Learn About Photosynthesis Formula

Some organisms need to create the energy they need to survive. These organisms are capable of absorbing energy from sunlight and using it to produce sugar and other organic compounds such as lipids and proteins. The sugars are then used to provide energy for the organism. This process, called photosynthesis, is used by photosynthetic organisms including plants, algae, and cyanobacteria. Photosynthesis Equation In photosynthesis, solar energy is converted to chemical energy. The chemical energy is stored in the form of glucose (sugar). Carbon dioxide, water, and sunlight are used to produce glucose, oxygen, and water. The chemical equation for this process is: 6CO2 12H2O light → C6H12O6 6O2 6H2O Six molecules of carbon dioxide (6CO2) and twelve molecules of water (12H2O) are consumed in the process, while glucose (C6H12O6), six molecules of oxygen (6O2), and six molecules of water (6H2O) are produced. This equation may be simplified as: 6CO2 6H2O light → C6H12O6 6O2. Photosynthesis in Plants In plants, photosynthesis occurs mainly within the leaves. Since photosynthesis requires carbon dioxide, water, and sunlight, all of these substances must be obtained by or transported to the leaves. Carbon dioxide is obtained through tiny pores in plant leaves called stomata. Oxygen is also released through the stomata. Water is obtained by the plant through the roots and delivered to the leaves through vascular plant tissue systems. Sunlight is absorbed by chlorophyll, a green pigment located in plant cell structures called chloroplasts. Chloroplasts are the sites of photosynthesis. Chloroplasts contain several structures, each having specific functions: Outer and inner membranes— protective coverings that keep chloroplast structures enclosed.Stroma—dense fluid within the chloroplast. The site of conversion of carbon dioxide to sugar.Thylakoid—flattened sac-like membrane structures. The site of conversion of light energy to chemical energy.Grana—densely layered stacks of thylakoid sacs. Sites of conversion of light energy to chemical energy.Chlorophyll—a green pigment within the chloroplast. Absorbs light energy. Stages of Photosynthesis Photosynthesis occurs in two stages. These stages are called the light reactions and the dark reactions. The light reactions take place in the presence of light. The dark reactions do not require direct light, however dark reactions in most plants occur during the day. Light reactions occur mostly in the thylakoid stacks of the grana. Here, sunlight is converted to chemical energy in the form of ATP (free energy containing molecule) and NADPH (high energy electron carrying molecule). Chlorophyll absorbs light energy and starts a chain of steps that result in the production of ATP, NADPH, and oxygen (through the splitting of water). Oxygen is released through the stomata. Both ATP and NADPH are used in the dark reactions to produce sugar. Dark reactions occur in the stroma. Carbon dioxide is converted to sugar using ATP and NADPH. This process is known as carbon fixation or the Calvin cycle. The Calvin cycle has three main stages: carbon fixation, reduction, and regeneration. In carbon fixation, carbon dioxide is combined with a 5-carbon sugar [ribulose1,5-biphosphate (RuBP)] creating a 6-carbon sugar. In the reduction stage, ATP and NADPH produced in the light reaction stage are used to convert the 6-carbon sugar into two molecules of a 3-carbon carbohydrate, glyceraldehyde 3-phosphate. Glyceraldehyde 3-phosphate is used to make glucose and fructose. These two molecules (glucose and fructose) combine to make sucrose or sugar. In the regeneration stage, some molecules of glyceraldehyde 3-phosphate are combined with ATP and are converted back into the 5-carbon sugar RuBP. With the cycle complete, RuBP is available to be combined with carbon dioxide to begin the cycle over again. Photosynthesis Summary In summary, photosynthesis is a process in which light energy is converted to chemical energy and used to produce organic compounds. In plants, photosynthesis typically occurs within the chloroplasts located in plant leaves. Photosynthesis consists of two stages, the light reactions, and the dark reactions. The light reactions convert light into energy (ATP and NADHP) and the dark reactions use the energy and carbon dioxide to produce sugar. For a review of photosynthesis, take the Photosynthesis Quiz.

The, Part E Race And Colonialism - 1849 Words

Theoretical Concepts Found Helpful The theoretical concepts that was helpful ware â€Å"Part E: Race and Colonialism†, this section was an eye opener to the realities aboriginal people endured in colonial and post colonial times. However, to be more specific the topics on Aboriginal Social Work Education in Canada: Decolonizing Pedagogy for the Seventh Generation, writer by Raven Sinclair helped me see these realities more in depth. For instance, in the first page of the article Sinclair reminds us that 150 – 300 children were lost in British Columbia through child welfare. (2004) This particular article was helpful because it deconstructed the notion I had from the dominant group here in Canada. When Raven Sinclair also goes on explaining that western hegemony is being taught from top to bottom and always alienating aboriginal teachings and beliefs. (2004) Gave me a clear understanding as to how important is for us as Social Workers to become agents of change, by learning about the true historical backgrounds of aboriginals and in turn become an ally and bring forth changes in social perspectives, in essence to deconstruct that western hegemony and as Sinclair asserts as â€Å"Conscientization is a critical approach†¨to liberatory education† (2004) The reading that also captivated me and had a big impact was â€Å"Indigenous Wholistic Theory: A Knowledge Set for Practice† by Kathy Absolona (2010) as it brought light to the knowledge indigenous people have. Moreover, on the second pageShow MoreRelatedThe Earliest Movements For Repatriation By Black Americans1421 Words   |  6 Pagesthe late nineteenth-century reflected the ways in which the gratuity of violence of both colonialism and slavery created a dialectical tension between Black Americans and Continental Africans. 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Extensive research in the United States has documented implicit and explicit bias based not just on race, but also skin tone — what anthropologists call â€Å"colorism,† described by the researchers Keith Maddox and Stephanie Gray as â€Å"the tendency to perceive or behave toward members of a racial category based on the lightness or darkness of their skin toneRead MoreNationalism, Industrialization, And Colonialism On Wwi And Its Occurrence1353 Words   |  6 Pagesmillions was shed. The war had far-reaching consequences for all nations that were involved in the conflict such that the entire boundaries of many countries were remarked. This paper will discuss the impact of nationalism, industrialization, and colonialism on WWI and its occurrence. Nationalism’s Effect on World War I The political and social conditions of Europe before the onset of the World War I were extremely unstable and undergoing a rapid transition that was driven by national interests ofRead MoreAfrican Vs. 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Night Creature Dark Moon Chapter Seven Free Essays

Nic sat up, groaned, fell back. I caught him before he cracked his head against the ground again. â€Å"Maybe we should get you to a doctor. We will write a custom essay sample on Night Creature: Dark Moon Chapter Seven or any similar topic only for you Order Now † â€Å"You’re a doctor.† â€Å"Not that kind.† â€Å"I’m all – † His voice faded, and his eyes closed as he slumped in my arms. Concerned, I leaned close. He was out cold, so I gave in to the urge that had been haunting me since I’d first seen him in the doorway. Pressing my lips to his forehead, I breathed in the familiar scent of his hair. All the feelings rushed back with a force that staggered me. I’d known I still loved him, but I hadn’t realized that I always would. Once we’d dreamed of sharing a life: marriage, careers, family. Together, we would never be alone again. I longed for that normal life – a normal me. But I’d come to understand that even if I cured myself, there were things I’d done in the interim for which there could be no forgiveness. Nic was as lost to me now as he’d been the first night I changed. The wind slapped snow against my face. The drop in temperature had turned the fluffy flakes into icy needles. I smelled death – probably just Billy’s. Nevertheless, we had to keep moving. With the clouds covering the moon, the road was dark. Though there wasn’t much of a chance a car would come along and run over us†¦ then again, one might. Taking advantage of Nic’s momentary lapse of consciousness, I lowered him gently to the ground and hurried to the ATV. After a quick glance to make sure he was still out, I picked up the machine and set it back on the road. There was a dent in one side, a bit of dirt on the other, but when I started the engine, it worked. Nic began to come around. I tugged on his arm, grunting as if he were â€Å"oh, so heavy,† though I could have lifted him with one hand. â€Å"Wanna help me out a little?† â€Å"Sorry, I’m – â€Å" â€Å"Hurt,† I supplied when he seemed to lose his thought again. Thankfully, he was too spacey to notice how much I helped him as he got to his feet, too woozy to see that my clothes were torn and I had flecks of blood in my hair. I hoisted Nic onto the ATY, crawled behind, then adjusted his body so that I could see, drive, and hold on to him. If I hadn’t had superpowers, I wouldn’t have been able to manage, making this one of the first times I was glad to be what I was. Nic drifted in and out of consciousness. I’d wondered how to make him stop asking questions. I’d have preferred another method. The wind shifted, or we were able to get ahead of the storm, because the highway outside of Clear Lake was dry, the forest surrounding it devoid of white. Most of the businesses on the main drag were closed, probably had been for a while. The town was small, innocent, clueless. I’d been toying with the idea of dumping Nic with a doctor – they had to have one – then disappearing again. But an hour on the ATV with little to do beyond think had nixed that idea. Billy might be dead, but Billy hadn’t blown up the compound. Whoever had, could be right behind us. I let my gaze wander over Nic’s still face. He’d say he was a highly trained FBI agent; he could take care of himself. But I knew better. To werewolves he’d be an easy lunch. No matter how dangerous it was for us both, I was going to have to take him along to Wisconsin. I pulled into the only gas station in Clear Lake. The attendant stepped outside. His gaze wandered over my torn suit, the spatters of blood and the leaves in my hair, then flicked to Nic’s lolling head. With the typical understatement that characterized inhabitants west of the Mississippi, he murmured, â€Å"Trouble?† â€Å"Nearly hit a†¦ deer. We flipped.† The story, close enough to the truth to be believable, explained Nic’s injuries and my appearance. â€Å"Need a doctor?† he asked. â€Å"No.† Nic struggled to sit up. â€Å"I’m okay.† The attendant’s brows drew together. â€Å"If you say so.† Nic tried to prove it by climbing off the ATV. He wobbled, but he didn’t fall down. â€Å"You know where I can buy some clothes?† As the word buy left my mouth, I realized I had no money. I glanced at Nic; he was already extracting his wallet. â€Å"And a car,† he added, pulling out an obscene amount of cash. â€Å"Got some T-shirts and sweatpants for sale inside.† The man scratched his head as he contemplated the money. â€Å"Car we’ll have to talk about.† I hesitated, prepared to deal, but Nic waved me away. â€Å"I’ll handle the car.† I let him. The less time we hung around, the better. Inside I snagged a pair of gray sweatpants and an equally cheery gray T-shirt. Making use of the restroom, I stripped off my torn and dirty suit. After extracting the wolf totem, I tossed the clothes into the nearly full garbage can. Holding the tiny bit of plastic between two fingers, I stared into the sparkly blue eyes. The idea that something this small, this tacky, could carry enough power to make me superduper wolf was laughable. But standing in a dirty women’s restroom in the middle of nowhere, I didn’t feel like laughing. I shoved the talisman into the pocket of my new sweatpants just as I remembered the little wolf wasn’t the only thing that had been in my skirt. Both the list of names Nic had given me and his .38 were missing. I must have dropped them somewhere along the road. I didn’t care about the list, but the gun might have been good for a bluff or two. Since I couldn’t go back for the weapon now, I shoved my bare feet into my tennies and picked one last flake of blood from my hair. My nails looked as if I’d been burying dead bodies in the woods, which was close enough to the truth to make me worry. I could only hope that the people we met between here and Wisconsin were less concerned with personal hygiene than I was. When I exited the bathroom, I found the attendant behind the register. I peered around the station, which was packed ceiling to floor with chips, soda, candy, and borderline pornography. But no Nic. â€Å"I sold your friend a car.† From the man’s grin, the deal had been sweet. Of course, we couldn’t exactly be choosy. We had to get out of here, and we couldn’t do that on an ATV. â€Å"He went across the street to pick it up.† Though I didn’t like Nic being out of my sight for more than a minute, his absence did give me time to do something I should have done before now. â€Å"Do you have a phone?† He pointed to the wall behind me. I considered the risks. I doubted anyone would have thought to put a bug on this particular phone, and Edward always had his own lines meticulously swept for listening devices. By the time someone traced the call, Nic and I should be long gone. I punched in the numbers as the clerk moved off to refill a potato chip display. Edward answered on the second ring. â€Å"Elise?† How did he do that? The caller ID should have read â€Å"Joe’s Gas Station,† not â€Å"Elise Hanover.† Sometimes the old man was spookier than everything he hunted. My response – â€Å"Yes, sir!† – was rewarded with a vicious stream of German curse words. â€Å"I know you aren’t often glad to hear from me,† I muttered, â€Å"but is that necessary?† â€Å"I have been calling the compound every half an hour, and the line is dead. If we are having a malfunction, Elise, it is your job to inform me.† â€Å"It’s a little bit more than a malfunction.† â€Å"Be specific.† I’d known Edward all of my life. He’d practically raised me – although paying various nannies, shipping me off to the best schools, then recruiting me to be his right-hand woman was hardly raising someone. There was little warmth between us, no matter how much I might want there to be. â€Å"Specifically†¦Ã¢â‚¬  I glanced around. No one was in the gas station but me and the attendant, who was more interested in straightening the Hustler supply than listening to me. Nevertheless, I lowered my voice. â€Å"There’s a crater where the compound should be.† Silence greeted my statement. â€Å"Sir?† â€Å"Sabotage?† I thought of the shadow, the shot, the silver. â€Å"Definitely.† â€Å"The guard?† â€Å"Dead.† â€Å"Subjects?† â€Å"Could be alive.† Edward’s grunt told me he understood the ramifications of that as much as I did. â€Å"Except for Billy.† â€Å"And Billy is not alive because†¦ ?† â€Å"He pissed me off.† Though his sigh traveled hundreds of miles before reaching me, the sound lost none of its power to belittle. â€Å"Your temper is, as always, a problem.† Only Edward would think that I had a temper. Everyone else considered my personality one step removed from ice bitch of the universe. Except Nic, but then, he didn’t really know me as well as he thought. â€Å"I will send someone to Montana,† Edward said. â€Å"Someone who can take care of things.† Taking care of things being a J-S euphemism for cover-up. Even if Nic managed to send some of his pals into the woods, by the time they got there, there’d be nothing left to see. â€Å"Who is responsible for this travesty?† Edward continued. â€Å"Bad guys?† The line went silent again, and I waited for the inevitable set-down. But instead of a lecture, I was rewarded with a dry chuckle, which made my heart stutter. â€Å"Who is this?† I demanded. He had the heavy German accent down to a T, but there was no humor in Edward – never had been. Which was understandable. His life had not exactly been one laugh riot after another. â€Å"What have you done with my boss?† â€Å"It is me, Elise. I have just lightened up in my old age.† Lightened up ? Okay, the world had stopped turning, and I had been too busy to notice. â€Å"So much time with Jessie and Leigh†¦Ã¢â‚¬  I could almost see him shrug in that way he had that implied both nonchalance and Old World European manners. â€Å"They are amusing.† My teeth ground together at the reminder of Edward’s favorite Jger-Suchers. I had known him the longest, had helped him the most, yet when Edward had chosen pets, I was not one of them. Jessie McQuade and Leigh Tyler-Fitzgerald were Edward’s darlings as well as bosom buddies. Not that they hadn’t tried to kill each other on occasion – when you released hunters into the same field you got explosions more often than tea parties – but they were two of a kind, and I didn’t fit in. I wasn’t the type to banter and snipe. I didn’t dare participate in the physical scuffles they relished. Sarcasm wasn’t my venue. Nevertheless, having them take the place that I’d always wanted in Edward’s affections made me a lot less enamored of them than he was. â€Å"If this is Edward,† I continued, â€Å"then tell me something only we would know.† Another long swell of silence drifted over the line. For a minute I thought I was right, maybe someone was impersonating my boss. I should have known that no one got the better of the old man, including me. â€Å"By that,† he said in a hard, cool voice that made me straighten even though he wasn’t there to see, â€Å"I suspect you’re referring to the fact that I killed your mother.† How to cite Night Creature: Dark Moon Chapter Seven, Essay examples

Hemingway Essay Example For Students

Hemingway Essay Ernest Hemingway’s tough, Terse prose and short, declarative sentences did more to change the style of written English that any other writing in the twentieth century. II. Ernest Hemingway has had many great accomplishments in his historical life but just one event has hardly sticks out from the rest. The Old Man and the Sea is one of Hemingway’s most enduring works. Told in Language of great simplicity and power, it is the story of an old Cuban fisherman, agonizing battle with a giant marlin far out in the Gulf Stream. Here Hemingway recasts, in strikingly contemporary style, the classic theme of courage in the face of defeat, of personal triumph won from loss. Written in 1952, this hugely successful novel confirmed his power and presence in the literacy world and played a large part in his winning the 1954 Nobel Prize for Literature. This novel also won the Pulitzer Prize award. III. July 21st, 1899, Ernest Hemingway was born. He was born to DR Clarence Edmonds and Gr ace Hall Hemingway. He grew up in a small conservative town called Oak Park, Illinois. His father, a practicing doctor, taught him how to hunt and fish, while his mother, wished to make him a professional musician. His upbringing was very conservative and somewhat religious. He attended Oak Park and River Forest High School, where he distinguished himself in English. His main activities where swimming, boxing, and of course writing. In 1917, turning his back on University, he decided to move to booming Kansas City where he got a job as a cub reporter on the Kansas City Star. At the train station, his father, who later on disgusted Ernest by committing suicide, kissed his son tenderly good-bye with tears in his eyes. This moment was eventually captured in For Whom the Bell Tolls. Hemingway wrote that he felt so much older than his father that he could hardly bear it. The Star was the first to introduce Ernest to news writing which demands brief, to the point sentences, that contain a smooth easy following of ideas. He would later adapt this style to his fiction. In May of 1918, Hemingway became an honorary second lieutenant in the Red Cross. He could not join the army due to a defective left eye (resentfully inherited from his mother). On his first day of service across seas, he and other ambulance drivers were assigned the horrific duty of picking up body parts from an exploded munitions factory. Death, mostly of women, on such a scale was most definitely another very shocking moment in Hemingways young life. But he soon recovered from this experience and became known as the man who was always where the action is. He would often sneak cigarettes and chocolate to soldiers on the Italian front. It was on one of these occasions that he was severely wounded by an Austrian trench mortar. Even with over a hundred pieces of shrapnel and an Austrian machine gun bullet logged in his leg he managed to carry a wounded soldier a hundred yards to safety. He got the Italian Medal of Valor for his courageous action. He spent his recovery time at the Ospedale Croce Rossa Americana, in Milan. It is there that he met and fell for a thirty year-old nurse called Agnes Hannah. To Ernests disappointment, Agnes was not willing to embark in a relationship. Ernest, who had not yet turned twenty, who was a war hero, a journalist and a wounded soldier, was too young for beautiful Agnes . With the will to write fiction, he moved to Chicago where most of his work was refused. He lived by writing for the Toronto Star and working as a sparing partner for boxers. It was in Chicago that Hemingway met Elizabeth Hadley Richardson. She was an innocent young woman with graceful features and a strong attraction for the eight year younger Hemingway. Not having much income and wanting to marry Hadley, Ernest chose to move to Paris. Hemingway managed to convince the Toronto Star to accept a series of Letters from Europe. The young couple also received money from Hadleys trust f und while Ernest continued to work as a sparing partner for boxers. In Paris, Hemingway encountered many of the greats (historically known as The Expatriates). He met Gertrude Stein, Ezra Pound, James Joyce, Scott Fitzgerald , Ford Madox Ford and John Dos Passos. It was Stein who took him under her wing. She had been working to renew literary writing by removing useless gothic, Victorian and archaic forms. She was the first to point Hemingway in the direction of the simple declarative sentence, an attempt to make words communicate concretely and efficiently. It was also during this period of his life that Hemingway discovered the bull fight, the Pamplona bull run and the famous San Fermin July Fiesta. He would later write several books and short stories about bull fighting and the many events that surround this tragic ritual. Among these are Death in the Afternoon and The Dangerous Summer. Quickly after Patricks birth, they moved on to what would remain Hemingways only true residenc e in the United States Key West, Florida. It was there that a whole new world broke itself open to the sportsman in Ernest. Fishing the deep sea for great fish like the tarpon and the barracuda was his newest love. But even in Key West, a heavenly earth, tragedy struck Ernest. His father, struggling with diabetes and angina pectoris, had put a bullet through his head. Hemingway was very ashamed of this. He had always felt that life was for the testing of death. Suicide was the surrendering of life to death. This was forbidden in his code of courage. From that day on, Ernest turned his back on his father. 1929 marked the release of A Farewell to Arms. It was instantly accepted as a great work by critics and the public. With the success of this novel, Hemingway became a true American writer. To many, he also became a hot headed fool. He would make loud remarks about some of his fellow writers. He would make proclamations about artistic integrity that he himself would often not respect . He clearly was no longer the shy young journalist he had been for the Kansas City Star. He had become Papa. Even with the beautiful surroundings of the Key West, Hemingway still longed for Spain. At the time he was tediously working on Death in the Afternoon, a marvelous, intriguing and powerful look at the bullfight. Although at times overdone, Death in the Afternoon will capture greatness and power in the minds of its readers, even those that are most disgusted by the bullfight. After Ernest finished Death in the Afternoon and Pauline gave birth to another boy, they set off for Africa. It was there that Hemingway hopped to find the true meaning of heroism. Three stories would result from the events of Africa. The Green Hills of Africa, which lacked effective meaning and carried a false tone of masculine hunting spirit, was the least successful. The Snows of Kilamanjaro was a much more potent tale about the hunt. Arguably one of Hemingways best, it drew from the troubles of a bro ken Scott Fitzgerald to depict the guilt of a talented yet unacomplished artist as he faced death. The last short story to result from Africa was The Short and Happy Life of Francis Macomber, which seeks the meaning of courage. The Spanish Civil War became official in July, 1936. Hemingway was offered a liaisons job by the North American Newspaper Alliance (NANA). He accepted, much to Paulines opposition. Being a newsman, officially he remained neutral throughout the war. Despite this, Hemingway could often be overheard raising funds at social gatherings in America to fight the fascists back in Spain. In 1940, after the end of the Spanish Civil war Hemingway published For Whom the Bell Tolls. His long divorce with Pauline came to an end, and he married Martha Gellhorn. This would turn out to be the shortest and least understandable of his four marriages. During the Second World War, he equipped the Pilar with grenades and sub conning towers for the purpose of hunting Nazi submarines . This got it recognized as an official Q-ship. Of course its unshaven crooked crew never sunk a sub, they simply spent their days fishing off the Cuban coast. Martha, who was very involved in the war, was as always quick to criticize Ernest. She accused his personal navy of fraudulent use of gasoline rations. One night, when returning form a drunken party, Hemingway had a severe car accident. He was hospitalized with serious head trauma . Martha returned from the front to see him. Instead of comforting the banged up Ernest, she simply laughed at his sad state as he lay in the hospital. In June 1944, Hemingway finally set foot in Normandy. He made his way to the front, compiling a small army of undesirables by his side. With his small guerrilla force and a few bottles of scotch Hemingway marched in to Paris on the 25th of August, five days before the city was officially freed. He proceeded, by his own authority, to liberate the Travelers Club and the Ritz, in which he took a room as well as the bar. He was eventually awarded the Bronze Star for his part in the invasion. On another occasion, while Mary was staying with him at the Ritz, he shot a portrait of her husband. He placed the frame over the toilette in his room at the Ritz and discharged his hand gun into it. This flooded the room and several floors below it. Mary got a taste of Hemingways madness. Yet, in 1946, they were married. The war was over and they returned happily to La Finca. Across the River and into the Trees was the fist of a fictional three part saga about earth, sea and air. It takes place in Venice. It is about an old soldier, who is no longer at war and who falls for the sweet beauty of the much younger contessa Renata. Some said that this novel had a strong Shakespearean quality, many others only saw a pathetic tale about an old man infatuate with a young lady. Hemingway outdoes himself with charming descriptions of Venice in this book. Yet he fails in making his protagonist soldier sy mpathetic, a sign that he was self-conscious of his boisterous behavior. This book marked a turning point in Hemingways life, it stood for his passage into middle age, something he had not been willing to accept easily. In 1950, after having been dubbed as a burnout, Hemingway put himself to work on his greatest story ever. The Old Man and the Sea was published in 1952. It was a very touching tale about an old man who finds grandness of life and death while battling the great marlin. He is ready to heal down before the fish, when it finally gives in. While towing the animal back to shore, its beauty is destroyed by sharks. The humility of the old man, his handshake with grandeur, all make this tale truly beautiful. The Old Man and the Sea was Hemingways second entry in his triad about land, seas, and air. It got him the Pulitzer Prize, and in 1954, the greatest literary award of all, the Nobel Prize. Hemingway had three true phobias in his life: telephone conversations, the taxman, and public speaking. Yet he wrote a very touching speech that was read. It was at that moment that the end had begun for Papa Hemingway. Before the Nobel Prize in 1954, Ernest and Mary had sought out his fifth African safari. This time he was much less boisterous. He maintained a clear mind. He shot very well, and demonstrated great ability. Yet the safari ended badly with two plane crashes. The first had not been too serious. The second, although, had distraught Hemingway quit badly. His injuries included concussion, paralysis of the sphincter, first degree burns on his face, arm and head, a sprained right arm and shoulder, a crushed vertebra, and a ruptured liver, spleen and kidney. He was in continuos pain for quite a while. It was in Pamplona that Hemingway celebrated his sixtieth birthday. Mary had spent two months preparing for the event. She ordered champagne from Paris, Chinese foods from London, codfish from Madrid. She hired a shooting booth, fireworks specialist, flamenco dancers, waiters, barmen and cooks from all over the world. Guests included General C.T. Lanham from Washington, Ernests old Paris pals, Italian Royalty and the Maharajah of Behar. The party went twenty-four hours strait, from noon of July 21st to noon July 22nd. Seeking a calm place to recuperate and continue work on The Dangerous Summer, Ernest and Mary relocated to their cabin in Ketchum. But for Hemingway, Idaho was a far cry from the comforts of Cuba. His eyes were failing him to the point of not being able to write anymore. Even if he did manage to put down words, they often were incoherent, lacking any logical meaning. He could not drink anymore due to his kidney injury. Slowly but surely Ernest degenerated physically and psychologically. Even while still living in Cuba, Ernest began showing signs of paranoia and delusion. He would often say that FBI agents were following him. Although he had large enough savings to cover any immediate financial problems, he was constantly a fraid of being hunted down by the IRS for tax evasion. It soon became evident to those around him that psychiatric help would be necessary. They managed to convince Ernest to institutionalize himself. Fearing he would refuse, it was agreed upon to tell him that the treatment was for his high blood pressure (Hemingway had always been wary of his blood pressure). On November 30th in 1960, Ernest Hemingway was committed to the Mayo Clinic in Rochester, Minnesota. During the month of December he was given electroshock therapy. In January of 1961, Ernest was released. At first all seemed well again. He had even managed to write a few coherent words for the jacket of George Plimptons new book: On April 23rd, Ernest Hemingway tried to take his life for the first time. He had tried to put a shotgun to his head. It had failed the first time but he then later went on to put that one tragic bullet in his head. IV. My opinion of this book is phenomenal. It taught me a sensational amount of inte resting facts about Ernest Hemingway. He went through so much in his life. A lot of events in his life interested me which made me keep on reading the book! Hemingway had may happy events but yet he went through some really hard times. All this added up in which he followed in his father’s foot steps and killed himself. Overall Ernest Hemingway should be a world figure for his excellence and commitment to writing. V. V. 1. In 1926 Ernest wrote the novel The Sun Also Rises. 2. In 1929 Hemingway wrote the novel A Farewell to Arms. 3. In 1932 Ernest wrote the novel Death in the Afternoon. 4. In 1940 Hemingway wrote the novel For Whom the Bell Tolls. 5. In 1950 Ernest wrote the novel Across the River and into the Trees. 6. In 1953 Hemingway wrote the most famous of his novels called The Old Man and the Sea. 7. In 1953 Ernest Hemingway won the Pulitzer Prize for is novel The Old Man and the Sea. 8. In 1954 he won the Nobel Prize in Literature. 9. On November 30th in 1960, Ernest H emingway was committed to the Mayo Clinic in Rochester, Minnesota. During the month of December he was given electroshock therapy. 10. In 1961 Ernest Hemingway took his own life by committing suicide. Frees - Sarcasm and Irony in A Modest Propos Essay Santiago, the main character in the story, does not divulge in any pleasures what so ever. It almost seems as though he is trying to make himself suffer. Everyday, Santiago hardly eats anything but a little fish or coffee. He does not have any relationships with women in the story, as many Hemingway novels have included. While Santiago is out on the boat, he does not let himself stray from the task at hand even though it is very uncomfortable. The Hemingway code hero would be the exact opposite of Santiago. He would eat large meals every day, make love to many women, and never put himself in a position that he did not like. The code hero would do everything as though it was the last time he was doing it because he did not believe strongly in the presence of God. Santiago was different because he believed in God, and prayed to him for help throughout the story. While he was at sea, he often prayed that he would get the fish or that he would live to see the fish brought to the village. Santiago did not fear death and the reader senses that Santiago believes that if he dies, he will go to heaven. The story is also filled with many biblical references and the whole book has a religious theme. Hemingway does not usually have his code heroes be religious, and most of them feel that they only have this time on earth and they had better make the best out of it. Finally , Hemingways code hero differs in The Old Man and the Sea because of the presence of the boy that is Santiagos companion. Known only as the boy, he is constant throughout the novel as Santiagos student, who takes care of Santiago. It seems that everything that Santiago does is for the boy, and not himself. In the normal code hero, the hero would do everything for the benefit of himself and for no one else. The hero is usually not a fatherly, gentle figure, but a robust, energetic manly man. Therefore, it can be seen that Santiago is different than other Hemingway characters because of the distinct actions he made. And although Santiago does not meet the standards of the Hemingway code hero, it is his disinterest in physical pleasures, his need for a companion, and the presence of religion that makes him a special hero in his own right. Bibliography:

An Inquiry Into Hamlets Madness Essays - Characters In Hamlet

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

Atonement Ian Mc Ewan Essay Example

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

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

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

America and the end of the Cold War Research Paper

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

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

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

Advances in DNA Sequencing Technologies

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