Thursday, December 26, 2019

Essay on Arguments for and against Lowering the Drinking Age

The controversy on the proper drinking age is one that has been repeatedly discussed and researched over the years. Its common to hear the argument â€Å"If someone is old enough to take a bullet for their country, they should be allowed to drink alcohol.† But is that enough justification? Some would say no. â€Å"According to the National Institute on Alcohol Abuse and Alcoholism (NIAAA) it is estimated that in 2004 there were more than 1,700 student deaths, 599,000 injuries, and 696,000 assaults annually associated with excessive drinking† (Fennell 247). Given these numbers, would lowering the drinking age really be the best thing for America’s youth? In fact, the state and federal laws for consuming alcohol are different. â€Å"The federal law†¦show more content†¦Was it a good decision to choose 21 as the legal drinking age? Research shows that â€Å"alcohol consumption, heavy drinking, and daily alcohol use has declined among young adults age 18-2 0 since the 1980s† (Wechsler). Along with the decline in consumption, the percentage of alcohol-related traffic fatalities has also dramatically declined from the 1980s until 1997 when it leveled off. (Wechsler). (Wechsler). Despite the positive results that have come about since this law was passed, it is pretty clear that it has not prevented people under 21 from obtaining and drinking alcohol. College campuses across the country report problems with students binge drinking and students who drink deliberately to get drunk (Smith). â€Å"Today, the generally accepted definition of binge drinking is the consumption of five or more drinks in a row at least once in the past 2 weeks† (Binge). Some of the reasons given for binge drinking range from being curious to trying to escape from the ever-present stress in our lives. The latter reason is more prevalent in college students. â€Å"College students are more likely to engage in heavy drinking than their peers who do not attend college with 2 in 5 students nationally engaging in binge drinking on at least 1 occasion in the past 2 weeks† (Wechsler). Some believe that making the drinking age 21 is one cause of the currentShow MoreRelatedThe Leg al Drinking Age Should Be During The United States1387 Words   |  6 Pagesdebate about what the legal drinking age should be in the United States has been ubiquitous. People of all ages advocate both for and against lowering the age, and some people do not even have an opinion. What has led people to their specific convictions, and what facts do they possess that support these convictions? In the following paragraphs, this popular controversy will be addressed as each position is thoroughly analyzed. Many people today advocate for the drinking age to remain where it is, suchRead MoreMinimum Legal Drinking Agre1173 Words   |  5 PagesThe legal age of adulthood in the United States for most purposes is 18. At the age of 18, a person enters the realm of adulthood and is assigned the rights and responsibilities associated with this legal status. For example, an 18 year old can legally sign a contract and is bound by the terms and conditions of the contract. An 18 year old can marry without parental consent, serve on a jury, and vote in state and federal elections. An 18 year old who is charged with a crime is not tried in theRead MoreShould the Drinking Age be Lowered in the United States?1017 Words   |  5 Pagesdebate on the drinking age? The United States can take a look at other States such as: Germany, the Netherlands and France, and see how successful they are. Many teenagers would jump on the bandwagon of lowering the drinking age just because they want to have the a bility to drink, the argument of being able to die for the United States but can’t drink and it would take away the â€Å"Forbidden Fruit† of drinking. Much of the older generations would jump on the bandwagon of keeping the drinking age the sameRead MoreDrinking at 18 Essay1243 Words   |  5 PagesDrinking at 18 If you look around at college parties it seems as if everyone is drinking. Actually you are probably right, but over half of those people drinking are also under the legal drinking age. Drinking is one of the main forms of entertainment for the typical college student. The only problem with drinking being the main form of entertainment is that half of the students in college or 20 years or younger. This seems to be a problem all over theRead MoreThe Drinking Age Of The United States Should Be Lowered929 Words   |  4 PagesAn argument that many tend to dispute today, whether the drinking age of the United States should be lowered from 21 to 18. The drinking age for people to drink alcoholic beverages was made into law by the National Minimum Drinking Age Act. This ant enforced all states to raise their legal drinking age to 21. To get this law pass, the congress tried to strongarm the states, if the states did not comply, the government would take away their highway funds. Both arg uments for it to be lowered and toRead MoreFavors for and Against Lowering the Legal Drinking Age771 Words   |  4 PagesFactors In Favor of and Against Khimley Young Critical Thinking and Problem Solving/ Hum 200 AOS Instructor Dr. Steven Mathews October 24, 2012 Lowering the Legal Drinking Age to 18: Yea or Nay Argument in Favor of Lowering the Legal Drinking Age The age of 18 is a transitional point in life. An 18-year=old can vote, marry, enlist in the military and buy cigarettes. To some it’s absurd that an 18-year-old can vote politicians into office and fight wars for the country but cannotRead MoreLowering the Drinking Age1523 Words   |  7 Pages According to Andrew Herman, â€Å"Each year, 14,000 die from drinking too much. 600,000 are victims of alcohol related physical assault and 17,000 are a result of drunken driving deaths, many being innocent bystanders† (470). These massive numbers bring about an important realization: alcohol is a huge issue in America today. Although the problem is evident in Americans of all ages, the biggest issue is present in young adults and teens. In fact, teens begin to feel the effects of alcohol twice asRead MoreThe Legal Drinking Age ( Mlda )1428 Words   |  6 Pagesthem? Since 1984 the legal drinking age has been 21, but that hasn’t stopped many of the younger adults, ag es 18-20, from partying with their friends, and consuming alcoholic beverages. It has lasted over the years as a popular way to rebel against parents, or other authority. Alcohol has a sort of â€Å"forbidden fruit† quality for young adults and teens because it is made out to be such a big deal by the government. Lowering it will get rid of this quality. The drinking age is set too high and I believeRead MoreNot Lowering the Drinking Age1642 Words   |  7 Pages28 July 2011 Not Lowering the Drinking Age Many teenage deaths in the United States are caused in some way by the influence of alcohol; however, many people still believe that the legal drinking age should be reduced to eighteen. This issue has been going on for years, but the law has not been changed since the change to twenty-one in 1980. States have become stricter about preventing under-age drinking, but teenagers have no problem getting alcohol. There are many arguments in favor of changingRead MoreThe Drinking Age Should Be Legal974 Words   |  4 PagesThe Drinking Age For many reasons, the drinking age has been set at twenty-one years old, but has the time come to lower the drinking age? Many argue that the drinking age needs to be lowered back to eighteen for many reasons; however, studies and statistics show that lowering the drinking age is harmful and even deadly. Some people believe that binge drinking can be solved by lowering the drinking age, but lowering the drinking age is not the solution to binge drinking. Many teenagers spend their

Wednesday, December 18, 2019

Evaluation Of Animal Farm By George Orwell - 1101 Words

Animal Farm by George Orwell The book Animal Farm by George Orwell was first published in 1945, and the context when it was written plays a huge role in shaping it. It was after the Russian Revolution in which we see Joseph Stalin became the new leader of the Soviet Union. Stalin’s idea and leadership clearly doesn’t impress George Orwell, as this book shows the dark side of Stalin’s system through a group of fictional characters, the animals. Not only that, the book was also able to foresee the situation every government in the world is facing: the lethal side of power. The plot of the book is pretty straightforward; it resembles the Russian Revolution and the aftermath of that event a lot. Starting with a barn called Manor Farm where†¦show more content†¦At the meeting about the project, Snowball has a great and passionate speech about his vision, but when it’s Napoleon’s turn to speak, he signals nine attack dogs to chase Snowball off the farm. He then declares his leadership and declares that the pigs will make all the decisions, no more meeting required. He also uses Snowball as a scapegoat for everything bad that happens to them. As time goes on, Napoleon and the pigs behave more and more like humans: they move into Mr. Jones’ house and live in prosperity while the other animals have less and less food. As more of the Seven Commandments are broken by the pigs, the commandments are slightly changed so the pigs technically don’t break any of them. As the years pass, Napoleon purchases more land from a neighbor ing farmer, Mr. Pilkington. Life for all animals is harsh, except for the pigs. Eventually, the pigs start walking on two legs and behave like a human. The Seven Commandments are later reduced to a single law: All Animals Are Equal / But Some Are More Equal Than Others. As we connect the book to Russia in the post-Russian Revolution era, we find the resemblance between the fictional and historical character. The boar Old Major is the representation of the Marx-Lenin idea of a better life after the suffering they’ve had under Mr. Jones, who represents Tsar Nicholas II (the last Tsar of Russia). Napoleon is the â€Å"necessary† leader toShow MoreRelatedAnimal Farm by George Orwell1175 Words   |  5 PagesAn enthusiastic participant in the Spanish civil war in 1936, George Orwell had a great understanding of the political world and made his strong opinions known through his enlightening literary works, many of which are still read in our modern era. Inspired by the 1917 Russian Revolution and the failed society it resulted in, Animal Farm by George Orwell is an encapsulating tale that epitomises how a free utopian society so idealistic can never be accomplished. The novella exemplifies how influencesRead MoreGeorge Orwell s Animal Fa rm1496 Words   |  6 Pages Introduction In a perfect world, everybody is equal. People s race, gender, culture, intelligence wouldn’t matter everyone would be the same. Sadly this is not a perfect world and in George Orwell s novel Animal Farm he explores the reason total equality is nearly impossible to obtain. George Orwell was born Eric Arthur Blair on June 21, 1903 in Motihari, India to a British civil servant. He started to write at a young age publishing his first poem in a newspaper at the age of eleven. InRead MoreReview Of George Orwell s The Road 1923 Words   |  8 PagesReview on George Orwell – The Road to Wigan Pier Course – BA Hons (With foundation) Community studies. Health, youth, and community Module – Reading Modern Society Tutor – Wendy Bateman Student ID – 1608296 Submission Date – Tuesday 6th December 2016 Describe and illustrate an informed opinion based on research and analysis of evidence Analyse information, experiences, and article reasoned arguments through reflection, review and evaluation. Demonstrate an introductoryRead MoreCritical Analysis and Evaluation of 1984, by George Orwell.1487 Words   |  6 PagesGeorge Orwell 1984 The New American Library Copyright 1961 George Orwell George Orwell, whose real name was Eric Blair, was born in Bengal, India, in 1903. When he was eight years old, as it was customary, his mother brought him back to England to be educated. He was sent to a boarding school on the south coast, a school whose students were sons of the upper class. He was allowed in with lower tuition and not being from a wealthy background, he was subject to snobbery of the others at the schoolRead More Shooting An Elephant Essay1374 Words   |  6 Pages The story that my evaluation will be based on is Shooting an Elephant written in 1936. The author George Orwell was born in 1903 in India to a British officer raised in England. He attended Eton College, which introduced him to England’s middle and upper classes. He was denied a scholarship, which led him to become a police officer for the Indian Imperial in 1922. He served in Burma until resigning in 1927 due to the lack of respect for the justice of British Imperialism in Burma and India. HeRead MoreCritical Review of Animal Farm2575 Words   |  11 Pagesâ€Å"Animal Farm† Bibliography: Orwell, George. â€Å"Animal Farm.† New York: Penguin Books Ltd, 1989 Introduction and Summary: Animal farm is an animal fable with a deliberate purpose. It is very realistic about society and its politics.  There are a number of conflicts in Animal Farm: the animals versus Mr. Jones, Snowball versus Napoleon, the common animals versus the pigs, Animal Farm versus the neighbouring humans, but all of them are expressions of the underlying tension between the oppressorsRead MoreThe Purpose of a Justice System1828 Words   |  8 Pagesoppression of the individuals. For example, in George Orwell’s classic novel Animal Farm, the act of rebellion that can diminish growth and maturity when individuals seek justice is evident through Mr. Jones, Napoleon, Snowball, Squealer, and Mollie. Mr. Jones owns Manor Farm and is a hard master to his animals. Napoleon, Snowball, and Squealer are three pigs that live on Manor Farm. Mollie is a foolish white mare, who enjoys the attention of other animals. In a similar fashion, in Guy Vanderhaeghe’sRead MoreGrammar: Figures of Speec h5410 Words   |  22 Pagesliteral meaning. In some allegories, for example, an author may intend the characters to personify an abstraction lie hope or freedom. The allegorical meaning usually deals with moral truth or a generalization about human existence. Ex. â€Å"Animal Farm† George Orwell Alliteration - The repetition of sounds, especially initial consonants in tow or more neighboring words (as in â€Å"she sells sea shells). Although the term is not used frequently in the multiple-choice section, you can look for alliterationRead MoreHistory of Social Work18530 Words   |  75 Pages.........................................................................28 Mary Richmond.......................................................................................................................................................29 George Orwell, John Howard Griffin, Pat Moore, Tolly Toynbee, Gà ¼nther Wallraff, Barbara Ehrenreich ............30 Sir William Beveridge .........................................................................................................................Read MoreANALIZ TEXT INTERPRETATION AND ANALYSIS28843 Words   |  116 Pagesmoral qualities. Second, we are concerned with the techniques an author uses to create, develop, and present chara cters to the reader. Third, we are concerned with whether the characters so presented are credible and convincing. Naturally, such an evaluation can only take place within the context of the work as a whole, which inevitably links character to the other elements of fiction. Characters in Fiction The term character applies to any individual in a literary work. For purposes of analysis, characters

Tuesday, December 10, 2019

Revolution Essay Example For Students

Revolution Essay RevolutionDestruction of statues, screaming in the streets, rash actions, hasty decisions, and adrenaline-influenced outbursts. Prim and proper, fancy meetings, organized schedules, time for tea, and the thought of perfection. Total opposites are bound to clash at sometime or another, and for America, that time was now. The movie Revolution shows us movingly and realistically how the Revolutionary War was led up to, how the years of battles continued, and how finally victory was attained. Poor King George III had no idea what hit him. All of the colonies now had their own governments to lean on, their own Declaration of Independence already being passed out among the people, and their own volunteer army. The famous Liberty or Death was their cry. No more repression for the people of America, they believed that God was on their side, and it was time for freedom. When the battles began, first they were tiny squabbles that were simple and not messy. But as time passed, so did being civilized, anything and everything that would hurt the enemy in any way that could be done, was done. For that reason, on top of others, hospitals were needed. They were lacking in every department except for amputation. But since sanitary conditions were impossible, almost all had their wounds infected, and would die from the aftermath of that. During battles there was always a flag present. No matter what happened, there was always someone carrying the flag. As soon as someone would get take n out, another would run and pick it up to show that you just cant keep a good man down. Also, when people traveled in and out of battle areas, they would need flags to show their business and whom they supported. For example, if a wagon came in that brought rations for the colonists, they would first need to pay a toll, have an American flag, and a white flag to show they werent in battle. But usually, and unfortunately, in the heat of madness and testosterone, those battle codes were not heeded. The fighting tactics were primitive. It usually was to make the other army move back a smidgen, take a break, then try again to kill, kill, kill. For the British, when a battle was won over a colony, they would parade through the streets with the remaining soldiers, the wounded and captured American soldiers. Any person who didnt support the British was taken prisoner and was used as examples for the others. They would have no privileges, no rights, so the British would use them for anythi ng, to kill as entertainment, become a servant of some kind, or even be the fox in a jolly-good time of hound hunting. While speaking of hunting, Native Americans also played a great part in the Revolutionary War. Either the British or the Americans hired them. They were asked to help track and kill enemies or escaped prisoners and also were excellent scouts. It may have taken a while, but another ally of the Americans was the French. With both helping, the optimum outcome was achieved. In the end of our war, America celebrated her victories with packed streets and happy returns. The dreams of the colonists, a place to be, no one to bow down to, you can say what you feel, you can make a home for your family, they had come true. A fitting and appropriate quote that the movie embodied, Thats the idea of America a revolution.

Monday, December 2, 2019

Its The Weekend, You Have Nothing To Do So You Decide To Essays

It's the weekend, you have nothing to do so you decide to play around on your computer. You turn it on and then start up, you start calling people with your modem, connecting to another world, with people just like you at a button press away. This is all fine but what happens when you start getting into other peoples computer files. Then it becomes a crime, but what is a computer crime really, obviously it involves the use of a computer but what are these crimes. Well they are: Hacking, Phreaking, & Software Piracy. To begin I will start with Hacking, what is hacking. Hacking is basically using your computer to "Hack" your way into another. They use programs called scanners which randomly dials numbers any generating tones or carriers are recorded. These numbers are looked at by hackers and then used again, when the hacker calls up the number and gets on he's presented with a logon prompt, this is where the hacking really begins, the hacker tries to bypass this anyway he knows how to and tries to gain access to the system. Why do they do it, well lets go to a book and see "Avid young computer hackers in their preteens and teens are frequently involved in computer crimes that take the form of trespassing, invasion of privacy, or vandalism. Quite often they are mearly out for a fun and games evening, and they get entangled in the illegal use of their machines without realizing the full import of what they are doing" , I have a hard time believing that so lets see what a "hacker" has to say about what he does "Just as they were enthraled with their pursuit of information, so are we. The thrill of the hack is not in breaking the law, it's in the pursuit and capture of knowledge." , as you can see the "hacker" doesn't go out to do destroy things although some do. It's in the pursuit of knowledge. Of course this is still against the law. But where did all of this start, MIT is where hacking started the people there would learn and explore computer systems all around the world. In the views of professional hacking is like drugs or any other addictive substance, it's an addiction for the mind and once started it's difficult to stop. This could be true, as hackers know what they are doing is wrong and they know odds are they will be caught. But as I mentioned some hackers are just above average criminals, using there skills to break in banks and other places where they can get money, or where they can destroy information. What a hacker does at a bank is take a few cents or even a few fractions of a cents from many different accounts this may seem like nothing but when all compiled can be alot. A stick up robber averages about $8,000 each "job", and he has to put his life and personal freedom on the line to do it while the computer hacker in the comfort of his own living room averages $500,000 a "job". As for people destroying information, this is for taking some one down, destruction of data could end a business which for some is very attractive. It can cost a company thousands of dollars to restore the damage done. Now that you have an understanding of what a "hacker" is, it time to move on to someone closely associates with a hacker. This is a Phreak, but what is that. For the answer we turn to the what is known as the "Official" Phreakers Manual "Phreak [fr'eek] 1. The action of using mischievous and mostly illegal ways in order to not pay for some sort of telecommunications bill, order, transfer, or other service. It often involves usage of highly illegal boxes and machines in order to defeat the security that is set up to avoid this sort of happening. [fr'eaking] v. 2. A person who uses the above methods of destruction and chaos in order to make a better life for all. A true phreaker will not go against his fellows or narc on people who have ragged on him or do anything termed to be dishonourable to phreaks. [fr'eek] n. 3. A certain code or dialup useful in the action of being a phreak. (Example: "I hacked a new metro phreak last night.")" The latter 2 ideas of what a phreak is, is rather weird. A Phreak like the hacker likes to explore and experiment, however his choice of exploring is not

Wednesday, November 27, 2019

Christmas in America and Russia

Christmas in America and Russia Free Online Research Papers The article (Celebrate! Holidays In The U.S.A. Christmas Day tells us about how Americans celebrate Christmas holidays and what it means to them. Christmas in America is a very important holiday, especially for children who usually await the 25 of December with great excitement. Kids hang sock all around the house and Santa Claus comes down the chimney in the night and leaves presents and candy in them. On December 24, Christmas Eve, people have Christmas dinner which includes turkey potatoes, pie and lots of deserts. Christmas, while remaining one of the main Christian holidays in Russia, is celebrated on the 7th of January by the Russian Orthodox calendar, but not on the 25th of December. Traditionally, these the girls were telling fortunes, sang mysterious songs, children listened to Christmas fairy tales, frightening stories. Fortune-telling is is still very popular among young ladies as well as in old times both in cities and in the country. The most popular kind of fortune-telling involves the future husband appearing in the mirror. But still, nowadays the 7th of January is more of a religious holiday than a national one and isn’t usually even celebrated by non religious people. There is much more similarity between Catholic Christmas and New Year in Russia. Russians celebrate New Year twice: on the 1st of January, according to the New style calendar, and on the 14th of January, Old style. But the main fun is on the 31st of December. Usually Russians give each other presents on New Years and celebrate all night long, having champagne, the â€Å"Olivie† salad and the TV on with all the different New Year shows along with many singers and TV stars. There is also â€Å"Ded Moroz†, the Russian Santa Claus. But he, unlike Santa, does not use the chimney and does’t usually visit people’s homes in the night. He comes – along with his granddaughter Snegurochka to special parties called â€Å"Iolka† (Christmas tree) which can be held in schools, kinder gardens or just organize in some other places like theatres, so people cam just buy tickets and take their children to see Ded Moroz. All in all we can say, that Christmas is celebrated very differently in Russia and America. Both of the countries have their long going traditions, but, nevertheless, both countries have lots of magical fun and get lots of presents during the winter holidays, no matter how they are called and when exactly they are celebrated. Research Papers on Christmas in America and RussiaLifes What IfsAssess the importance of Nationalism 1815-1850 EuropeNever Been Kicked Out of a Place This NiceHarry Potter and the Deathly Hallows EssayPersonal Experience with Teen PregnancyHip-Hop is ArtThe Effects of Illegal ImmigrationEffects of Television Violence on ChildrenPETSTEL analysis of IndiaRelationship between Media Coverage and Social and

Saturday, November 23, 2019

Biography of James Hutton, Founder of Modern Geology

Biography of James Hutton, Founder of Modern Geology James Hutton (June 3, 1726–March 26, 1797) was a Scottish doctor and geologist who had ideas about the formation of the Earth that became known as Uniformitarianism. Although not an accredited geologist, he spent much time hypothesizing that the Earths processes and formation had been going on for eons and were continuing to the present. Charles Darwin was well-acquainted with Hutton’s ideas, which provided a framework for his work in biological evolution and natural selection. Fast Facts: James Hutton Known For: Founder of modern geologyBorn: June 3, 1726 in Edinburgh, United KingdomParents: William Hutton, Sarah BalfourDied: March 26, 1797 in Edinburgh, United KingdomEducation: University of Edinburgh, University of Paris, University of LeidenPublished Works: Theory of the EarthChildren: James Smeaton Hutton Early Life James Hutton was born on June 3, 1726, in Edinburgh, Scotland, one of five children born to William Hutton and Sarah Balfour. His father, who was a merchant and treasurer for the city of Edinburgh, died in 1729, when James was only 3 years old. He also lost an older brother at a very young age. His mother did not remarry and was able to raise Hutton and his three sisters on her own, thanks to the wealth his father had built before his death. When Hutton was old enough, his mother sent him to the High School of Edinburgh, where he discovered his love of chemistry and mathematics. Education At the young age of 14, Hutton was sent off to the University of Edinburgh to study Latin and other humanities courses. He was made the apprentice of a lawyer at age 17, but his employer did not believe that he was well-suited for a career in law. Hutton decided to become a physician to be able to continue his studies in chemistry. After three years in the medical program at the University of Edinburgh, Hutton finished his medical studies in Paris before receiving his degree from the University of Leiden in the Netherlands in 1749. Personal Life While studying medicine at the University of Edinburgh, Hutton fathered an illegitimate son with a woman who lived in the area. He named his son James Smeaton Hutton. Although he financially supported his son, who was raised by his mother, Hutton did not take an active role in raising the boy. Following the birth in 1747, Hutton moved to Paris to continue his medical studies. After finishing his degree, instead of moving back to Scotland, the young doctor practiced medicine in London for a few years. It is not known whether this move to London was prompted by the fact that his son was living in Edinburgh, but it is often assumed that is why he chose not to move back to Scotland. Soon, however, Hutton decided that practicing medicine was not for him. Before he had started his medical studies, Hutton and a partner had become interested in sal ammoniac, or ammonium chloride, a chemical used in making medicines as well as fertilizers and dyes. They developed an inexpensive method of manufacturing the chemical that became financially rewarding, enabling Hutton in the early 1750s to move to a large plot of land he had inherited from his father and become a farmer. Here he began to study geology and came up with some of his best-known ideas. By 1765, the farm and the sal ammoniac manufacturing company were providing enough income that he could give up farming and move to Edinburgh, where he could pursue his scientific interests. Geological Studies Hutton did not have a degree in geology, but his experiences on the farm gave him the focus to form theories about the formation of the Earth that were novel at the time. Hutton hypothesized that the interior of the Earth was very hot and that the processes that changed the Earth long ago were still at work millenniums later. He published his ideas in his book, The Theory of the Earth, in 1795. Hutton asserted in the book that life also followed this long-term pattern. The concepts in the book about life changing gradually by these same mechanisms since the beginning of time were in line with the principles of evolution well before Charles Darwin came up with his theory of natural selection. Huttons ideas drew much criticism from most geologists of his time, who followed a more religious line in their findings. The prevailing theory at the time of how rock formations had occurred on Earth was that they were a product of a series of catastrophes, such as the Great Flood, that accounted for the form and nature of an Earth that was thought to be only 6,000 years old. Hutton disagreed and was mocked for his anti-Biblical account of the Earths formation. He was working on a follow-up to the book when he died. Death James Hutton died in Edinburgh on March 26, 1797, at age 70 after suffering poor health and pain for a number of years caused by bladder stones. He was buried in Edinburgh’s Greyfriars Churchyard. He left no will, so his estate passed to his sister and, on her death, to Huttons grandchildren, the children of his son, James Smeaton Hutton. Legacy In 1830, geologist Charles Lyell rephrased and republished many of Huttons ideas in his book Principles of Geology and called them Uniformitarianism, which became a cornerstone of modern geology. Lyell was an acquaintance of Robert FitzRoy, captain of the  HMS Beagle  on Darwins voyages. FitzRoy gave Darwin a copy of  Principles of Geology, which Darwin studied as he traveled and collected data for his work. It was Lyells book, but Huttons ideas, that inspired Darwin to incorporate the concept of an ancient mechanism that had been at work since the beginning of the Earth in his own world-changing book, The Origin of the Species. Thus, Huttons concepts indirectly sparked the idea of natural selection for Darwin. Sources James Hutton: Scottish Geologist. Encyclopedia Brittanica.James Hutton: The Founder of Modern Geology. The American Museum of Natural History.James Hutton. Famous Scientists.

Thursday, November 21, 2019

Business Essay Example | Topics and Well Written Essays - 1250 words - 5

Business - Essay Example In simple terms, diversity can be viewed as difference. In the workplace, diversity is defined by Bell (2007) as attracting, recruiting and retaining persons from a wide talent base regardless of their religious affiliation, race, class, gender, national or ethnic origin, sexual orientation, age, disability, marital status and any other groupings. This ensures the organizations recruits individuals with a wide range of skills and from different economic, social and cultural backgrounds. There are often group conflicts in organizations that hinder a good working relationship and attainment of company objectives yet diversity is highly valued in organizations. The big question is, â€Å"how can diversity work for organizations?† To answer this question, this paper will compare and contrast various views from different authors regarding diversity in the workplace. The trends that have necessitated diversity will also be discussed. It will also evaluate the diversity in practice i n two organizations: HSBC and Wells Fargo. It will discuss the importance of diversity in the two companies. Some companies develop a diverse workforce to comply with laws but for other organizations, diversity is much more than just a policy as it is the key to success. Besides compliance, companies that have an inclusive workplace environment enjoy a lot of benefits which will be discussed later. The challenges facing implementation are also worth noting. Global Trends Various global demographic, economic and legislative trends have over the years necessitated the development of diversity in workplaces. According to Mor Barak (2011) there has been a workforce decline in various countries thus the need to engage immigrant workers to fill the employment gap. Italy workforce for example, is expected to decline from 60 million to 56 million by 2050 while that of Germany is expected to decline from 82 million to 69 million (P. 4). These are countries which have been known not to entert ain immigrant workers but the demographic trend forces them to. On the other hand, developing countries are faced with the problem of the youth who comprise more than half of the population (Kirton & Green, 2004). Since these economies are growing at a slow pace they cannot accommodate all those youths hence they look for jobs outside borders. Another trend is the growing number of women in the workforce and individualized migration to look for better opportunities without relying on their husbands or family (Bibard, 2011). This has changed the workforce dynamics to a great extent prompting the need to embrace diversity. Various legislations have also been emerging that press the companies to become diverse workplaces. These range from the universal declaration of human rights to the legislations on equal employment opportunity. Inclusive workplace programs have therefore, been instituted in many organizations to tap the benefits of a diverse workforce (Findler, Wind & Mor Barak, 20 07). Companies which do not know how to manage diversity in the workplace risk losing business due to high turnover, absenteeism, and low earnings. Managing diversity is not a simple task. On one hand companies need to appreciate the importance of diversity for organization success and on the other hand, diversity brings about group conflicts which can lead to disharmony and even violence in the workplace (Powell, 2004). Service companies rely so much on diverse customers thus needs to

Tuesday, November 19, 2019

Public Relations Plan for Merger and Acquisition Essay

Public Relations Plan for Merger and Acquisition - Essay Example Furthermore, a business may acquire another so that it expands its coverage of a given market, giving it a competitive edge over its competitors. Besides enabling a business to maintain and expand its grip on its customers or markets, mergers and acquisitions assist businesses to venture into new locations, thereby increasing productivity and profitability. Although they are closely related terms, merger and acquisition are two distinct concepts that should be easily distinguished from each other. In essence, acquisition refers to a scenario in which one business buys another business entity. Although any business may opt to merge with others, those seeking to expand their markets and those facing negative publicity due to their monopolistic characters, political influences or lack of public trust are found to be more likely to merge in order to survive in the competitive markets (Oliver, 2004). For such a company or a corporate organization, it is imperative that effective public re lations strategies, plans and campaigns are established, implemented, evaluated and reformed in cases where they are not effective enough. It is therefore essential that an elaborate public relations plan

Sunday, November 17, 2019

Universal rules of marketing Essay Example for Free

Universal rules of marketing Essay Describe any universal rules of marketing that might be applied to most products, markets, customers and situations.  All marketing activities have one thing in common and that is to give customers a reason to buy the companys product. One of the most important universal rules of marketing is that marketers need to find a way to break out of commodity status to meet customers needs better than competing firms. All organizations both for profit and non-profit require effective planning and a sound marketing strategy to do this effectively. The Internet has shifted the power to customers not marketers. The reason for that is, the customer has access to more information and is now able to do comparison shopping. Marketers often conduct and analyze research to see the needs, opinions, and attitudes of the customers. Marketing strategy helps companies to evaluate the usage of their strengths and capabilities to meet the needs and requirements of the market. Due to the customers constant new needs and wants, marketing is forced to continuously change and adapt to its dynamic environment. Having the right information is just as important as having the right product. Marketers have learned that by establishing a long-term customer relationship, they can increase customer sales and gain important marketing information about their customers. Having a good relationship with customers is a great benefit to have, then marketing is people driven and it is all fulfilling the needs of customers, shareholders, partners, society and the organization itself.

Thursday, November 14, 2019

Researching Titles for the Enumerative Bibliography Essay -- Personal

Researching Titles for the Enumerative Bibliography Fantasy literature was always something that I had read outside of class, almost guiltily, as if it didn’t constitute â€Å"real literature.† Faced with an opportunity, however, in which I could research anything I chose, the prospect of critically engaging a work I had enjoyed since I was in junior high proved too tempting to resist. I chose a topic, then, based on a favorite series of books, Tolkien’s The Lord of the Rings. I knew that critics spoke of the series as an allegorical reference to biblical events, but to limit the theology to just Christianity seemed too narrow to me. For the enumerative bibliography, therefore, I researched the idea that, while understanding the Christian undertones to The Lord of the Rings is one key to illuminating the text, other, more pagan ideas are also apparent in Tolkien’s writing. To begin my research, I went first to the note cards that I had prepared for many of the reference work in the library. After a significant amount of winnowing down, I picked five of the cards and began to search for criticism that would help me understand and build my argument. Unfortunately, I was forced to eliminate two of the cards rather quickly, as they provided no information useful to locating what I needed. Robert Reginald’s Science Fiction and Fiction and Fantasy Literature 1975-1991: A Bibliography of Science Fiction, Fantasy, and Horror Fiction Books and Nonfiction Monographs was simply a list of primary works and awards, with no critical works included. And while The Cambridge Bibliography of English Literature did include scholarship and criticism relevant to the works it covered, there was no available volume that cover... ...ersonal Inquiry. Chicago: Henry Regnery, 1964. Reynolds, Patricia. â€Å"Funeral Customs in Tolkien’s Fiction.† Mythlore 72 (1993): 45-53. Roche, Norma. â€Å"Sailing West: Tolkien, the Saint Brendan Story, and the Idea of Paradise in the West.† Mythlore. 66 (1991): 16-20. Rosebury, Brian. Tolkien: A Critical Assessment. New York: St. Martin’s, 1992. Sanford, Len. â€Å"The Fall from Grace – Decline and Fall in Middle Earth: Metaphors for Nordic and Christian Theology in The Lord of the Rings and The Silmarillion.† Mallorn 32 (1995): 5-14. Stanton, Michael N. Hobbits, Elves, and Wizards: Exploring the Wonders and Worlds of J.R.R. Tolkien’s The Lord of the Rings. New York: St. Martin’s, 2001. Tolkien, J.R.R. The Monsters and the Critics, and Other Essays. Ed. Christopher Tolkien. London: HarperCollins, 1997. Tree and Leaf. Boston: Houghton Mifflin, 1965. Researching Titles for the Enumerative Bibliography Essay -- Personal Researching Titles for the Enumerative Bibliography Fantasy literature was always something that I had read outside of class, almost guiltily, as if it didn’t constitute â€Å"real literature.† Faced with an opportunity, however, in which I could research anything I chose, the prospect of critically engaging a work I had enjoyed since I was in junior high proved too tempting to resist. I chose a topic, then, based on a favorite series of books, Tolkien’s The Lord of the Rings. I knew that critics spoke of the series as an allegorical reference to biblical events, but to limit the theology to just Christianity seemed too narrow to me. For the enumerative bibliography, therefore, I researched the idea that, while understanding the Christian undertones to The Lord of the Rings is one key to illuminating the text, other, more pagan ideas are also apparent in Tolkien’s writing. To begin my research, I went first to the note cards that I had prepared for many of the reference work in the library. After a significant amount of winnowing down, I picked five of the cards and began to search for criticism that would help me understand and build my argument. Unfortunately, I was forced to eliminate two of the cards rather quickly, as they provided no information useful to locating what I needed. Robert Reginald’s Science Fiction and Fiction and Fantasy Literature 1975-1991: A Bibliography of Science Fiction, Fantasy, and Horror Fiction Books and Nonfiction Monographs was simply a list of primary works and awards, with no critical works included. And while The Cambridge Bibliography of English Literature did include scholarship and criticism relevant to the works it covered, there was no available volume that cover... ...ersonal Inquiry. Chicago: Henry Regnery, 1964. Reynolds, Patricia. â€Å"Funeral Customs in Tolkien’s Fiction.† Mythlore 72 (1993): 45-53. Roche, Norma. â€Å"Sailing West: Tolkien, the Saint Brendan Story, and the Idea of Paradise in the West.† Mythlore. 66 (1991): 16-20. Rosebury, Brian. Tolkien: A Critical Assessment. New York: St. Martin’s, 1992. Sanford, Len. â€Å"The Fall from Grace – Decline and Fall in Middle Earth: Metaphors for Nordic and Christian Theology in The Lord of the Rings and The Silmarillion.† Mallorn 32 (1995): 5-14. Stanton, Michael N. Hobbits, Elves, and Wizards: Exploring the Wonders and Worlds of J.R.R. Tolkien’s The Lord of the Rings. New York: St. Martin’s, 2001. Tolkien, J.R.R. The Monsters and the Critics, and Other Essays. Ed. Christopher Tolkien. London: HarperCollins, 1997. Tree and Leaf. Boston: Houghton Mifflin, 1965.

Tuesday, November 12, 2019

Abolitionism †African American Essay

With abolition is found the gateway towards freedom. The African American influence in this area was of great authority especially in rural districts such as Lancaster and Chester Counties (Pennsylvania). In these groups sometimes the blacks worked alone and sometimes they partnered with whites. With this movement, African Americans used brute force to gain what they wanted (Bordewich, 138). Abolitionism was a great aid in spearheading the rights of blacks serving as soldiers. The abolitionism movement was fueled with evangelical religion, which deemed slavery as a sin. With this motto, members of the movement (both white and black) demanded that slavery be done away with, and terminated completely (Glatthaar, 15). The abolition movement is one in which its essential existence is tied integrally with that of African Americans, for it is their freedom which is at stake and is the goal of the movement. In the abolition movement is found the beginnings of the Anti-Slavery Society that has this as its constitution, This Society shall aim to elevate the character and condition of the people of color, by encouraging their intellectual, moral and religious improvement, and by removing public prejudice, that thus they may, according to their intellectual and moral worth, share an equality with the whites, of civil and religious privileges; but this Society will never, in any way, countenance the oppressed in vindicating their rights by resorting to physical force. Here is established the beating heart of the movement, to liberate the black community, and restore to them their God-given rights as humans to live freely, without adversity, without a fundamental challenge to their worth as part of humanity. Conclusion The myriad of influences the African American culture and people had on the Civil War is vast in its subjects, from black soldiers, the abolitionists, to their role in religion, African Americans have proven that their participation in the Civil War is essential. Black soldiers were only given praise and trustworthiness after they had proven themselves in the field of battle as equal compatriots to the Northern white soldier. During the clandestine times of the Underground Railroad, African Americans showed their dedication, and their strength of will through traveling thousands of miles to be free, and then they traversed the same paths in order to allow for other fugitives to find their way to the North. In Frederick Douglass there was found a man who stood for what he believed, not only in speech, but also in action. His deliberate animosity to ignorance in owning slaves helped to fuel the fires of the abolition movement, and thus the public awareness and knowledge of what slavery truly is: a vile creature, distorted with hate, and allowed to live only through dictatorship, and autocracy. McPherson states of the Civil War, â€Å"The Lincoln administration and the Republican press, even antislavery newspapers such as the new York Tribune, declared emphatically that the purpose of the war was the restoration of the Union, and that the issues of slavery and the Negro had nothing to do with the conflict† (22). Without the establishment of the Underground Railroad, the Abolitionist movement, and the fight for freed blacks to become soldiers, the Civil War would not hold for a history about the emancipation of a race, but the unification of a country instead. The ultimate influence that the African Americans had in the Civil War was their participation in all aspects of it; they were not going to be denied their human right to be their own masters, and without their voices and contributions in the war, slavery might not be an old issue. Without the personal stories of African Americans such as Frederick Douglass then the war would be empty of freedom. As McPherson quotes of Susie King Taylor, In this ‘land of the free’ we are burned, tortured, and denied a fair trial, murdered for any imaginary wrong conceived in the brain of the negro-hating white man. There is no redress for us from a government which promised to protect all under its flag. It seems a mystery to me. They say, ‘One flag, one nation, one country indivisible. ’ Is this true? Can we say this truthfully, when one race is allowed to burn, hang, and inflict the most horrible torture weekly, monthly, on another? No, we cannot sing, ‘My country, ‘t is of thee, Sweet land of Liberty’! It is hollow mockery. The Southland laws are all on the side of the white, and they do just as they like to the negro, whether in the right or not†¦(313). African Americans made this their war. Through fortitude and strength of will, they placed their faith in the decency of the Northern states and abolitionists to see the truth of the hate and prejudice in the country. The Civil War would not be about freedom, and the extraction of the activity of slavery in America if not for African Americans. African Americans paved the way for their own rebellion by speaking up, by acting, by using their talents in the field of battle and fighting for themselves, for liberation, for their sisters, brothers, mothers, and fathers. Without the influence of African Americans, the Civil War would have been just about unification. Work Cited Bordewich, Fergus M.Bound for Canaan. The Underground Railroad and the War for The Soul of America. HarperCollins, New York. 2005. Elkins, Stanely. Slavery. University of Chicago Press. 1976. Glatthaar, Joseph T. Forged in Battle: The Civil War Alliance of Black Soldiers and White Officers. The Free Press. New York, 1991. McPherson, James M. The Negro’s Civil War. Pantheon Books. New York, 1965. McPherson, James M. Ordeal by Fire. McGraw Hill. New York. 2001 Tracy, O. 2005. http://www. teacheroz. com/index. htm.

Sunday, November 10, 2019

Organizing Researching and Illustrating Material Essay

Step 1 1. Interview the administration groups, employees, and clients of Phoenix advertising, Roanoke Branch. * In order to understand the background, the process, and the internal situation of Roanoke Branch, interviews to different people that are connected to the company must be made. 2. Conducting surveys to both employees and clients of the company. * There must be surveys to conduct in order to gather information from the people who are connected in the company and to have specific cases that would give probability to the proposed actions to solve the problem. 3. Using print and online resources as added materials to the research. * Along with the interviews and surveys, print and online resources should also take into account to identify the company based on historical cases that print and online sources could provide. Print source will be used as historical data while online sources are used for contemporary and future data of the company. Step 2A: Surveys Employees 1. As employees, are you being paid by the company with the right benefits that the company has imposed? * This is asked in order to determine the compensation of the employee that can be the cause of employees’ work distraction. 2. Is there any process of account review in the company? * This is asked to identify the strengths and weaknesses of the company when it comes to the accounts of the employees and management itself. 3. Is Roanoke Branch the same with other branches when it comes to mode of payments? * This is asked to compare and contrast the situation of Roanoke Branch to other branches of Phoenix Advertising. Clients 1. Are you satisfied with the work of the company that was given to you? * This is asked to determine the stand of the client when it comes to the quality of the company’s work. 2. Do you know anything about the current situation of the company? If yes, kindly state the situation in brief. * This is asked to know if the clients are sensitive to the issues and  situation of the company. 3. Will you still use the company (specifically the branch) despite of the fact that there are internal problems? * This is asked if the clients will still be loyal to the company even if there are problems within it and to also determine if the problem of the company do not manifest within their production of products. To: The CEO of the Roanoke Branch Phoenix Advertising Mr. Gregory S. Forest Dear Mr. Gregory S. Forest: As the Vice President of Human Resources in Phoenix, I send you this letter to ask your good office to assist me on your company. I would like to make a visit to your company on August 18, 2010 to conduct some interviews and surveys. In order to fulfill the study for the probable causes and further effects of the problems and circumstances within your branch, I would like to ask you some questions about the company and its current situation in both internal and external forces. I would also like to interview some of your employees and clients in relation to these issues. The coverage of my interviews and surveys are based on the policies, employee performance reviews, project designs, internal and external agendas, and administrative configuration when it comes to company issues. I hope to hear positive feedback from this letter in order to conduct the interviews and surveys as part of the research. Thank you so much. Problems: 1. Quality of work 2. Loyalty to the customer 3. Issues within the company a. Do we need to share with the client b. Will this affect our relationship Facts and Causes: 1. Loyalty to the customer a. Answer any questions the customer might have b. Address the issues Impacts and Effects: 1. Loyalty to the customer a. Since this has been addressed production has increased 28% b. Turnover is down 15% c. Absenteeism is down 29% Morale has improved significantly which shows in production Solutions: 1. Incentive program a. This will increase morale b. Decrease morale for employees not receiving an award c. Encourage other employees to participate Illustrations: I chose to use the bar graph so you can clearly see the decrease in turnover, increase in production and decrease in absenteeism. It is simple and clear so that you can understand why this is so important.

Thursday, November 7, 2019

5 Tips for Handling Clients

5 Tips for Handling Clients 5 Tips for Handling Clients 5 Tips for Handling Clients By Colin Running a home-based writing business is a great way to make a living. It allows one to be creative, flexible, and above all, it allows for a certain amount of freedom. There remains however, some things that anybody who runs any size of business can get out of; client management. Without clients you have no business, and without your business, it’s back to the drawing board. There are several key points all freelance writers should remember, in order to stay organised, stress-free, and legally covered. None are hard to implement, but one should work hard at sticking to the following basic guidelines: Get It In Writing First It goes without saying that contracts are a vital tool if you want to be a successful freelance writer. Having a standard contract detailing your terms of work, deliverables, and billing procedures, sets client expectations and means you will be taken seriously. Always ask for the contract to be signed and dated, and provide a copy for your client for their own records. Any further agreements should be placed in a superseding contract. Template contracts are readily available from the Internet, but a good one can be adapted from the example provided by Peter Bowerman in his book, The Well-Fed Writer. Set Your Payment Schedule In Advance There’s nothing more unprofessional than an unprepared freelance writer. When asked how much a job will cost, a client wants to hear confidence, reliability, and professionalism, more than they do a bottom-rate charge. Good clients know how much good writers cost, so set your rate card in advance and stick to it. Working for free or severely discounted rates not only damages your reputation, but it leaves you open to being taken advantage of. Nobody will take you seriously, and it hurts the industry as a whole, especially for those writers who do charge market rates for work that you have offered to do for next to nothing. Clients try many tricks to get payments down to a minimum, so always remain aware of slick persuasive tactics. Don’t become over friendly, and keep the relationship business-like and professional. This includes when asking for payment, and sticking to the terms of the contract they have already signed. Set Reasonable Deadlines Never be pressurised into agreeing to work at a shortened timescale, when you know you will struggle to complete it. It’s far better to complete a project well within an agreed deadline than after it, because the client will likely not use you again. Until you are very experienced, always be prudent with your time estimates for work to completion, and incorporate revision and research time within the original estimate. If a client has a non-debatable deadline in which he is looking for your help to meet, it may be a good tactical move to rearrange other work to accommodate him. If there is room for altering one or two other deadlines to meet a client’s urgent request, they will be delighted when you are seen to be bending over backwards to help. Obviously, this scenario will result in a higher percentage fee for the client, so have a line detailing this in your contract. Be Comfortable Saying ‘No’ Sometimes it’s all too easy to agree to take work, especially when you start to do well and the money begins to roll in. But it’s not always a good idea to take on too much work if you don’t want to hurt the relationships you have built up with your clients. Not only will you end up working 20-hour days, but the quality of your work will deteriorate, you will lose your focus, your clients, and probably lose your head. Money isn’t everything, and the business won’t grow any faster. Saying ‘No’ is as important as saying ‘Yes,’ and further down the line you will be glad you struck a balance. Working for oneself is supposed to permit a certain amount of freedom, so don’t blow that by agreeing to every project that comes along. Your body will thank you for it, and believe it or not, clients will respect you for it. If they really want you, they will wait until you can schedule them in or pay you to reschedule them in. Allow Downtime for Administration Being a self-employed freelance writer means more than typing out articles, sales copy, or web content. You are the director, the manager, the employee, the cleaner, the accountant, the marketing executive, the secretary, and even the cleaner. In short, the success of your business depends on you! In order to keep your business running smoothly and efficiently, you must build in a certain amount of time each week for administrative tasks. It helps if you can develop as smooth a process as possible for keeping track of all your work, looking for more work, and managing cash flow. Whatever process you settle on, stick to it religiously but don’t be afraid to adapt it if it needs fixing. Falling behind will get you into a mess very quickly, and you will only spend more time than you can afford untangling the mess and fixing all the problems. An unorganised freelance writer rarely gets work, is never taken seriously, and loses clients faster than hot cakes from a baker’s shop. Want to improve your English in five minutes a day? Get a subscription and start receiving our writing tips and exercises daily! Keep learning! Browse the Freelance Writing category, check our popular posts, or choose a related post below:Types of Rhyme3 Types of HeadingsComment, Suggestion, and Feedback

Tuesday, November 5, 2019

Four Common Idioms from Shakespeare

Four Common Idioms from Shakespeare Four Common Idioms from Shakespeare Four Common Idioms from Shakespeare By Maeve Maddox What do the following examples from the Web have in common? Changing my mind  is not something that happens often.  Its a simple case of  me  stating  my  point and  refusing to budge an inch  from it.   US Recovery Cold Comfort for Unemployed Are your kids  eating  you  out of house and home  during the summer? . I made the mistake of buying him an egg salad sandwich, even though  in my heart of hearts I knew  he wouldnt like or eat it. Each one contains a phrase from Shakespeare that is still in widespread use. refuse to budge an inch In the frame story of The Taming of the Shrew, drunken Christopher Sly has been thrown out of an inn. An inn employee threatens to call the law on him, but Sly refuses to be intimidated by the threat. He tells the employee to call whom he will, but that he’ll â€Å"not budge an inch.† Sly uses the expression literally: he will not physically move from the place where he immediately falls asleep. In modern usage, the idiom is usually used figuratively with the meaning, â€Å"stand firm,† â€Å"refuse to change one’s mind on a matter.† cold comfort Shakespeare uses this expression in two plays: The Taming of the Shrew and King John. In the Shrew, Grumio uses the expression in a lengthy and bawdy punning exchange with another servant. In King John, the king, dying of poison, suffers from a burning fever. When his attendants inquire how he feels, he responds hyperbolically, personifying Winter and chiding them for not asking winter: to make his bleak winds kiss my parched lips And comfort me with cold. I do not ask you much; I beg cold comfort; and you are so strait And so ingrateful you deny me that. In modern usage, â€Å"cold comfort† is used figuratively in contexts in which something that is good in one sense is not adequate consolation for those who do not benefit from it. For example, the news of a drop in unemployment is â€Å"cold comfort† to people who remain unemployed. to eat one out of house and home In Henry IV, Part 2, Hostess Quickly of the Boar’s Head tavern has called the law on Falstaff because he has run up an unpaid bill of 100 marks. When the Lord Chief Justice asks for details, she says, â€Å"He hath eaten me out of house and home; he hath put all my substance into that fat belly of his.† In modern usage, the expression seems to be especially common in reference to teenagers. in my heart of hearts Shakespeare puts the expression in Hamlet’s mouth, although without a plural: Give me that man That is not passion’s slave, and I will wear him In my heart’s core, ay, in my heart of heart. Hamlet is praising Horatio for being the kind of man who can be trusted. In modern usage the phrase â€Å"heart of hearts† means, â€Å"the seat of one’s truest feelings.† The expression is especially popular on dating sites. For example: The most important question to ask yourself is this:  In your heart of hearts, do you believe that he or she is the one and only? Happy Birthday, Shakespeare! He was not of an age, but for all time!- Ben Jonson (1572-1637) William Shakespeare Born: April 23, 1564 Died: April 23, 1616 Related posts Shakespeare’s Vocabulary Book Titles From Shakespeare Sources of Titles Drawn from Shakespeare 20 Movies Based on Shakespeare Plays The Most Unkindest Cut of All Thou Lily-livered Boy Want to improve your English in five minutes a day? Get a subscription and start receiving our writing tips and exercises daily! Keep learning! Browse the Expressions category, check our popular posts, or choose a related post below:Fly, Flew, (has) FlownFlied?44 Resume Writing Tips7 Proofreading Steps

Sunday, November 3, 2019

Short case study Example | Topics and Well Written Essays - 750 words

Short - Case Study Example This article seeks to explore some of the problematic details in the relationship with specific reference to the value of unions to the Canadian economy. The general thinking behind the establishment of trade unions is the safeguarding of the welfare of workers. In the rush to make profits and enhance their corporate profile, companies may engage in trade practices that contravene their moral obligations to the workers. Proponents of trade unions including United Food and Commercial Workers (UFCW) work under the philosophy of protecting workers from the excesses of corporate practices (Business Case 8). Comparative evidence, case reviews and situation analyses indicate a gap in trade union activity between the United States and Canada. Unlike Canada, there has been a significant decline among workers in the United States to join trade unions (Bronfenbrenner, 2007). Differences in corporate culture and working practices have been cited a determining factor of the differences between the United States and Canada. Critical questions continue to attend to the question regarding the value of unions in the Canadian economy. Although collective bargaining agreement are meant to secure the interest of workers, past incidences show and precedence shows that companies would not easily cede to the demands by workers, which may lead to dire consequences on the economic front (Segerlund, 2010). Basically, collective bargaining denies the corporate world of the individual initiative and competition within the work force. This is because the workers are conditioned to operate under some common laws, which do not inspire the nurturing and growth of individual enterprise. One case that is easily recalled was the tussle that pitted Walmart and UFCW in 2009. Walmart refused to accept the condition set by the arbitrator and chose to close down its stores. Walmart’s action illustrated a growing resistance by the

Friday, November 1, 2019

Treatment versus Punishment - That is the Question Research Paper

Treatment versus Punishment - That is the Question - Research Paper Example The juvenile courts, in large part, exist so as to rehabilitate the youth who have done wrong. To that end, the paper will discuss treatment as the most effective juvenile intervention strategy to counter crime since it bests support the over arching concept of social justice. Based on the current population reports, there are more than 75 million children who are under the age of 18 years in the U.S. This is more that 25% of the total population. This number is projected to rise to over 100 million in 2050. These indicate that there are various issues that affect the American children, and there an increased risk of these children falling into the juvenile justice system. The Federal Bureau of Investigations in its 2013 report, Crime in the United States, reported that about 2 million youths below the age of 18 are apprehended every year for crimes ranging from loitering, to kidnappings, to arson, to drug dealing, to murder, and even terrorism. Besides that, the report also found that more than 850,000 youths belong to street gangs. The statistics also indicate that most youths were arrested for arson attacks and crime on property with 1% having driven a car after drinking alcohol, 25% arrested for robbery, and 41% were arrested for vandalism. Most states and cities across the nation have enacted laws that automatically bypass the Juvenile Justice System. In Boston, New York and Chicago, there are higher rates of detention as well as probation within the minority ethnic and minority groups. Averagely, 57% were Black, 22% Hispanic, 10% White, 5% Asian, and 1% American-Indian. According the National Criminal Justice Reference Service (2012), there are over 7 million youths in Massachusetts. Youths aged 19 years and below make up 27% of the population in the state of Massachusetts. In Boston, 89% of the youths apprehended in 2010 were charged with nonviolent crimes.

Wednesday, October 30, 2019

On the Moral and Legal Status of Abortion Essay

On the Moral and Legal Status of Abortion - Essay Example Methods of induction have ranged from utilizing sharpened tools, herbal medicines and physical trauma. Opinions, both culturally and legally differ worldwide, with public debate over legal ramifications and ethics of abortion being a very emotive issue. Abortion and the debate ranging around it have birthed activism, debates and controversy in equal measure. It is in fact a norm for people to refer to themselves as pro-life or pro-choice. Personal beliefs touching on responsibility, morality, government role in public policy and religion all affect ones view on abortion. Mary-Anne Warren, an American philosopher, put forward in her article â€Å"On the Moral and Legal Status of Abortion†, the perspective that abortion is acceptable morally. This paper seeks to delve into her article, especially her arguments and form an objection to her work. Warren begins her argument by debating how permissibility of abortion morally is dependent on whether the subject fetus is indeed a pers on. She argues that while a satisfactory defense of the right of a woman has to an abortion without proof of a fetus not being a human being is not satisfactorily possible, this difficulty in conclusion of a fetus’s status should not make it impossibility in the production of a solution to the moral status of the abortion problem (Warren 2). Pro-abortionists, due to not coming to grips with issues surrounding abortion, have had most of their arguments fall flat, failing to weaken traditional arguments on antiabortion. Their arguments are of two sorts, they either state that denial of a woman’s right to abortion is a deprivation of her rights to have control over her own body, or that deaths induced by illegal abortions, especially by poor women, is as a consequence of this law. This is obviously flawed since the fact that access restriction to abortion has such tragic side effects is not a pointer to the unjustified restrictions, as murder is still wrong. She cites J. Noonan saying, â€Å"The fundamental question in the long history of abortion is; how you determine the humanity of a human being?† (Warren 1). She goes on to argue that once the assumption of a fetus’ moral rights is allowed in full, the question of whether abortion is justifiable becomes a difficult and complex question. Warren seeks to push her abortion agenda via discussion of the five characteristics she believes are central to being a person. Warren tends to view persons as entities who have consciousness, self motivation, reasoning, communication capacity and presence of self awareness and self concepts (Warren 2). The first issue she tackles is the Definition of a human. She argues that â€Å"human† has two meanings, distinct but not easily distinguishable. Since killing of innocent humans is wrong and fetuses are an example of humans that are innocent, killing them is wrong. She shoots down this argument by claiming that the usage of human in similar se nse in both conclusions, whichever use is meant of the two, one of them begs questions. If used in two senses that are different, then the conclusion is still wrong (Warren 5). She claims that the presence of the human genome in fetuses does not point to it being morally human. Her second characteristic deals with the definition of moral community. She asks if it is indeed possible to establish if moral humanity can be sufficiently defined by genetic humanity. She describes the moral community as all people and only people, rather than all human beings and only human beings. Her argument continues to debate what characteristics enable any entity qualify for consideration as a person. She puts these characteristics down as consciousness and capacity to detect pain, acting out of self motivation,

Sunday, October 27, 2019

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