed to maintain his popularity where other presidents would have fallen from grace. I may add that media has come to influence change in society because without it, the Americans would not have been able to elect a black president. Essay Media has experienced plenty of changes, especially from the mid twentieth century to the present day. It has not only led to the development of better means of communication between people but has

Leadership Experience Essay Example | Topics and Well Written Essays - 1500 words

Leadership Experience - Essay Example The team managed the lowest price and finished the task within the three minutes. This paper seeks to describe the activity, what was learnt, and the solutions provided with a focus on communication, team dynamics, influencing other team members, sharing information, agreeing on tasks and objectives. 1. Leadership, strategy and upward communication Strategic leadership can be defined as the ability to maintain flexibility, anticipate, and empower the other team members to form necessary strategic change. It involves coping with increasing changes, management of an entire team rather than functional subunits, and management of activities through the other team members. As a team leader, I had to affect the behavior of the other team members in environments that were sometimes uncertain via personal example and ability to envision the requirements for the next stage in the task (Yukl, 2010 p34). It is not feasible to formulate strategies which will give returns without strategic leader s. Therefore, it is necessary that as, a team leader, I had the ability to be a strategic leader to avoid over managing and poorly leading the team. Effective, strategic leadership shapes the formation of strategic mission and intent which influence successful strategic actions. These actions result in the formulation and implementation of these strategies which then yield strategic competitiveness and above average returns. For a leader to prepare the team’s minds towards one vision and direction, it is necessary to transform the team’s strategic direction. Directing the team towards the set goals, and having the ability to adapt concurrently ensures that the strategy delivers the aim that the leader wants (Yukl, 2010 p35). It is essential to have a different way of thought on how resources can be marshaled in the formulation and execution of the strategy in order to be successful. This balances strategy making’s human dimension with analytical perspectives tha t are focused. Coupled with time management commitments in engaging the team in dialogue on strategy, this strategy lays the foundation for building a foundation for creation of winning teams that define, adjust, commit, and adapt quickly to strategy. I learnt that in order to shape the strategic direction, I had to know what my primary role was as the chief strategist, my role as a leader during the strategy making process, the team required for the strategy making, and the point at which the making of the strategy can be considered finished. A leader requires to incorporate human and analytical dimensions, in order to create winning outcomes, and for the team to comprehend and commit to the set common agenda, and must also be clear about each member’s strategic capacity. Using feedback from the team members helps to provide a holistic view on the team’s and its individual member’s effectiveness. Ratings by the team members themselves and their peers can be use d to evaluate the efficiency of the team and the individual (Yukl, 2010 p35). The members rate the following personal traits and competencies: Communication which is the ability to objectively, supportively, and openly share opportunities using nonverbal and verbal behaviors while listening actively. Conflict resolution, which involves the

Australian capital market Assignment Example | Topics and Well Written Essays - 2500 words

Australian capital market - Assignment Example This paper will discuss the Australian Capital Market and will also focus on the impact of the global financial crisis on the country. The capital markets have been faced with challenges especially during this period of global financial crisis. The global financial crisis has negatively affected the Australian capital markets and therefore there has been a pressing need for a serious counter action to raise the country to its usual state according to RICS Research (2007). In the last three months, positive change has been recorded in Australia’s money market. More initial public offers by private companies have been noted and indications show that this trend will most likely continue. This has increased the share market performance of the various industries. Indications show that banks are reluctant to provide financing in terms of debt financing. Companies have adapted merger and acquisitions to survive through the recession like Tolhurst Noall which merged with Patersons securities (RICS Research, 2007). This has prevented them from dropping out of the industry. Companies are diverting attention to the ecommerce/digital media companies that’s taking a significant market share from newspapers and magazines. The country has seen a lot of reluctance in debt financing since financial institutions have preferred to fund businesses through equity as opposed to debt. Companies that seek to increase their capital, one viable option has always been to give up a generous portion of their shareholdings. While IPOs were a common way of cashing out investments in the last couple of years, this is no longer the case as many investors have not been able to venture into new businesses due to restrictions on IPO’s according to ResearchWhitePaper (2010). The country has however continued to enjoy a lot in terms of issues. In 2007 for example, Ivanhoe Australia

Efficient Markets Theory and Behavioural Finance Essay

Efficient Markets Theory and Behavioural Finance - Essay Example In this theory, therefore, assumptions are done perpetuating that the information organisation and the behaviour of market participants systematically control individuals’ decisions in investment and the outcomes of the market. According to (Malkiel, 2003) the efficient market theory, has implications of theoretical perspectives to the market trends, while it ignores or underestimates the practical perception of the market. On the other hand, the behavioural finance theory has been thought of being more practical based and focused on people’s behaviour (Ashta & Patil, 2007). Following the event of the financial crises in 2007 to 2010, contention has developed amongst various authors on its implication to the popularity of the already criticised efficient markets theory and its contribution to the upsurge of the prevailing interest in behavioural finance theory. This paper compares and contrasts the explanations of efficient market theory and behavioural finance with reg ard to the financial crisis 2007 to 2010 and identifies the explanation considered to be strongest. The efficient market theory upholds the notion of the randomness in stock prices, based on short-run serial relationships amid successive changes in stock prices (Malkiel, 2003). Such was the case in the year 2001 when the US economy experienced a recession, followed by a boom that led to the dotcom bubble, and accounting scandals. The behavioural market theory regarded such occurrences in a different way from that of the efficient market theory, in that, the fears in individuals’ mind of a recession were considerable. Therefore, in regard to the recession in 2001 being disregarded, the stock market was thought of as not having a memory of the way the price of a stock behaved in the past, so as to determine its future behaviour. The randomness inefficient market theory is questionable, due to the high frequencies with which successive moves towards the same direction occurred in the period of 2001 and 2003 when subprime mortgage grew from 2001 to 2005.  Ã‚  

The Consumer and Their Relation to the Commodity According To Marx, De Essay

The Consumer and Their Relation to the Commodity According To Marx, De Certeau, Horkheimer and Adorno - Essay Example Also, in the cases when an individual goes to a store and purchases a shirt, toy, a beverage or any other purchased commodity, they are arriving at the decision to do so as a consumer. On the other hand, a commodity is any physical substance, these including metals, foods, and grains – which is exchangeable with another commodity of the same kind, which traders buy or sell, typically though futures agreements Another characteristic of any commodity is that the price of the given product at any given time is subject to the law of supply and demand. Notably, risk is actually the fundamental reason on the basis of which – the barter trading of the basic farming commodities began (Ozanne & Jeff 129-144). According to the review of De Certeau’s publication: the practice of everyday life: walking in the city, the tactics and the strategies discussed are in operation during the process of making product choices among customers. These choices are, however, based on the c onsumers’ relationship to the different commodities that consumers encounter on a daily basis. The expressed viewpoint is drawn from the experiences of every person within the community. The argument is deliberately presented in a poetic manner. The book centers upon the uses that the consumers make of, from the things that they purchase, or that which they decide to buy. The book also talks of the different networks that are operating within the society, which play the role of helping people oppose and avoid the order presented by institutions. De Certeau goes further to argue that the disciplinary constructions contained by the institutions within the society are deflected by developed tactics: the participants within the society in question offer an anti-discipline. Further, the consumers within the society, through the same tactical development, make their own tactically developed paths, which are somewhat unpredictable. These paths that are formulated by the consumers ca nnot be described fully, from a formal, official or a statistical point of view (Certeau 76). These strategies, according to De Certeau, are predictive of the following: the Strategies themselves – require that the city or the subjects act as alienated from an environment, in that they have an appropriate place, an officially recognized place, which is seen externally. The tactics, on the other hand, have no properly established localization, and not necessarily alienated from each other, which means that they often mingle and interrelate with each other. Further, the tactics take place within the territory of the strategies. These tactics, also, tend to be opportunistic, in that they combine a diverse range of elements towards gaining momentary advantage. Everyday practices, are most times some of these tactics – which include the small maneuvers that allow for the realization of a momentary victory over the obstacle facing the subject at the time of need (Ozanne & Je ff 129-144). According to the account of Marx, in the account, ‘idealism and materialism,’ the account holds that, humans are made to exist in physical organizations – where these organizations are helpful to the man, as they help him create their own means of subsistence. However, the subsistence models depend upon the available means – which they find existent as well as tat which they have do develop. Further, the productive efforts of these individuals are reflective of their life, expressing themselves –

Was World War Two a Good War Essay Example | Topics and Well Written Essays - 1000 words

Was World War Two a Good War - Essay Example This is evident with the commencement of WWII, which historically despite cited as a â€Å"good war† in terms of ending repressive regimes, is far from it due to then its associated mass extermination of human life and crippling of allies’ economies. Contrary to numerous scholars’ works not citing detrimental effects of the war, it yielded to immense loss of lives to both civilians and those who engaged autocratic or â€Å"evil forces† as commonly preferred. As Mooney states, â€Å"roughly 6 million Jews and approximately 4 million others were murdered in the â€Å"Holocaust† (Mooney, 110).† Hence, revealing the utter retaliations of autocratic forces after realizing their victims of the day had support from external world, which was ready for engagements. However, numerous history scholars despite noticing this utter humiliation tend to incline towards adopting political perspective while ignoring scars the war left in people’s hearts . Most of the retaliations made by the then â€Å"evil forces/powers† of the day encompassed unleashing terror on those who could not defend themselves. The core reason was to irritate other global blocs who through their actions of inducing peace seemed to defend victims despite most of them being not their citizens. However, these efforts by sympathizing blocs instead of bringing relieve to the suffering continued to worsen while trying to eliminate totalitarian regimes. According to Mooney’s sentiments, â€Å"100,000 more eventually died from burns and radiation sickness†¦ On 9 August a second atomic bomb was dropped on Nagasaki, killing 80,000 more Japanese (Mooney, 119).† This was more than what opposing peaceful blocs together with their allies of the then campaigned while in the quest of inducing peace and saving life. It is irrefutable WWII despite attributed with overthrowing and containing the then tyrannical powers, it yielded to the destruction and stagnation of numerous global economies. This is in terms of numerous resources that went into supporting the military forces against their autocratic enemies, which was not an easy task. According to Ernie, â€Å"For a mile out from the beach there were scores of tanks and trucks and boats that you could no longer see, for they were at the bottom of the water - swamped by overloading, or hit by shells, or sunk by mines (Ernie, â€Å"The Horrible Waste of War†).† This was one of the numerous destructions evidenced on both sides as each pioneering state solicited monetary and weaponry support from respective allies. Hence, rendering the war as the most expensive the world has ever had, which somehow crippled entire global economy. It also led to the disruption of family structure whose effect is still evident to date. The female gender ended up assuming male’s occupations where they worked in industries to support the then ongoing combat. During then, almost all men comprising the energetic gap ganged together compelled by their respective states to wage war against tyrannical powers. This is evident in Sauer’s confession citing, â€Å"I worked seven days a week† (Sauer, â€Å"The War Changed My Life Completely†). Hence, revealing utter situation she had to undergo due to the lack of labor force in industrial firms where

Truman Capote Essay Example for Free

Truman Capote Essay Capote, the 2005 movie directed by Bennett Miller, starring Philip Seymour Hoffman, Catherine Keener, Clifton Collins, Jr., Chris Cooper and Bruce Greenwood, is the story of the making of Truman Capote’s 1966 non-fiction novel, In Cold Blood. Hoffman, playing Capote as a fish-out-of-water, a mincing homosexual researching his opus in a small Kansas town in 1959, shares screen time with Keener’s Harper Lee, a childhood friend of the author. The true story of how Capote researched his book is not as compelling as the story he actually wrote. Keener, playing Harper Lee, the author of To Kill a Mockingbird makes a more interesting figure and the viewer sometimes wonders why the movie isn’t about her and the making of her own opus.   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   Keener, as Lee, is the key to Capote being able to access the rural people of the little Kansas town. They are amused by, and wary of the little man with the lisp and extravagant dress. It is she who opens the doors for him and permits the work to be researched. Hoffman, as Capote, is technically on the mark, but his portrayal is still that of a hollow man. The audience is supposed to feel empathy for this tragic individual but there is little compassionate or passionate about him. The tragedy of the story is, in reality, the Kansas farm family who was slaughtered like animals by the socio-paths who will be exploited by Capote. The audience is supposed to care that the soulless sophisticated and dapper homosexual is attracted to the cold-blooded killer, and, most likely, is in love with him.   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   The film is bleak and also runs out of steam by midpoint. The acting is good but the subject is borderline disgusting. Capote exploited the community and the slain family.   The film version of the actual Capote work, In Cold Blood, is a much better movie.

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