EMG muscle activity in various rehabilitation exercises Research Proposal

EMG muscle activity in various rehabilitation exercises - Research Proposal Example In their analysis, the pressure an individual suffers on the joint of his or her knees is four times the body weight of an individual (Rubin, 2000; Sing, 2012). This is an indication that an everyday pressure on the knees can result to pain (Vogt, 2011). However, there are a number of muscle activities or exercises that can help in the reduction of this knee joint pain. This research aims at identifying these exercises and coming up with information that explains their effectiveness in the reduction of pain within the knees of an individual. This research will use the quantitative methods of data collection. The specific method to be used is the electromyography (Creswell, 2003). Electromyography is a diagnostic procedure that medical experts use for purposes of assessing the health of individual muscles, and the nerve cells which have the capability of controlling the motor neurons. The EMG relies on the motor neurons for purposes of transmitting signals that have the capability of causing muscles to contract (Pons, 2013). The EMG will thereafter transmit these signals into sounds, graphs and numerical values which a specialist will interpret. It is these results that the researcher will use in analyzing and coming up with a conclusion on the best exercise that can be used for purposes of controlling and treating knee joint pains (Subic, 2000). This research will help in understanding this concept of knee joint pains. It will identify different exercises that can be used for purposes of reducing knee pains. In this regard, this research is important because its recommendations can be used for purposes of treating pains on the joints of a

An Analysis Of Cultural Heritage Tourism Tourism Essay

An Analysis Of Cultural Heritage Tourism Tourism Essay Heritage is property of the world. It has important evidences of past incidents and changes and it is the necessity to conserve it without political involvement and racial discrimination (UNESCO, 2004). So far Graham et al. (2000, p40) suggested Heritage is tremendously concerned in the construction and legitimating of collective constructs of uniqueness, such as group, gender, religion, civilization and nationalism. When we talk about the relation of heritage into tourism industry, at first, holy cities such as Rome are acknowledged as a highly multifunctional and even multi heritage place, as a result, the heritage attractions positively become the feature of Italy for tourist. To manifest the implication of heritage is consequently the key point of managing a heritage sites. As cultural heritage tourism is mostly depending on the history, the event in the past has abundant evidence that how the past travels had been changing the entire pages of each century and affected our past life. Much of it is also passed on form age to age occasionally in the form in which it actually happen but more often as myth or fable. In whichever form it is of prime importance to a tourism professional, particularly in the circumstances such as the one obtaining in India with wealthy cultural heritage having continuity form the ancient (IGNOU, 2002). The process of finding will continue to the future because no one predict that how many real stories have buried in the earth or still has not being found. But somehow scholars has been able to find the real incident of past with the help of evidences of literature which still present all over the world as well as the role of science and researchers. An archaeology department of each country is encouraging the researchers to fin d out about our past. These particular reasons also encouraged to the selection of the dissertation topic, so at first this will focus on past history and development of Ajanta and Ellora caves and then it will look at the past record of tourist information and the with the help of available secondary data from Books, Journal articles, news papers past surveys and the information available from the internet, respective governments and non government agencies such as UNESCO, ICOMOS, IUCN, GHF, etc. Introduction: Cultural heritage conservation is always a centre of discussion in the form of developing economy of various nations. Cultural heritage tourism product is becoming a prime source for develop and developing countries. Countries like India where every tourist gets glimpses of diverse culture in his/her entire journey. India is primarily a cultural destination in international tourism in these are the features of India marketed as tourism product in international as well as domestic circuit. Domestic tourism competition already has begun in India from past decades every states government has been developing their strategies with the help of international organization to improve the tourism. In result due to sudden changes the competition can be seen among service providers as from hawkers to a large scale stakeholders. In scenario market everyones mind has been diverting toward the heritage sites in India, thus its creating trafficking of tourists as well as service providers. So it is affecting the quality of services and facilities. So it shows a different picture of hospitality and tourism industry in India. Visitors/ tourists these are the main driver of tourism industries. Firstly, the different demands and requirement of individual is affecting on tourism industry in India such as tailor made tours and the class of tourist. Also the issues and understanding of heritage tourism by people it may be visitors/tourists or local communities are different. There are two types of tourists are who has interested to visit a cultural sites around the world and the second one who Most of the peoples travel interest is to watch heritage sites around the world and some leisure activity thus it boosting the economy of respective destination. The most important part of tourism is a cultural as well as natural heritage property. So it always remains prime attraction to most of the people to come and enjoy their holidays. This dissertation will focus on the every aspects of cultural heritage tourism industry such as supply, demand, conservation management, interpretation, authenticity and politics of cultural heritage site. The primary data can receive with the help of some questionnaire to understand the real nature and find out what can do more to achieve the overall development at Ajanta and Ellora Caves at Aurangabad. To understand the World cultural heritage site here the UNESCO considers that as a monuments, architectural works, works of monumental sculpture and painting, elements or structures of an archaeological nature, inscriptions, cave dwellings and combinations of features, which are of outstanding universal value from the point of view of history, art or science; groups of buildings: groups of separate or connected buildings which, because of their architecture, their homogeneity or their place in the landscape, are of exceptional universal importance from the point of view of history, art or science; sites: works of man or the combined works of nature and man, and areas including archaeological sites which are of excellent universal value from the historical, aesthetic, ethnological or anthropological point of view. In contrast it is suggested that World Heritage Sites should not only be exemplary situations for the pursuit of research but also be closely identified with the creation and maintenance of different kinds of knowledge (Darvill, T., 2007). People always willing to learn new things or exchange the knowledge between each other, it is nothing but to become aware about our surroundings and changing of trends in scenario market. Here, In the Krakow Charter (2000), a monument is defined as a clearly determined entity, the bearer of values, which represent a support to memory. In it, memory recognises the aspects that are relevant to human performance and feelings, associated with the historic time-line (Vecco, M 2010). Economic In the Times of India there was an article about heritage tourism and in that the Atul Sethi has mentioned that Heritage can become a serious economic driver for India, if the country is able to get its act together. If we take an example as future 2025, so the picture of the heritage site will give different view as no beggars or touts in particular area like Maluti temple, a world heritage site in Jharkhand India. So tourist can enjoy the holistic experience of the temples and can savour the living legacy of the region. The heritage tourism in India is attracting hordes of international and domestic tourist and top of the line contributors to the countrys foreign exchange and GDP. The global heritage fund organizations 2010 report says that developing countries like India can tap a $ 100 billion a year opportunity by 2025, if they make sincere attempt to preserve and responsibly develop their heritage sites. Literature Review: Introduction: This chapter will investigate the perception of cultural heritage tourism and visitor management as well as development of tourism at site by reviewing several concerned literatures. The review will start with the overview of cultural heritage tourism including the concept of Culture, heritage, cultural heritage tourism and cave architecture from different World Heritage Sites to understand the their current scenario of cultural heritage tourism management. The review will be followed by the discussion on visitor impacts, their experience and management. Also it will focus on the intermediaries of cultural heritage tourism. Cultural heritage tourism: Cultural Tourism is the subset of tourism that is defined as travel directed toward experiencing the traditional and contemporary culture, arts, and special character of a place. This includes the performing, visual and literary arts, language, museums, heritage, crafts, architecture, design, film and broadcasting. The primary benefit of cultural tourism is economic impact. There are plenty of statistics that talk about the fact that travellers who participate in cultural activities spend more money and stay longer than leisure travellers. A good cultural tourism plan shapes and defines a communitys image, both to itself and to the outside world Cultural tourism and cultural heritage management work as equivalent activities in most places with really little conversation between the two (Mckercher and Du Cros, 2002). As it shows common interest between the cultural and cultural heritage is the management, conservation, and preservation of the cultural and heritage properties. So the results of this many lost opportunities to provide value to visitor experiences even though managing rare and weak resources in a social context, environmentally and ethically responsible and sustainable manner. Sometimes this loss results in some (and scholars stress some) unprincipled tourism operator exploring local culture and heritage assets for their own personal gain (Mckercher and Du Cros, 2002). Hall and Zeppel (1990a:87 in Timothy and Boyd, 2003) stated that relation between cultural and heritage tourism is: Cultural heritage is experiential tourism based on being involved in and stimulated by the performing arts and festivals. Heritage tourism, whether in the form of visiting preferred landscapes, historic sites, buildings or monuments is also experiential tourism in the sense of seeking an encounter with nature or feeling part of the history of a place. Most researchers believe that heritage is linked to the past which symbolizes some sort of gift to be passed down to current as well as future generations, both in terms of cultural traditions and physical objects (Hardy, 1988 cited in Timothy and Boyd 2003). But in contrast many authors have pointed out that what elements of past a society need to maintain (Fladmark 1998; Graham et al., 2000; Hall and McArthur 1998 in Timothy and Boyd 2003). As there are many incidents all cannot link to the cultural aspects. This makes selective sort of heritage it assumes some aspect of value, that which is of personal value is labelled as personal or family heritage, whereas those values dictated by nations or communities become our heritage (Hall and McArthur 1998 cited in Timothy and Boyd 2003). Hall and Zappel (1990) observed differently that the connections between cultural and heritage tourism, stating that Cultural tourism is experiential tourism based on being involved in and stimulated by the performing arts, visual arts and festivals. Heritage tourism whether in the form of visiting preferred landscapes, historic sites, building or monuments, is also experiential tourism in the sense of seeking an encounter with nature or feeling part of the history of a place. However the most internationally accepted definition of heritage was defined by UNESCO that Heritage is our legacy from the past, what we live with today, and what we pass on to the future generations (UNESCO 2008a, p.5). Furthermore Timothy and Boyd (2003) stated that heritage can be divided into tangible immovable resources (e.g. national park, sanctuaries, monuments, buildings), tangible movable resources (e.g. objects in museums) and intangible resources including values, customs, ceremonies, lifestyles and experiences such as cultural events, currently UNESCO focusing on folk dances of south India as a cultural heritage( ). It defines that heritage tourism as an immersion in the natural history human heritage, arts, philosophy and institutions of another region or country (Timothy and Boyd 2003). The importance of Heritage The importance of heritage and reasons for preserving heritage can be described into four aspects which are social, economical, scientific and political (Hall and McArthur 1993a). In social significance, heritage refers to personal and collective identity people and society have which can lead to create a sense of belonging. The sense of belonging and social conscience is a driver to consider preservation in the first place. In terms of economic importance, heritage is preserved because of its value for tourism and recreation. As being a large scale business, heritage tourism can generate incomes for the sites either from visitor spending or sponsorship from various stakeholders (Hall and McArthur 1993a). Further mainly Heritage have scientific and educational importance since heritage attractions especially natural heritage sites such as national parks may have rare habitat and endangered species which will be useful for scientific researches as well as the monuments and wonders of the worlds creates more opportunities to the scientist and researchers to find out the exact meaning of the past it may be by digging by the archaeological department of each country or the scientific analysis of wall paintings at various world heritage sites (Hall and McArthur 1993a). In terms of political importance, Hall and McArthur (1993a, p. 9) state that the importance and demonstration of heritage may serve political ends as the conservation and interpretation of certain heritage attractions may serve to underline a particular version of history or to promote existing political values. Current tourist demand: According to (Pavliv, 2009) up gradation in the standard of living is become a main cause of rises in fluctuation of tourists spending for example the income generating from the modern tourism commodities. Modern tourists behaviour is the main factor in current tourism industry. As currently many of travel operations that could be from demand side or supply side are in boom because ease of accessing Information through internet. So the importance of information technology in the current industry is the main factor while considering the supply and demand of tourism industry. Also it can be assumed that the personal needs of the modern tourism have more influence compare to old masses of tourism. The current tourism mostly depended on economic mass and production to consume mass, standardized and rigidly packaged holidays. The new tourists are dictating the pace and direction of industry changes (Poon, 1993 in Pavlic, 2009). New tourists behaviour is the most important factor in the modern tourism development. Consequently, here the intention of the research is to find out the main factors and consequences of tourism demand changes. The researcher wants to find out if there is the universal type of tourist for the universal tourism supply or there are different types that require different marketing approach for example the e-tourism method to attract the tourists. Also the goals of the paper are to research the main causes of changes in modern tourism. The stated phenomenon has exerted a great influence onto the behavioural changes, as well as on the change in structure of the existing needs of tourist demand compare with the previous period. While the old mass tourism, apparently identical tourists were forced by the economic and mass production to consume mass, standardized and rigidly packaged holidays of every group of people and individual, new tourist consider the changes in this industry are and it affecting on their individual decision (Poon, 1993 cited in Pavlic, 2009). For ex: tour packages. Vellas Becherel (1997 in Pavlic, 2009) the modern tourist can be classified in various factors and in that three main factors which particularly in demand of travel of tourist firstly, demographic and communal changes. These factors mainly control by traditional and existing outbound market respectively these are two main markets in this industry so as far as concern it has enormous influence on the individual tourist as well as on international tourism. Secondly, leisure time and duration of holidays is greater than before as. Segmentation of holidays and market segmentation. Chapter 3 A case study of Ajanta and Ellora caves The consideration of architectural heritage had been a matter of national concern only at most the laws regarding protection of historic building in Europe date back to that period of their national borders. There are numerous cultural groups live in each country, but their scope never went beyond the boundaries. The cultural internationalism was a result of the First World War, with the formation of the confederation of Nations, and most of all of the Second World War, with the formation of the United Nations Organization and the establishment of the UNECO (ICOMOS, 2010). The chapter provides a literature review of cultural heritage tourism in India, Particularly at Ajanta and Ellora Caves World Heritage sites inscribed in 1983 by UNESCO at Aurangabad, India. These caves are great example of cave architecture from the 2nd to 6th century. It is mainly demonstrate the Buddhist, Jain and Hindu religion cultures. So, these studies will focuses on the religious diversity of cultures in India as well as it involve the importance of heritage visitors management at those sites. And then it will compare with other world heritage sites. As what can do more to maintain/sustain the balance between demand and supply side of heritage tourism industry in India. Increasing tourists and the conservation of heritage sites these are the main issues now days. So, as subsidizing product of tourism industry what are the main significance and characteristics of cultural heritage tourism and its importance around the world and in India particularly at Ajanta and Ellora caves . According to the UNESCO, the Ajanta Caves are the masterwork of the Archaeological rock cut caves. The first Buddhist cave monuments at Ajanta date from the 2nd and 1st centuries B.C. During the Gupta dynasty period (5th and 6th centuries A.D.), highly abundantly decorated caves were added to the original group. The paintings and sculptures of Ajanta, considered as a stunning success of Buddhist religious art, have had a considerable artistic influence. As it mentioned above the Ajanta caves depict the role of the Buddhist community, intellectual and religious halls, schools for self development based on the teaching of Vipassana. In the  Buddhist  tradition which means insight  into the nature of reality. Vipassana is one of worlds most ancient techniques of meditation; it is a practice of self-transformation through self-observation and  introspection. In English, vipassana  is often referred to simply as insight meditation. The caves also was a reception centres in the India of the Gupta and their immediate successors. The caves are located 100  km north-east of Ellora, 104  km from Aurangabad, Maharashtra India and 52  km from Jalgaon Railway Station. They are cut into the volcanic lava of the Deccan in the forest ravines of the Sahyadri Hills and are set in beautiful sylvan surroundings. The most important part is these magnificent caves containing carvings that depict the life of Buddha, and their carvings and sculptures are considered to be the beginning of classical Indian art. And this is main reason why tourist and researchers attract to those sites. The description of the caves is, the total number of caves are 30 but one is unfinished caves so the 29 caves were excavated beginning around 200 BC, but they were abandoned in AD 650 in favour of Ellora. In that five of the caves were temples and 24 were monasteries, thought to have been occupied by some 200 monks and artisans. After 7th century to 18th century there was no any supportive record has found, it may be because of increased forest surrounding of the site and that it was forgotten until their rediscovery by a British tiger-hunting party in 1819. The Ajanta site consists of 29 caves cut into the side of a cliff which rises above a wander in the Waghora River. Today the caves are reached by a road which runs along a terrace mid-way up the cliff; however each cave was once linked by a stairway to the edge of the water. This is a Buddhist community, comprises five sanctuaries or Chaitya-grihas  (caves number 9, 10, 19, 26 and 29) and monastic complex  sangharamas  or viharas. A first group of caves was formed in the 2nd century BC: the Chaitya-grihas open into the rock wall by doorways surmounted by a horse-shoe shaped bay. The ground plan is a basilical one: piers separate the principal nave from the side walkway which joins in the apsis to permit the ritual circumambulation behind the (commemorative monument). This rupestral architecture scrupulously reproduces the forms and elements visible in wooden constructions. A second group of caves was created at a later date, the 5th and 6th centuries AD, during the Gupta and post-Gupta periods. These caves were excavated during the supremacy of the Vakatakas and Guptas. According to inscriptions, Varahadeva, the minister of the Vakataka king, Harishena (c. AD 475-500), dedicated Cave 16 to the Buddhist sangha while Cave 17 was the gift of the prince, a feudatory. An inscription records that the Buddha image in Cave 4 was the gift of some Abhayanandi who hailed from Mathura. The earlier architectural formulas were re-employed but treated in an infinitely richer and more ample manner. The decoration attained, at this time, an unequalled splendour: the statuary is numerous (it was already permissible to represent Buddha as a human; these representations are found both on the facades and in the interior). Finally, the wall painting, profuse and sensitive, constitutes, no doubt, the most striking artistic achievement of Ajanta. Under the impulse of the Gupta dynasty, Indian art in effect reached its apogee. The Ajanta Caves are generally decorated with painted or sculpted figures of supple form and classic balance with which the name of the dynasty has remained synonymous. The refined lightness of the decoration, the balance of the compositions, the marvellous beauty of the feminine figures place the paintings of Ajanta among the major achievements of the Gupta and post-Gupta style and confer on them the ranking of a masterpiece of universal pictorial art. Here needs to understand the history of India as how the past events has changed its culture first major civilisation Indus River valley was found early five thousand years back. The twin cities of Mohenjodaro and Harappa World heritage site UNESCO now in Pakistan were ruled by priests and held the fundamentals of Hinduism. These civilisations are known to possess a sophisticated way of life, a highly developed sense of aesthetics, an astounding knowledge of town planning and an unreadable script language. The Indus culture at one point of time extended nearly a million square kilometres across the Indus river valley. It existed at the same time as the ancient civilizations of Egypt and Sumer but far outlasted them. Surviving for nearly a thousand years the Indus valley civilisation fell to tectonic upheavals in about 1700 BC, which caused a series of floods.   The Aryans came around 1500 BC, and that was the reason to the collapsing Indus Valley culture. At the dawn of Vedic ages the Aryans came in from the North and spread through large parts of India bringing with them their culture and religious beliefs. Pleased In 567 B.C. Siddhartha Gautama was born. After asceticism and  meditation, Siddhartha Gautama discovered the Buddhist  Middle Way-a path of moderation away from the extremes of self-indulgence and  self-mortification. Siddhartha Gautama attained enlightenment sitting under a  Pipal  tree, now known as the  Bodhi tree  in  Bodh Gaya, (A name of place) India Gautama, from then on, was known as  The Perfectly Self-Awakened One,  the  Samyaksambuddha. There are lots of paintings and sculptures Vipassana. During this time lived Mahavira, who founded the Jain Religion. The Indian subcontinent is full of caves and monuments devoted to these religions and are worth a visit.   After two hundred years, in the 4th century B.C., Emperor Ashoka, one of the greatest King of Indian histories, led the Mauryan Empire to take over almost all of what is now modern India. This great leader embraced Buddhism and built the group of monuments at Sanchi (a UNESCO world heritage site). The Ashoka pillar (four lions are seated back to back on single cut rock pillar) at Sarnath has been adopted by India as its national emblem and the  Dharma Chakra  on the Ashoka Pillar adorns the National Flag. They were followed by the Guptas in the north, while in the south part of India quite a few different Hindu empires, the Cholas, the Pandyas and the Cheras spread and grew, did business with Europe and other parts of Asia till the end of the 1100s. Christianity came in India at about the same time from Europe. Legend has it that St. Thomas the Apostle arrived in India in 52 A.D. Even earlier than that people of the Jewish religion arrived on Indias shores. In about the 7th century A.D. a group of Zoroastrians, or Parsees, landed in Gujarat and became a part of the large mix of religions in India today, each of which adds its important and distinctive flavour. In the 15th century Guru Nanak laid the foundation of the Sikh religion in Punjab. In 1192, Mohammed of Ghori, a ruler from Afghanistan, came into India and captured several places in the north including Delhi. When he went home he left one of his generals in charge who became the first Sultan of Delhi. During this time Islam, was introduced into a major part of Northern India. It can be mentioned that even before that, just after the period of the prophet, Islam was brought to the western coast of India by Arab traders and flourished in what is now Kerala. The Dehli Sultanate gradually took control of more and more of North India over the next 200 years, till Timur, who was called Timur the Lame or Tamberlane came from Turkey in 1398 to attack India. He and his army stole all the valuables that they could carry and left again, Soon the Mughals, who were from Iran, came in and took control of the north. In the meantime south, in 1336, the Hindu Vijayanagar empire was set up and became very strong. The Europeans Portuguese, French, Dutch, Danish and British started arriving in the early 1600s. The above people held territories in India and made a good friends as well as enemies among Indias rulers as they got more and more involved, with the Indian politics, however the British who eventually controlled most of India and finally made it one of their colonies. Two hundred years later, in the 4th century B.C., Emperor Ashoka, one of the greatest King of Indian histories, led the Mauryan Empire to take over almost all of what is now modern India. This great leader embraced Buddhism and built the group of monuments at Sanchi (a UNESCO world heritage site). The Ashoka pillar at Sarnath has been adopted by India as its national emblem and the  Dharma Chakra  on the Ashoka Pillar adorns the National Flag. They were followed by the Guptas in the north, while in the south part of India several different Hindu empires, the Cholas, the Pandyas and the Cheras spread and grew, trading with Europe and other parts of Asia till the end of the 1100s. Christianity entered India at about the same time from Europe. Legend has it that St. Thomas the Apostle arrived in India in 52 A.D. Even earlier than that people of the Jewish religion arrived on Indias shores. In approximately the 7th century A.D. a group of Zoroastrians, or Parsees, landed in Gujarat and became a part of the large mix of religions in India today, each of which adds its important and distinctive flavour. In the 15th century Guru Nanak laid the foundation of the Sikh religion in Punjab. To Top In 1192, Mohammed of Ghori, a ruler from Afghanistan, came into India and captured several places in the north including Delhi. When he went home he left one of his generals in charge who became the first Sultan of Delhi. During this time Islam, was introduced into a major part of Northern India. It may be mentioned that even before that, just after the period of the prophet, Islam was brought to the western coast of India by Arab traders and flourished in what is now Kerala. The Dehli Sultanate gradually took control of more and more of North India over the next 200 years, till Timur, who was called Timur the Lame or Tamberlane came from Turkey in 1398 to attack India. He and his army stole all the valuables that they could carry and left again, and after that the Delhi Sultanate was never so strong again. Soon the Mughals, who were from Iran, came in and took control of the north. In the meantime south, in 1336, the Hindu Vijayanagar empire was set up and became very strong. The Europeans Portuguese, French, Dutch, Danish and British started arriving in the early 1600s. All of them held territories in India and made friends and enemies among Indias rulers as they got more and more involved, with the Indian politics, but it was the British who eventually controlled most of India and finally made it one of their colonies. India got its independence from Britain in 1947 after a long struggle led mostly by Mahatma Gandhi. In the process of becoming independent, India became two countries instead of one. In the years since independence India has made huge progress and coped with great problems, and has developed its industry and its agriculture, and has maintained a system of government which makes it the largest democracy in the world. India got its independence from Britain in 1947. Since independence India has made huge evolution and muddle through with great problems, and has maintained a system of government which makes it the largest democracy in the world. Every community and nation try to protect, conserve and develop heritage as an asset, particularly to make them important tourist sites by developing better infrastructure and facilities, which offer a rich cultural experience to tourists and ensure cultural and natural heritage of the destination to be preserved and conserved. To extend INTACHs mandate beyond conservation, the Heritage Tourism Division was set up in December 2005. A cogent system of sustenance of heritage sites was evolved in the activities of the Division. This would allow people to have access to and understand heritage sites in complete detail. The Heritage Tourism Divisions primary work is to synergize heritage with tourism. The Division works for development of sustainable tourism connected with heritage as an asset. A development of tourist facilities is in harmony with the local eco-system and heritage architecture, and regulates sensitivity of design in architectural style of construction of any new tourist facilities. The aim of developing tourism is to conserve and utilize buildings to sustain their maintenance. Heritage Tourism development aspects are: Community Development of tourist receiving destination Socio-economic Growth of the receiving community Preservation and Conservation of cultural and natural heritage sites Income Generation

Essays --

Education is very important for human life. It becomes main necessity for human beings. Almost all aspects of life are influenced by education. A human is introduced to education started from born, and it keeps going on until the death. If a human has a good basic education, it will be easy for the next development. Human will be easily accept and adapt their self with new education which more advanced. They can accept values in social life, school, family, and environment. Moreover, of course they will be smart and be respectful human. Sometimes, education is regarded as a sign whether a human is in high class or not. It influences a lot of areas in human life, for example; in the world of work, in getting money, in communicating with others, and in adapting human in this more modern era and advanced technology. First, education influences the world of work. Human are demanded to have skill in its sector. Usually a company conduct interview before recruiting employee. In recruitment process, there are some questions that related to level of education background. Having good quality in education will help a human to get wanted job and of course with balance income. A human who has high level education will get good job easier than a human who has not good education. Moreover, to get a job is very difficult. It shows that education has important role in the world of work. Secondly, education helps human in getting money. Educated human are more useful and easier in getting money because they have something that can be sold. Just by using the skill, they can get money. Furthermore, they do not need to work powerfully because they work use their brain. It is different with an uneducated human. Uneducated human usually work by using ... ...ality of education because that is very helpful in increasing human’s quality. Therefore, education is very important in the survival of a human because it affects many aspects of life. It can be gotten from formal institute and informal institute. However, many human prefer to choose formal education. Usually it is started from elementary school until university. It has big role in forming good personality, such as; responsibility, honesty, and attitude. Responsibility is important thing that must be belonged to every human. Then, honesty comes from the heart. The price of it is very expensive and it cannot be changed by everything. Next is about attitude, it relates to human’s behavior. Attitude cannot be kept hiding because it comes out spontaneously. Human do not need to be worried if they have good habit in behavior, because a good habit shows a good attitude.

Iron Crowned Chapter 20

I didn't know what the flowers meant. Nothing like that had ever happened when I'd meditated in the Thorn Land. Over the next few days, I just kept thinking about what Rurik had said, that no other monarch save my father had ruled more than one kingdom in recent history. It had taken great power and magic for me to exert my dominance over the lands†¦. Were they feeding it back to me in return? I certainly felt stronger with them, but I'd never expected any sort of unconscious physical manifestation. What else was I capable of? What could I make the land do? I didn't mention the matter to anyone, not even Kiyo. He'd seen the red flowers but brushed them off. If I told him about the Thorn Land, I feared he'd grow upset about the thought of my magic increasing. He grudgingly accepted what I already possessed but still feared it would turn me into my father, no heir needed. And although I'd felt physically better in the Otherworld, I grew weak again after a day or so back in Tucson. I didn't mention this to Kiyo either, but Jasmine was around enough to pick up on it. â€Å"Are they calling to you again?† she asked over breakfast one day. She was devouring Pop-Tarts, another love we apparently shared. I was too worried to have an appetite and simply watched. â€Å"You look like crap.† â€Å"I don't know,† I said, drumming my fingers against a glass of water. â€Å"There's no precedent for this – at least not anymore. No one knows what to expect from me having two kingdoms.† â€Å"I bet Dorian would know.† I bet he would too, but I shook my head. â€Å"He's not all-knowing no matter how much he wants to be,† I countered. â€Å"And I'm done with him.† â€Å"Okay.† She didn't fight it. For a while, she'd kept telling me I'd made a mistake in breaking up with Dorian, but Kiyo had been growing on her. I still wasn't sure if she approved, but at least I didn't have to listen to teen advice about my love life anymore. â€Å"But you might just have to go back soon. I mean, think about it. You're bound to two lands in the Otherworld. Aren't the lands and the monarch one? Part of you's there. It makes sense you'd have to be there twice as much.† I winced at the idea, though it had been on my mind too. â€Å"If I were there any more, I'd be living there permanently.† She swallowed the last of some crust. â€Å"You may not have a choice.† Her flippant tone irritated me. â€Å"There's always a choice. I rule them. They don't rule me.† I stood up abruptly and briefly became dizzy. It felt like the lands were mocking me. Damn it, I thought. You will not call me back so quickly. I'm staying in this world for a while. I'll come and go when I please. â€Å"I just need to stop thinking about it. I'm going to see if Lara's got a job.† â€Å"Yeah,† said Jasmine dryly. â€Å"That'll fix everything.† Lara did have a job for me, several actually. Even though she was all but living with Tim – in my house – she still kept meticulous records and took all my calls. She looked disappointed that I only accepted one from her growing list of jobs, a small one at that: a simple haunting that would probably take about five minutes. She said nothing, but I knew that she worried if I didn't make any money, she wouldn't either. So, remembering Enrique's comment about needing help but not being able to trust anyone, I gave her his card with the suggestion she call about part-time work. â€Å"Are you firing me?† she asked. I smiled as I gathered up all my weapons. â€Å"No, but I want you to have a backup plan in case you get laid off.† Her eyes widened in alarm at the joke. Or, I suddenly wondered, was it a joke? I brought Jasmine with me to the job because I still felt uneasy about leaving her alone. Besides, she was finally getting her fill of the human world, and I had a feeling her insistence on me returning to the Otherworld was partly selfish. Later, after I'd finished the job, I kind of regretted bringing a witness. â€Å"Wow,† she said, as we drove home. â€Å"You got your ass kicked.† â€Å"I did not.† â€Å"Did too.† So. This was what it was like having a sister. â€Å"I banished it, didn't I? You saw it go to the Underworld.† â€Å"Yeah,† she admitted, â€Å"but it sure did take a long time. I felt like I could have done it, and I've never banished anything before.† I gritted my teeth, refraining from commenting that I still had her chains. The troubling thing was, I had kind of sucked. I'd been in no real danger – not with a ghost that minor – but it had beaten me up more than it should have. I was off my game, a little slower, a little weaker. I'd walked away with some bruises and now noticed as we drove that my shoulder itched. For a moment, I thought the ghost must have hit me there, but there was no pain. The stitches. I'd nearly forgotten about them, now that they'd finally been able to heal. My skin had probably started to grow over the threads. I needed them out. No one was at my house, much to my disappointment. I'd hoped Kiyo had stopped by and could remove the stitches. Trying to be optimistic, I decided he must be pulling a shift at the veterinary hospital and wasn't with Maiwenn. Thus far, I'd heard no official word from her about my new double-queen status. Other monarchs had weighed in, though. Some had responded by showering me with congratulatory gifts and groveling. Others had let me know – in an amiable way – about other monarchs they were pals with, monarchs with big armies. It turned out everyone did fear the Iron Crown. I called my regular doctor, hoping to get an appointment this week as backup, in case Kiyo stayed absent. To my pleasant surprise, they'd had a cancellation that afternoon and could remove the stitches right away. It was good news for me but an annoyance for Jasmine, who'd just gotten comfortable on the couch. â€Å"Oh, come on,† she said, stretching out. â€Å"We just got home. Can't you please leave me here? I promise not to conquer the world or get pregnant while you're gone.† â€Å"You know,† I said, â€Å"Lara and Tim had sex right where you're lying.† She jumped up. A half hour later, we arrived at my doctor's office. I left Jasmine in the waiting room, deeming her safe enough with her iPod and magazines for the five minutes it would take to remove my stitches. Maybe she'd read some contraception pamphlets to pass the time. â€Å"They did this in the ER?† the doctor asked when I was admitted to an examination room and had taken off my shirt. I'd been seeing Dr. Moore for a couple years now. She was a pleasant, mid-fortyish woman who had eventually learned not to ask too many questions about my injuries. She thought I was a â€Å"contractor† who practiced martial arts on the side. â€Å"Not exactly,† I said. â€Å"I tore the ones the ER did, so my boyfriend had to redo them.† She took hold of tweezers and a tiny pair of scissors and leaned over. â€Å"Well, his work's neat, and it didn't get infected. If I'd seen you when this happened, I would have confined you to your bed. I would have known better than to assume you wouldn't promptly rip these out.† â€Å"Yeah, I really pulled one over on the other doctor.† She snorted a small laugh and proceeded to pull the stitches out. They stung where they tugged the skin, but honestly, it was nothing compared to my normal wear and tear. â€Å"There you go,† she said, stepping back. â€Å"You'll have a scar.† I put my shirt back on and faced her. â€Å"Battle trophy.† She rolled her eyes, leaning against the wall with crossed arms. â€Å"You shouldn't joke about that.† â€Å"Sorry.† I picked up my purse, but her expression said we weren't done. â€Å"Eugenie †¦ I don't ask many questions, not any more than I need to treat you, but I'm worried about how often you come in with these kinds of injuries.† If only she knew how many I didn't come in for. â€Å"I – â€Å" â€Å"No, no,† she interrupted. â€Å"I don't need to know all the details of your life. I try not to judge – but you might need to. There are jobs out there that are physical in nature. That's life. But whatever you're doing †¦ maybe you should reevaluate it. To be blunt, you look terrible today.† â€Å"Oh, that.† Crap. I could hardly explain that it was the residual aftereffects of a magical battle in the Otherworld, during which I'd fought for dominion of a fairy kingdom and become its new master, thus doubling my reign. â€Å"I'm just, uh, coming down with something. Just kind of tired, you know.† She arched her eyebrows. Double crap. â€Å"Then let's do some quick blood and urine tests,† she said, straightening up. â€Å"Check your electrolytes, thyroid †¦Ã¢â‚¬  I fumbled for an excuse. I'd never been comfortable with those kinds of tests since discovering I had gentry blood. I was pretty sure human medicine couldn't detect that sort of thing, but I didn't want to take any chances. â€Å"I don't have time. My sister's waiting for me in the lobby.† â€Å"I'm sure she'll be okay,† said Dr. Moore. â€Å"This'll take five minutes.† â€Å"Fine.† I sat back on the table, defeated. â€Å"But can you send someone to make sure she's still out there? She's the sullen one.† Dr. Moore's nurse returned to send me to the bathroom and then drew blood when I came back. She was in the middle of telling me they would send the tests out to a lab, when Dr. Moore herself stuck her head back in. â€Å"Can we talk for a moment?† she asked. The nurse discreetly left, and once we were alone, I braced for another lecture about my lifestyle. â€Å"I really need to get back to my sister,† I told her. â€Å"You don't know what she's capable of.† â€Å"Eugenie.† Dr. Moore's voice was kind but firm. â€Å"Most of those tests we have to wait on, but there are a few we do right here with urine.† â€Å"And?† â€Å"And, you're pregnant.† I thought about this for a moment and then enlightened her. â€Å"No. I'm not.† Those eyebrows rose again. â€Å"Your test came back positive. Now, we can't tell how far just from a urine test, but based on – â€Å" â€Å"Your test is wrong!† I sprang up from the table. My world was starting to reel again. â€Å"I can't be pregnant!† To her credit, she took my outburst calmly, but that was probably part of her training. â€Å"The test is very accurate, and it would explain why you aren't feeling well.† â€Å"I can't be pregnant,† I repeated adamantly. There was a mistake here. A terrible, terrible mistake, and she needed to understand that. Until she did, I refused even to process what she was claiming. â€Å"I take my birth control pills. Every day. Same time. Just like I'm supposed to. I'm not going to lie: I do other stupid shit all the time. But not with pills. I take them perfectly. I did with the antibiotics too. I'm careless with stitches but not prescriptions.† That calm expression shifted to surprise. â€Å"Antibiotics? When were you taking antibiotics?† I pointed to my shoulder. â€Å"When I got this. The ER doctor gave me a prescription.† I frowned. â€Å"What? Why are you looking at me like that? I told you: I took them correctly, all of them.† â€Å"Antibiotics can negate birth control pills,† she said. â€Å"Didn't you know that?† â€Å"I †¦ What? No. That's not †¦ No.† A mistake. A terrible, terrible mistake. â€Å"Women taking both need to use some other form of contraception until the antibiotics have run their course.† A horrible, cold feeling began spreading over me. â€Å"How was I supposed to know that?† I asked in a small voice. â€Å"Your pharmacist should have told you when you got the antibiotics. The interaction would have shown up in your records.† I thought back to that night, how my mom and I had stopped at the place closest to the hospital. â€Å"I didn't go to my usual pharmacy†¦.† And I had gotten out of there as fast as I could, not bothering to talk to the pharmacist because I'd taken antibiotics lots of times in my life. I certainly hadn't bothered with the enclosed pamphlets. Dr. Moore seemed to think she'd gotten through to me. â€Å"Now, we can figure out how far along you are if you know when your last period – â€Å" â€Å"No,† I exclaimed. â€Å"No, no, no. I can't be pregnant! Don't you understand? I can't be. I can't have a baby. I can't!† I was shouting again and wondered if this place had security. â€Å"Calm down,† Dr. Moore said. â€Å"Everything will be all right.† No, no, it wouldn't. Everything wouldn't be all right. Nausea welled in me, nausea I'd felt for a few weeks or so – and that had nothing to do with inheriting the Rowan Land. After all this time, after all the planning and lofty talk, after all my fears about Jasmine †¦ it was me. Human medicine had screwed me over. No, I had screwed me over. I'd fucked up. My own carelessness had brought this about. Everything anyone had ever said about the Storm King prophecy began to run through my mind. Sformi, King's first grandson. An invasion of the human world. Led by his mother. Domination and blood. And I, I was bringing it about†¦. I was the instrument†¦. â€Å"Eugenie!† Dr. Moore was supporting me, and I had a feeling she'd said my name a few times. She glanced at the door and opened her mouth, about to call her nurse. â€Å"No!† I clutched at her white coat. â€Å"Don't. Listen to me.† My voice was raspy and desperate. â€Å"I can't. I can't have a baby. Don't you understand?† She peered at me through her glasses, regarding me knowingly. â€Å"Then you don't have to. There are options – â€Å" You can't have a boy, some voice inside me said. What if it's a girl? â€Å"Wait,† I interrupted her. â€Å"When can you tell the gender?† That got a shocked look. â€Å"You'd base an abortion on gender?† â€Å"I – no, wait.† Fuck. I couldn't think. I was panicked and scared and confused. I needed to get my head together. What did I do? I had to get rid of this baby, pure and simple. People did it all the time. It was easy in this day and age, right? â€Å"I meant, how long until you can tell gender and if †¦ if there's anything wrong.† I groped for something reasonable, something that wouldn't make me seem like a heartless woman who'd kill her son. â€Å"You can do those tests, right? Like, genetic tests? I †¦ I'm so afraid of having a baby and having there be something wrong. My family has a bad history. My cousins have had babies with birth defects, and I can't †¦ I can't handle that. I have to know. I have to know †¦ right away †¦ as early as possible because otherwise I'll †¦Ã¢â‚¬  The lies rolled easily off my lips. Anything. Anything to know the gender. Dr. Moore studied me again. I still sounded crazy and scattered, I knew, but a little less than before. â€Å"When was your last period?† she asked quietly. I turned to her wall calendar. The numbers swam before me. I couldn't focus. How the hell could I remember that when the fate of the world was on the line? I thought about my last period and tried to link it to some event, something that would trigger a date. â€Å"There.† I pointed. â€Å"It started on the fifth.† She nodded, doing mental calculations. â€Å"Which lines up with the antibiotics. You're almost nine weeks along, as the reckoning goes, though technically only seven since conception.† Seven. Seven weeks †¦ â€Å"You're almost in the range for chorionic villus sampling,† she said. Chorionic what? â€Å"They don't like to do it unless it's necessary, though. There are risks for the fetus. They almost never do it for someone your age, who's in good health†¦.† â€Å"But it can tell me?† I said urgently. â€Å"It can tell me what I need to know?† â€Å"It can tell you a lot. No test can tell you everything, but it can give you peace of mind †¦ especially if you really do have a bad family history †¦Ã¢â‚¬  Did I ever. â€Å"I do,† I said. â€Å"Please.† I held my breath, knowing she was wavering here. Finally, she turned to her filing cabinet, rifling through it until she found a carbon form. She scrawled something in doctor's handwriting on it and handed it over. â€Å"Here.† It was a referral to an OB-GYN's office nearby. The form had my name, some boxes checked, and a few illegible words. I did make out CVS and emergency. â€Å"Emergency?† I asked. I mean, it was, but I was surprised she'd nailed it. â€Å"It means you'll get scheduled in right away. Most of these tests are backed up – because they aren't done this early. Give it to my nurse when you leave.† She was writing something else as she spoke. â€Å"She'll call them and schedule you – but you need to be aware they may refuse it when you're there, based on their judgment. I meant it: this isn't routine.† My next words were hesitant. â€Å"Then why are you doing it?† â€Å"Because I believe that in pregnancy, the mother's health outweighs everything else.† Mother's health. I didn't like thinking of myself as a mother. Fuck. This shouldn't even be an issue at all! We should be discussing abortions. Why did I care about gender? I didn't want a baby. I wasn't ready for a baby. Certainly not one who'd fulfill a world-conquering prophecy. â€Å"In this case,† said Dr. Moore. â€Å"Your mental health is especially concerning. Which is what this is for.† She handed me the other piece of paper. It was a referral for a psychologist. â€Å"I don't need – â€Å" â€Å"Eugenie, shock over an unplanned pregnancy is normal. Expected. But it's clear †¦ you have some very serious issues around this.† She had no idea. â€Å"Have my nurse call for the test. Then schedule yourself a therapist appointment and a follow-up with me.† There was no way I could tell her I had no intention of going to therapy. I wasn't even sure about the follow-up. But I'd gotten away with something, and I knew it. I nodded meekly. â€Å"Thank you.† I left before she could change her mind. Jasmine's face was filled with irritation and impatience when I finally returned. â€Å"That took forever,† she said, tossing a magazine aside. â€Å"How deep were those stitches?† â€Å"Not that deep,† I murmured. I walked toward my car on autopilot, still stunned. â€Å"She was worried about how tired I was, that's all.† â€Å"Well, you can fix that when we go back to the Otherworld.† I started the car, staring off into space for a few ponderous moments as numbers floated around in my head. Nine weeks, seven weeks. Two days. That was how long until my test. Two days. I refocused on my surroundings so I wouldn't get us into an accident. â€Å"We aren't going to the Otherworld anytime soon,† I replied. Jasmine shot me a look that clearly expressed her feelings on that, but there must have been something in my own face that answered back because she didn't fight the issue anymore. When we returned to my house, I put my purse and paperwork in my bedroom before sitting with Jasmine in her usual spot on the couch. Mindless TV suddenly seemed like a good idea †¦ except, well, it didn't do a very good job of taking my mind off of my problems. Pregnant. Conqueror of worlds. Storm King's heir. Me. It was all on me: what had happened and what was to come. We hadn't been home long when Kiyo showed up. He gave me a cheerful grin and wore his white coat from work, meaning he must not have been cozying up with Maiwenn. Small blessing. His smile was enough to make Jasmine smile in return, but I couldn't muster one. There was nothing to smile about right now. Nothing good in this world. Nothing good in either world. He joined us on the couch, sandwiching me in between him and Jasmine, and caught hold of my hand. â€Å"Hey, how are you?† he asked. He peered at my face, even though I was pointedly not looking at him. â€Å"Are you okay?† â€Å"Fine,† I lied. â€Å"Tired.† Storm King's first grandson will conquer the human world. â€Å"She's been like that all day,† said Jasmine. â€Å"She needs to go back to the Otherworld but won't.† â€Å"Is that true?† he asked. â€Å"I didn't think you'd have a problem with that,† I said. â€Å"You've always wanted me to stay away.† â€Å"Yeah, but not if it's affecting you like this. You really look sick, Eug.† â€Å"She also got beat up by a ghost,† Jasmine added helpfully. â€Å"Hey!† I glared. â€Å"I did not!† Kiyo chuckled and pulled me closer. â€Å"Stop playing tough. Go to the Otherworld tomorrow. I'll come with you, so it won't be as bad.† He relaxed, and there was a finality in his voice that I didn't like. I didn't like his presumption. I also wasn't entirely sure I should be going to the Otherworld, in light of recent developments. Flowers. Flowers everywhere, everywhere I step. I'm the land, and the land is me. Where I bring life, the land does too†¦. Or death. I could bring death as well. It was my choice. Over and over. The words in my head were all I heard. I didn't hear the TV, or Kiyo and Jasmine's occasional comments. I didn't really hear when Kiyo said he'd make dinner and went to drop off his overnight bag in my bedroom. But I did hear him when he came raging back to the living room, waving my CVS referral form in the air. â€Å"Eugenie!† His voice was a roar, one that made Jasmine cringe and widen her eyes. â€Å"What the hell is this?† I stared up at him levelly, surprised I could be so calm in the face of that outrage, especially after the emotional upheaval I'd been through all day. My own despair and shock had never left, but now I was able to push it down and meet Kiyo's eyes, as I allowed myself to finally acknowledge the other thought that had been bouncing around in my mind. Because along with the choices I had and the consequences I faced, there was one other matter to consider. I'd looked at the numbers, at the calendar. I'd factored in the dates, the antibiotics, what had been done – or, perhaps most importantly, what hadn't been done. It was all very clear. There was no soap opera here. No talk show?Cworthy mystery. â€Å"Congratulations,† I told Kiyo. â€Å"You're going to be a father. Again.†

Study Plan for Chemical Engineering

Chemical Engineering and its importance Advertisements Chemical engineering has a number of applications in our day to day lives. This course is offered to students at the graduate and postgraduate levels. Upon the accomplishment of their studies, individuals can apply for jobs with firms of the private or public sector firms. Placement opportunities are available for aspirants within some of the prestigious Indian firms such as Reliance and Indian Oil etc. One can say that this sector is one of the many areas where one can get good jobs as well as other opportunities of the right type.In this article we are going to discuss about the importance of chemical engineering as well as its numerous applications. Importance of Chemical Engineering Areas where chemical engineering is applicable in our day to day lives include: Coal preparation and mineral processing Explosives manufacturing Fertilizer industry Food processing Glass and specialty chemicals Paints Steel and aluminum production In addition to the above mentioned areas, chemical engineering also has applications in production of electronics, clothing, paper and photographic equipment etc.The scope for individuals in the field of chemical engineering is bound to grow in time. This is mainly because of industrial growth as well as the related scarcity of the resources those are required. In future years, chemical engineers will be needed to develop synthetic replacement for those resources as well as materials that are low in supply. In overall, it can be said that chemical engineers will be able to make very crucial contributions to the improvement in addition to the maintenance of the quality of our lives.Areas where one can apply his knowledge: Although chemical engineering is relatively a new field, this field of engineering has shown a speedy expansion during the last few decades. This has in turn led to rise in importance of chemical engineering as well as the number of jobs. Career opportunities for t hese professionals are available with R&D departments, especially in the field of energy as well in developing fields such as nanotechnology and biotechnology. Chemical Plants Petrochemical Plants Pharmaceuticals Petroleum Refining PlantsMineral Based Industries Electronics Industry Photographic Equipment Units Clothing Units Pulp and Paper Manufacturing Aircraft Industry Some Job Types Supervisor Technical Specialist Project Manager Project Engineer Teacher Researcher Environmental, Safety & Regulatory Manager Quality Manager Senior Process Engineer Product Development Engineer Fuel Meter Calibration Technician In the government sector, chemical engineers can find jobs in areas where solutions for environmental problems like recycling, water treatment and others are needed.They can also get work with departments of energy conservation as well as with defense establishments. Sir William Wakeham on the Importance of Chemical Engineering Sep. 06, 2011 Sir William Wakeham, President, I ChemE Sir William Wakeham, President, IChemE more Sir William Wakeham, President, IChemE From Reliance Industries’ Mukesh Ambani to stand-up comedian and perpetual watermelon smasher †¦ A Wide Range – From Reliance Industries' Mukesh Ambani and SABIC's Mohammed al-Mady to stand-up comedian and perpetual watermelon smasher Gallagher, chemical engineers can be found in almost every walk of life. And if you have never heard of Gallagher, you can replace him with Dolph Lundgren, who forewent a career in chemical engineering when he found success as Ivan Drago in the movie Rocky IV). These days, chemical engineering is as diverse as the people who study it, covering areas from biotechnology to mineral processing, and its significance for the chemical industry is now more important than ever. Sir William Wakeham is currently president of the Institution of Chemical Engineers (IChemE), a global professional membership organization for people who have an interest in and r elevant experience in chemical engineering.He spoke with Brandi Schuster on how the field has evolved, what IChemE does to encourage students to study chemical engineering and the importance of having chemical engineers in all levels within chemical and pharmaceutical companies. CHEManager Europe: Sir William, the term â€Å"chemical engineering† doesn't have quite the same meaning as maybe 50 years ago. How do you think the profession has evolved? Sir William Wakeham: These days there is much more of a focus on the word â€Å"process engineering† rather than â€Å"chemical engineering. Often the processes involved are still chemical, but they now encompass many more things than we thought about 50 years ago. These days you have trained chemical engineers working in many process applications that aren't necessarily within the traditional realm of the chemical industry. One example of that is within the pharmaceutical business, in formulation engineering. This consists of the construction of pills, which goes hand-in-hand with the drug formulations.That involves quite a lot of chemical engineering, but wouldn't have been thought of as such 50 years ago. It's a similar situation within the water industry. There is a lot of activity which could be considered process engineering, and probably most of the reactions, if there are any, are biological reactions. All in all, I think the term has been broaden quite a bit over the last several decades in order to include many more aspects and technologies. In fact, the term â€Å"chemical engineer† is probably being replaced by â€Å"process engineer. Is the future of chemical engineering one with a very broad base? Sir William Wakeham: Yes, and in my own experience, trained chemical or process engineers are the kinds of engineers who are most able to work with other disciplines, because they have already quite a breadth in their formation as engineers. That is not quite the same for, let's say, ci vil engineers whose chemistry training is quite limited. Process engineers have a unique opportunity to bring scientists and other engineers together.Most of the big problems that the world is facing are a bit like that; people have to be brought together from different areas. What about diversity within the profession, particularly when it comes to women? Sir William Wakeham: In the UK, total chemical engineering undergraduate numbers are the most positive for women's recruitment of any engineering discipline. In the UK, about 27% of chemical engineering students are women; this is certainly a step in the right direction. What kind of activities does IChemE have to encourage more people to study chemical engineering?Sir William Wakeham: We have an enormous focus on bringing people into chemical engineering courses; this has, at least in the UK, pushed the numbers through the roof. We are particularly interested in attracting women, and one of the key elements of doing that is havin g women on the staff of chemical engineering departments who do the recruitment. Here in the UK, most departments have a substantial number of women on their faculty. In other areas of the world, such as in the Middle East, there are some cultural issues that are additional difficulty.However, in Malaysia, where we are also active, there are a significant number of women studying chemical engineering now. Apart from its European offices, IChemE is also represented in Asia, Africa and Australasia. Do you work towards promoting chemical engineering for women in these parts of the world as well? Sir William Wakeham: Yes. We have been using our activities in the UK as a basis, but fine-tuning it for the different cultural backgrounds. Clearly what needs to be done in Malaysia is not the same thing as what needs to be one in the UK. Our offices in these areas are usually staffed by local people, which is important for creating an understanding of the country's needs. What is a chemical e ngineer's role in an oil and gas industry? 2 years ago Report Abuse Shape Shape Best Answer – Chosen by Voters This is a very broad question as chemical engineers (as in someone with a chemical engineering degree) can do many different engineering jobs in the oil and gas industry, but other engineers can do them as well.For example I know people with mechanical and chemical engineering degrees that are maintenance engineers with the same job responsibilities. The same goes for a drilling fluid engineer which could be held by people of varying background and technical degrees. A chemical engineer can be involved in all parts of the oil and gas industry from building the oil rigs, drilling the wells, pumping it out of the ground, transferring it through pipelines, separating in into usable chemicals in a refinery, and finally making petroleum based products in a chemical plant.Source(s): Chemical Engineer Where Do Chemical Engineers Fit into the Upstream Oil and Gas Industry? B y Katie Horner | Comments (8) Before working for an upstream oil company, I was under the impression that chemical engineers working in oil and gas belonged in pipeline and downstream operations. For those of you not in the industry, most large, integrated oil companies consist of an upstream organization and a downstream organization. The former focuses on exploration and production and the latter refines crude petroleum into usable products (gasoline, lubricants, etc. . Within upstream, processes and departments are often separated by subsurface work and surface facility work. Generally, most ChemE’s in upstream are found on the facility side, managing projects related to tanks, pumps, pipelines and separators. Pumping Unit in Bakersfield, Ca You may be asking, what about subsurface? And, can chemical engineers contribute to a traditionally petroleum engineering realm? The answer is, most definitely! A reservoir is essentially a large tank filled with porous media and reser voir fluids – oil, gas and water.In order to recover oil or gas from a reservoir, chemical engineering fundamentals such as fluid mechanics, thermodynamics and heat transfer must be understood and applied. Petroleum engineering is not an exact science. Precise reservoir boundaries are often unknown, PVT samples are few and far between, recovery mechanisms are sometimes unclear, and original and current oil in place is determined probabilistically. The fact is, it wouldn’t be economical to collect all of the data to make it an exact science.Without having all of the data, oil companies still have been successful in recovering resources thus far. However, we’ve picked the low hanging fruit when it comes to oil and gas resources and are moving toward environments with increased complexity – heavy oil, challenging shale plays, tight gas, deepwater exploration, etc. It’s often said that the best place to find oil is within currently or previously produc ing reservoirs. As we go back in and try to capture the residual oil, chemical engineering concepts will be critical in designing processes to recover these resources.Many oil or gas recovery mechanisms are well understood, such as waterfloods or gas cap expansion. Fortunately for our profession, there are areas, such as steam and polymer floods, that still need the keen eyes of engineers to model and optimize. As we attempt to tackle the current global energy challenges, oil and gas will continue to be a key factor in the equation. While the focus of many chemical engineering graduates is in alternative energy solutions, there are still plenty of opportunities for a chemical engineer to make an impact in the world of upstream oil and gas.

Sixteen Candles essays

Sixteen Candles essays The 1986 film Sixteen Candles tells a timeless tale of growing up in suburban America. The films star, Sam, played by Molly Ringwald, wakes up with big expectations on her sweet sixteenth birthday only to be completely disappointed. Not only does she find that she looks exactly the same as when she was fifteen, but her family is so preoccupied with her older sisters wedding that they forget her birthday altogether. The film opens with Sam on the phone with her best girlfriend Randy. She is examining herself in her full length mirror and is totally horrified to find that her body didnt magically transform overnight. She was hoping to wake up with a body just like Carolines. Caroline is the head cheerleader, prom queen, and girlfriend of the most popular boy in school, Jake Ryan. Sam is hopelessly in love with Jake and is convinced that he wont know she exists until she is more developed, more mature, more like Caroline. Little does she know, Jake does notice her. He is intrigued by a certain mispassed note containing some very personal information about Sams sex life (or lack of one). In this note, Sam confesses that she is a virgin (she has never done it) and is saving herself not for marriage, but for Jake Ryan. Jake finds himself wanting to get to know Sam and wanting a real relationship with someone like her, rather than with someone like Caroline. He knows Caroline doesnt love him, and he doesnt love her either. The only real reason theyre together is because hes the most popular boy at school and shes the most popular girl. Throughout the movie, Sam is preoccupied with becoming more like Caroline, while the real reason Jake is interested in her is because she is not like Caroline. By the end of the movie Sam learns a valuable lesson about being her own person and even gets the guy along the way. ...

Dark Energy (Definition)

Dark Energy (Definition) Dark energy is a hypothetical form of energy that permeates space and exerts a negative pressure, which would have gravitational effects to account for the differences between the theoretical and observational results of gravitational effects on visible matter. Dark energy is not directly observed, but rather inferred from observations of gravitational interactions between astronomical objects. The term dark energy was coined by the theoretical cosmologist Michael S. Turner. Dark Energys Predecessor Before physicists knew about dark energy, a cosmological constant  was a feature of Einsteins original general relativity equations that caused the universe to be static. When it was realized the universe was expanding, the assumption was that the cosmological constant had a value of zeroan assumption that remained dominant among physicists and cosmologists for many years. Discovery of Dark Energy In 1998, two different teamsthe Supernova Cosmology Project and the High-z Supernova Search Teamboth failed at their goal of measuring the deceleration of the universes expansion. In fact, they measured not only a deceleration, but a totally unexpected acceleration  (Well, almost totally unexpected: Stephen Weinberg had once made such a prediction). Further evidence since 1998 has continued to support this finding, that distant regions of the universe are actually speeding up with respect to each other. Instead of a steady expansion, or a slowing expansion, the expansion rate is getting faster, which means that Einsteins original cosmological constant prediction manifests in todays theories in the form of dark energy. The latest findings indicate that over 70% of the universe is composed of dark energy. In fact, only about 4% is believed to be made up of ordinary, visible matter. Figuring out more details about the physical nature of dark energy is one of the major theoretical and observational goals of modern cosmologists. Also Known As: vacuum energy, vacuum pressure, negative pressure, cosmological constant

Management Essays by

Management Essays by What Is a Management Essay? The notion of management essay can be easily derived from the separate notions of management (i.e. the art of administrating all business activities that allows an organization/individual to achieve set goals within a specified time frame and with limited resources) and essay (a short piece of writing that is elucidates a point or question and may or may not include authors personal viewpoint). So, summing up a management essay can be understood as a piece of writing dedicated to one of the key management elements: planning, organizing, staffing, leading and controlling an organization. Management essays may be of two types: 1. Objective (particularly, when a scholarly investigation is required). 2. Subjective (providing students solution or explaining their understanding of a key issue required). Looking for an essay on management? Here is a good management essay example: Difference  between  Leadership and Management Management Paper Structure A management essay takes a structure that is generally prescribed to 5 paragraph essays: it must begin with an introductory sentence where a problem is introduced to the reader; then two or three body paragraphs should follow elaborating on the original question. The essay should be closed with a conclusion that echoes the introduction and sums up the body paragraphs. More information on essay structure: How to Write a Well-Structured Essay has a long history of completing essays on management for our customers. From day one has started receiving management related orders from its customers. This has led us to hire more management writers from various backgrounds and levels of expertise. Order your management essay from our professional writers.

Aviation - Eastern Airlines would need to have their business plan, Essay

Aviation - Eastern Airlines would need to have their business plan, marketing, route network, aircraft fleet or anything else look like what in order to be successful in todays current aviation market - Essay Example In this context, the example of Eastern Airlines can be taken into consideration. Eastern Airlines was one of the major airline companies based in the USA. The company was founded in the 1926 and headquartered at Miami International Airport, Florida. The company ceased its operations in January 18, 1991. The paper is directed to reveal some of critical reasons due to which Eastern Airlines had to be out of the business. For this purpose, the paper throws light of conditions of different crucial operations of airlines such as route structure, fleet, marketing, and business plan when they went out of business. In addition to this, the paper also provides some of the crucial recommendations to the company regarding different areas of business operations, which can allow the company to remain in the competition. There were a number of different reasons behind the demise of Eastern Airlines. Ill-business operations were one of them. There were several shortfalls in the different areas of its operations such as route structure, fleet, marketing, and business plan. From the perspective of route structure of the company, the span of business activities of the company was quite restricted. The international business of the company was not quite developed as it was expanded in Mexico, Caribbean and Canada only. The major hubs of the company were Charlotte/Douglas International Airport, Hartsfield Atlanta International Airport, John F. Kennedy International Airport (New York City), Kansas City International Airport, LaGuardia Airport (New York City), Luis Muà ±oz Marà ­n International Airport (San Juan), and Miami International Airport. In this way, the route structure of the company at time when they had to cease their operations was quite centered in the North American continent. The lack of international flights was major aspects of the business operations of the organization that made the company worthless for international passengers and tourists

CURRENT EVENT ARTICLE REVIEW ON ANYTHING FINANCE RELATED Essay

CURRENT EVENT ARTICLE REVIEW ON ANYTHING FINANCE RELATED - Essay Example The survey also found that consumer IT, health-care IT and business IT remaining as favorites for VCs to increase their investment dollars at the expense of clean technology, medical-device and biopharmaceutical companies. The paradox though is that both CEOs and VCs are optimistic about the increase in value of their investments (Basich, 2011). To adequately analyze this article it is first important to understand the role of Venture Capital for the startup businesses. Venture Capital is a finance that is provided to startups that are too small to raise the capital they need, either through bank loans or otherwise but that have a high potential for growth. The numerous benefits of VC is summarised by research conducted by Puri and Zarutskie (2010) as follows: VC-financed firms typically grow more quickly, tend to grow bigger and are less likely to fail in comparison to non-VC-financed firms. When we place this into the current American context it means that VC-financing would be critical for rapid job creation due to their typical quicker growth rate and given that most employees are employed by small businesses such as these rising startups. The higher potential for massive growth also portends well for the US economy as it predicts higher wages associated with large companies that will translate to higher disposable in comes and thus increased consumer spending. Finally, the decreased likelihood of failure of VC-financed firms is vital for increasing investor confidence in the US market in general and entrepreneurship in particular. The article’s prediction of decreased venture investing in 2012 therefore robs the US of the hope of quick recovery from its slow economy. Venture investing is often accompanied by other support services such as increased management capacity, industry networking and so on which are equally critical in supporting the growth of startups.

Effects of Globalization on Regional Security Essay

Effects of Globalization on Regional Security - Essay Example Communication and transportation have acted as a catalyst for globalization. Communication has enabled increased connectivity among the global communities. Transportation has facilitated physical movement of people and merchandise. This has resulted in a globe connected in a dynamic manner (McGrew ND). Therefore, an event that occurs in one part of the globe will have repercussion on other countries. A terrorist attack will lead to global panic since it may lead to closure of many airports internationally. Similarly, the plummeting of the value of stocks in one of the key security market may culminate in similar occurrences in other markets. This write up will evaluate the impact of globalization on the regional security across the globe (Bardhan 2005, p. 50). Southern Sudan conflict Globalization has numerous consequences, which include an increase in international trade, weapons proliferation and cultural exchange. Some of these impacts of this phenomenon have affected regional sec urity in some regions globally (McGrew ND). This write up will highlight some of those examples across the globe. The discovery of crude oil in Southern Sudan brought excitement to that nation. Consequently, leading nations across the globe wanted to partake in the exploitation of this resource. However, the Southern Sudanese community felt short-changed in the sharing of the resultant funds. This resulted in a 21-year conflict in the southern part of Sudan. The conflict required constant supply of weapons to sustain the conflict. Globalization has culminated in the proliferation of weaponry globally. Consequently, there are unscrupulous individuals who trade in this kind of arsenal. Rebels do not buy weaponry legally consequently; they seek black-market traders. These traders denote proliferation of weapons since they work under no regulations hence; they sell to any client that pays the right price for such artillery. Therefore, the proliferation of weapons was a factor, which fue lled the Southern Sudan conflict, which lasted over two decades with massive human casualty. The proliferation of weapon resulted directly from globalization. Consequently, this conflict was a directly related to globalization. This conflict had a massive impact on regional peace in East Africa and resulted in instability in other parts of the region such as northern Uganda. Weapon acquired from the black market were vital since they ensured that the rebels could sustain the conflict (Stiglitz 2002, p. 90). Niger delta conflict Nigeria has massive oil reserve in the Niger delta, which is a mangrove. The oil generates massive foreign currency, which has contributed massively to that nation’s wealth. Foreign companies are involved in the exploitation of oil in that nation which resulted in rebels attacks in the areas. The rebels claim that the central government is not apportioning the resultant resources appropriately. Globalization has allowed international companies to inves t in various nations across the globe. This kind of investment denotes the impacts of globalization on the economic front. However, this has culminated in the rise of a conflict. The rebels claim that the companies, which exploit oil, fail to invest the massive returns in the delta region. Globalization has resulted in the elite global companies being the chief beneficiary of the

Patents and Trade Secrets Assignment Example | Topics and Well Written Essays - 250 words

Patents and Trade Secrets - Assignment Example Targeted advertising is the most seen part during internet surfing. These advertisements can see connectedly to the user’s visited pages. Targeted advertising means unlimited reach to the internet users. This behavior is useful for the companies but an alarming sign for the users through privacy interventions (Belleflamme, 2013). The trackers are using different ways to see the privacy of any internet user, such as location tracking option. It is the easiest way for hackers to reach the target people on Facebook or google. It is sad to know that the technology is making our lives unsafe (Myhere, 2013). The internet users should be careful by not storing their important data on online devices to avoid the invading of their privacy. Another source of protection is to save the important files through old-fashioned techniques, e.g. deposit box etc. rather than depending on the new unproductive technologies (Myhere, 2013). Google has introduced ‘tool settings’ to block the advertisements by signing in to account, which is a useful option for the internet

Road Traffic Accident Essay Example | Topics and Well Written Essays - 2750 words - 1

Road Traffic Accident - Essay Example The case that is undertaken for discussion is a 26 year old pedestrian male who is struck by a high speeding car, consequent to which he suffers multiple injuries which includes deep 5cm laceration on his forehead, sharp pain in the back of the neck, unequal chest movements, abdominal injury and injury to the pelvis and right femur. The 3 main injuries which will be specifically discussed are the sharp pain in the back of the neck, unequal chest movement and injury to the pelvis. The sharp pain in the back of the neck of the patient hints at probable spine injury at that area. Most cervical spine fractures occur predominantly at 2 levels -at the level of C2 and at the level of C6 or C7 (Mueller 2006). The normal cervical spine has 3 distinct columns- the anterior column is composed of anterior longitudinal ligament and the anterior 2/3rds of the vertebral bodies, the middle column consists of the posterior longitudinal ligament and the posterior 1/3rd of the vertebral bodies, the annulus and intervertebral discs and the posterior column includes all of the bony elements formed by the pedicles, transverse processes, articulating facets, laminae, and spinous processes. Disruption of more than one of these columns makes the spine move as 2 separate units, increasing the likelihood of spinal cord injury. Based on the mechanism of injury, cervical spine injury can be classified into flexion, flexion-rotation, extension, extension-rotation, vertical compression, la teral flexion, and imprecisely understood mechanisms (Mueller 2006). The patient can present with the spinal shock which manifests as flaccidity, areflexia, loss of anal sphincter tone, fetal incontinence, priapism and loss of bulbocavernosus reflex. He can also manifest with signs of neurogenic shock like hypotension, paradoxical bradycardia and flushed but dry skin.  Ã‚        The 26-year-old man who met with a road traffic accident was admitted with multiple injuries involving the spine, chest and the pelvis. He was managed in the intensive care with continuous monitoring of his vital signs. He was given appropriate fluids, electrolytes, blood transfusion, diet, and medications. The main concern in this patient is the prognosis of spinal cord injury which is poor. Hence it is obvious that the patient and his family members are worried about his prospects in terms of recovery, day-to-day activities, employment, social and married life. The patient and the relatives will need emotional and psychological help before discharge. In addition, the patient will also need rehabilitation measures including physiotherapy and occupational therapy.            

Snakebites Research Paper Example | Topics and Well Written Essays - 250 words

Snakebites - Research Paper Example The use of these anti-venoms has become an effective cure for the infected body and these anti-venoms enable the victims to get back to life within 4-5 hours of the attack by minimizing effect of the poison. This method has proved to be quite successful in minimizing the death of people who suffer from this fatality (Shorter, 1999). In fact, the number of deaths occurring from snakebites has now become almost rare. These anti-venoms go through many processes, such as purification process to ensure their effectiveness. Even then, they may contain certain serums and proteins, which tend to have a reverse reaction on a person that is the reason of extreme supervision required during this method. There are several kinds of snakes’ anti-venoms present globally. Some of them are the â€Å"Tiger snake, Brown snake, Taipan, Black snake, Death adder, Sea snakes, etc† (Shorter, 1999). The proper supervision of these anti-venoms helps keep a check on the patient and prevent the po ison from completely spreading in the body. If the attacking snake is properly identifiable, it makes it easier for the experts to treat the poison accordingly, and it also make it easier for the patient to recover depending on intensity of the bite.

Road Traffic Accident Essay Example | Topics and Well Written Essays - 2750 words - 1

Road Traffic Accident - Essay Example The case that is undertaken for discussion is a 26 year old pedestrian male who is struck by a high speeding car, consequent to which he suffers multiple injuries which includes deep 5cm laceration on his forehead, sharp pain in the back of the neck, unequal chest movements, abdominal injury and injury to the pelvis and right femur. The 3 main injuries which will be specifically discussed are the sharp pain in the back of the neck, unequal chest movement and injury to the pelvis. The sharp pain in the back of the neck of the patient hints at probable spine injury at that area. Most cervical spine fractures occur predominantly at 2 levels -at the level of C2 and at the level of C6 or C7 (Mueller 2006). The normal cervical spine has 3 distinct columns- the anterior column is composed of anterior longitudinal ligament and the anterior 2/3rds of the vertebral bodies, the middle column consists of the posterior longitudinal ligament and the posterior 1/3rd of the vertebral bodies, the annulus and intervertebral discs and the posterior column includes all of the bony elements formed by the pedicles, transverse processes, articulating facets, laminae, and spinous processes. Disruption of more than one of these columns makes the spine move as 2 separate units, increasing the likelihood of spinal cord injury. Based on the mechanism of injury, cervical spine injury can be classified into flexion, flexion-rotation, extension, extension-rotation, vertical compression, la teral flexion, and imprecisely understood mechanisms (Mueller 2006). The patient can present with the spinal shock which manifests as flaccidity, areflexia, loss of anal sphincter tone, fetal incontinence, priapism and loss of bulbocavernosus reflex. He can also manifest with signs of neurogenic shock like hypotension, paradoxical bradycardia and flushed but dry skin.  Ã‚        The 26-year-old man who met with a road traffic accident was admitted with multiple injuries involving the spine, chest and the pelvis. He was managed in the intensive care with continuous monitoring of his vital signs. He was given appropriate fluids, electrolytes, blood transfusion, diet, and medications. The main concern in this patient is the prognosis of spinal cord injury which is poor. Hence it is obvious that the patient and his family members are worried about his prospects in terms of recovery, day-to-day activities, employment, social and married life. The patient and the relatives will need emotional and psychological help before discharge. In addition, the patient will also need rehabilitation measures including physiotherapy and occupational therapy.            

Health Care Reform Essay Example | Topics and Well Written Essays - 1000 words - 1

Health Care Reform - Essay Example The subsidies will enable the low earners to purchase private health cover. The legal provision also creates room for the development of exchanges for individuals that will be willing to buy cover. The bill also expands accessibility to health insurance by prohibiting insurance firms from denying cover to anyone based on pre-existent conditions (Andrews, 2012). The bill also makes provisions for the creation of an experts’ panel to limit reimbursements to only effective treatments and offer incentives to providers as a way to persuade them to â€Å"bundle† their services instead of charging by singled out procedures (Andrews, 2012). The reform redefines the way Americans purchase health cover by requiring all Americans without employee cover to buy privately provided health insurance cover or pay a tax percentage of 1%-2.5% (Andrews, 2012). Americans that cannot afford cover and do not have one from employers will either go into Medicaid/Medicare or receive tax credits to make the private purchases. Financing of the Affordable Care Act The Affordable Care act will be funded through government funding, taxes and budget cuts. The taxes will include 9% from Medicare and unearned income tax on earnings above $250 000. The taxes will be levied on a sliding scale. Therefore, the more one earns, the higher the taxation. Insurance companies and employers with over 50 fulltime employees will be taxed to fund the plan. Taxes will also be implemented for medical supply companies and pharmaceuticals. Modest estimates by the non-partisan congressional budget office from 2010 showed that health insurance companies would pay $2 billion, medical suppliers would pay $2.3 billion, and pharmaceuticals would pay $2 billion by 2011, and this amount was expected to go up to $10 billion by 2017. Reduction on wasteful spending is also expected to contribute to the budget. The taxes designated. The efficiency that the bill introduces is also expected to cut costs and incr ease efficiency, and therefore; contribute to the offset of the costs incurred. Influence in Legislation and Policy Making The policies relating to Federal changes on pre-existent conditions’ coverage in insurance made a significant part of the overarching initiative, which led to the legislation of the act. Generally, Democrats, liberals and physicians were supportive and still persist in supporting health reforms related to this major reform (Harrison & Gerard, 2010). On the other hand, insurance companies, Republicans and conservatives were opposed to the reform proposal, and they have been actively seeking to repeal on the act that contains the reform details. The opposing forces are still actively attempting to change the main elements of the act. In the initial stages of the proposed reforms, the public was overwhelmingly supportive based on statistics from public opinion polls. However, currently the public is fairly split on the issues surrounding the reform process ( Harrison & Gerard, 2010). Notably, there were also various advocacy organizations that supported the legislation of the act. These included the â€Å"American Association of Retired Persons† (AARP) (Roy, 2012). The Potential Effects of the Affordable Care Act on the Economy The act presents a number of welcome economic effects as well as some unwelcome economic e

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