Drama In Road Not Taken by Robert Frost Essay

A living and breathing show of life is painted in â€Å"Road Not Taken† that presents a circumstance wherein speaker is trapped in a dynamic problem. The setting of the sonnet presents the speaker t a bifurcation of street where he should choose which trail to take. He can not make out where either street leads. He takes a gander at the physical parts of the street and chooses to go on the less-voyaged street. The sonnet represents the basic subject of decision making and speaker’s independence in lean toward a less-regular way. It further suggests that dynamic must not be procrastinated in light of the fact that it is more unsafe than the result of an undertaking. Moreover, in show that there is no outright decision accessible to person and he needs to lean toward one decision over the other gave and administered by predetermination. The main differentiation that artist has, is his inclination of less-regular decision. Albeit entire sonnet is an all-inclusive representation of life where one runs over numerous events when he needs to settle on choices however Frost acquaints different figurative articulation with pass on the power of the circumstance. In the absolute first line, Robert Frost acquires the essential representation, the wandering streets. Clench hand line presents the situation of veering street while in line 2-3 writer shows the impediments of physical being and laments over human lack of ability to go on both street. â€Å"Long I stood† portrays that he took the imprudent choice and took as much time as necessary to settle on choice. Lines 4-10 delineate his similar assessment of the two streets. He again alludes to human insufficiency when he recognizes that his vision is insufficient. Next two lines reverberate the past inclination as he finds the both street â€Å"really about the same†. In 13-15, he settles on his choice though lines five lines, he imagines himself in future, discussing his choice to go on the less-voyaged way.  â â â â â â â â â â The tone of the sonnet is reflective on the grounds that speaker examines on his choices. He needs to consider the attributes of every street. There is a component of disappointment additionally as he can not go on both street however sonnets closes on an idealistic not as artist feels that going on less-voyaged street will have the effect. He means the significance of his uniqueness that propels him to proceed onward a less-trodden way. He doesn't receive the customary way in the everyday exercises of life.  â â â â â â â â â â The structure of the sonnet has an unconventional significance and is built on four refrains with five lines each. The main verse presents the issue; second refrain represents the decisions accessible to the speaker. Third verse shows his lament over not taking the other street while fourth street delineates the choice of the artist to take the less voyaged street. There is indication of outcry after line 13 that demonstrates the fervor on choice to save the primary way for one more day however this energy before long ceases to exist as he questions that whether he will return. In lines 18 and 19, there is reiteration of â€Å"I† that signifies the accentuation on â€Å"his† choice to proceed onward less voyaged trail. Rhyme plan of the sonnet is abaabcdccd†¦Frost utilizes basic lingual authority that passes on his message with far reaching.  â â â â â â â â â â So all in all, this sonnet utilizes diverse poetical apparatuses to depict the mental quandary of the artist in dynamic. His experience isn't abstract however is of general nature and he presents it in an unpretentious manner.

How to Write Opinion Essay That Will Capture the Readers Attention

How to Write Opinion Essay That Will Capture the Reader's AttentionIt seems that very few people get how to write an opinion essay. In this article, I will give you some good tips on how to improve the effectiveness of your writing and use it to write convincing essays. I will discuss some basic things that will help you improve your essay.First, you need to make a good amount of research. You need to know what is in the area that you are writing in. This is the first thing that you need to learn how to do if you want to be successful in this kind of writing.Second, you need to have a background of the current trend in public opinion. Knowing what the public think about something means that you can incorporate these ideas into your essay. Knowing about the current trend is very important because it will give you an idea on how to write the essay.The third step that you need to do is that you need to gain insight and knowledge about the topic on which you are writing your opinion essa y. This is very important if you want to create something that is convincing. This is the reason why you need to go online and see what is in demand. You need to gain knowledge about the topic in order to develop your essay.Fourth, you need to focus on developing your writing skills. You need to improve your writing skills and your composition skills if you want to be successful in writing your essay. When you write a good essay, you will be able to show your readers the proper manner. Make sure that you follow the rules and formats that the experts use.The fifth step that you need to do when learning how to write an opinion essay is that you need to get confidence. You need to understand that the essay should not only be successful but should also be powerful. You need to keep in mind that confidence is the key to success when writing essays. You need to build up your confidence so that you can finish your essays in a timely manner.The sixth step that you need to do is that you nee d to have a good understanding of the topic. You need to develop your understanding if you want to write a good essay. Understanding the subject in detail will make your essay more powerful. This is the reason why you need to go online and find out the different opinions that people have about a certain topic. This will give you a good idea of how to write the essay.The seventh step that you need to do when learning how to write an opinion essay is that you need to write persuasively. You need to make sure that you have an effective writing style in order to come up with a good essay.

The Meaning and Significance of Books to Three Characters in Dai Sijie’s Balzac and the Little Chinese Seamstress

Since the time the principal cave dweller dug to cut out pictures on rock, life has been changed, formed, shaped, and changed by the enchantment of composing. The composed wordâ€or pictures, as on account of the old cavemanâ€as made by the joined endeavors of understanding and the thoughts of the human psyche has opened entryways and ways for in any case encased spaces and impasses. One might be genuinely alone yet feel encompassed by an abundance of companions and outlandish areas brought upon by the accounts of talented writers.The experience of perusing, while regularly done on one’s own, has the ability to reinforce and grow the brain and the mentality, permitting passage to thoughts that might not have been accessible to the peruser already. Such was the aggregate impact of books on the youthful personalities of Luo, the Little Seamstress, and The Narrator; while they were each presented to for all intents and purposes indistinguishable considerations from gathered from the amazing compositions of notorious Western authorsâ€Balzac, in particularâ€their translations made a definitive difference.The enchantment of the words spun so engagingly and in a provocative way in the long run cast its spell on the particular need of every person. While The Narrator and Luo were set apart for re-training as an imperative of the Cultural Revolution, the Little Seamstress, then again, was in desperate need of instruction. The consummation uncovered the result of these objectives as far as companionship, love, life, force, and respect.II. Force and Life as Read By The Narrator Of every one of the three characters, The Narrator accomplishes the conventional degree of one who understands the nature and reason for perusing booksâ€which is the comparable force coming about because of learning new thoughts and investigating unknown domains. Books gave him the certainty to be what he never figured he could, and do things he would have never considered.The revelation of this recently discovered force unexpectedly implied new life amidst his preparation to wipe out intellectualism; in this way it was a non-debatable truth to guarantee the source, regardless of whether it implied breaking into Four-Eyes’ home, or having his â€Å"body as an energizing ground for multitudes of lice† (Dai Sijie 71) at the miller’s. The Narrator is a kid of delicate and unassuming character, making him the ideal foil to Luo’s forceful and flippant position, conceived out of his special background.The Narrator was of plentiful methods also, however Luo would exceed him in pretty much every angle. The Narrator’s information was the gained taste of violin music, while Luo’s fondness for narrating made him the more mainstream of the two. Indeed, even to their greatest advantage in The Little Seamstress, Luo developed as the victor. Subsequently when The Narrator found the force managed by Balzac, yet additionally of â €Å"Flaubert, Gogol, Melville, and even Romain Rolland† (Dai Sijie 119).The last author’s work, Jean-Christophe, end up being the most huge to The Narrator; it was maybe the solitary subject of â€Å"one man facing the entire world† (Dai Sijie 119) that resounded inside his own existence. The partition from his folks and the embarrassment that anticipated them as a major aspect of the alienated bourgeoisie, his constrained remain in Phoenix Mountain, and the guidelines that he needed to follow may have been the elements that The Narrator accepted he needed to fight.At the finish of the story, it was the estimations of affection and dedication conferred to him by the books he read that drove him to follow up on the best experience of his young life: ensuring The Little Seamstress as a guarantee to Luo. III. Experience and Conquest as Read by Luo The kid Luo had all the earmarks of being the most complete all things considered, explicitly since his disposition and interests were essentially inside the traditional idea of legends in books. A run of the mill saint was one who displayed remarkable mental fortitude, without shortcoming, and spared the lady in distress.While Luo read the books he and The Narrator got their hands on, he was especially focused with crafted by Balzac, the first was about a â€Å"French story of affection and miracles† (Dai Sijie 57). With this in his arms stockpile, Luo continued to utilize the book’s charm to catch the core of The Little Seamstress, his own variant of a storybook princess. Obviously, Luo’s relationship with books had more to do with his objective to overcome, as opposed to improve his mind.Luo previously had the endowment of talk and an intrinsic ability for turning stories, and going significant stretches to peruse Balzac’s stories to The Little Seamstress was a piece of his idea of experience. In the event that saints in books introduced gems and garments to their wome n, Luo’s offering was his obtained stories, expecting to instruct the young lady on culture, as he was of the brain that â€Å"’she’s not socialized, in any event insufficient for me! ’† (Dai Sijie 27). Much to his dismay that his steady sharing of information from Balzac’s books would confer culture, yet change the manner in which The Little Seamstress saw her own life and value.As an additional note, it is obvious that Luo, among all the characters in the novel, didn't experience a lot of progress or progress; what he was first and foremost was equivalent to at long last. Once more, this connects with the account of a legend, who consistently starts and finishes with a similar measure of solidarity and bluster. IV. Opportunity and Discovery as Read by The Little Seamstress The Little Seamstress, being a nation young lady, was the specific inverse of The Narrator and Luo; all she brought to the table were her sewing aptitudes, her feeling o f daughterly obligation, and her stunning beauty.The last quality had been elucidated upon by The Narrator finally, her face at one time he depicted as â€Å"oval†¦ and the radiance in her eyesâ€without question the loveliest pair of eyes in the area of Yong Jing, if not the whole region† (Dai Sijie 21). Being of no conventional instruction, The Little Seamstress couldn't peruse, and along these lines depended on Luo to take her through the intriguing scenes she was unable to get to. Her life, until the appearance of Luo and The Narrator, was dull, everyday, and repetitiveâ€as life in the nation during the Mao time was characterized.It could be accepted that her aptitudes in sewing were essentially obtained for absence of decision; her dad was a tailor, and an effective one at that. Ladies like The Little Seamstress, covered up in the mountains and entrusted to do female-situated occupations, had next to zero opportunity to develop mentally; and the prohibition on intellectualism during this period exacerbated this even. In this way her appreciation for Luo may be seen on the shallow level, yet in addition since she considered the to be as her lone wellspring of the sort of information she lacked.Ironically, it is her obtained information on her praised excellence that permitted her to push ahead and set out on another life; by acknowledging Balzac’s words, â€Å"a woman’s magnificence is a fortune past price† (Dai Sijie 184), The Little Seamstress set out to utilize the one quality she realized she had and investigate openings that would isolate her from the mechanical life she was destined to live. Writing offered her not simply the intriguing districts depicted to her by Luo, yet in addition the understanding that she must be a piece of such a world for her new dreams to be realized.Dai Sijie’s portrayal of her eyes as her best component had become an analogy for her new viewpoint. V. End The allotment of books as the impetus in the novel is something other than a specialized gadget to present learning new thoughts and methods of reasoning; the more vital viewpoint is the earth wherein they exist, a general public where scholarly development and investigation is considered illicit and corrupt. By making this setting, the yearn for information had gotten increasingly substantial, and its procurement, yet furtively, turned into the weapons required by the more powerless members.Having youngsters very nearly adulthood is splendidly appropriate for this contention, as they are the most fit for crossing the separations of new information. Unexpectedly, books and youngsters don't generally blend, in less prohibitive conditions; but since of the circumstance into which they had been constrained, books turned into their sole partner. Plainly, the creator took on a basic perspective on Communism and how it incredibly influenced China and its kin; by uncovering the act of ‘re-education’ , Dai Sijie set forth a credible conversation with respect to the normal human requirement for development, uniqueness, and information.

A Vet or MD Why This Student Chose Medical School

This interview is the latest in an Accepted blog series featuring  interviews with medical school applicants and students, offering readers a behind-the-scenes look at top medical schools and the med school application process. And now, introducing Abby†¦ Accepted: We’d like to get to know you! Where are you from? Where and what did you study as an undergrad? When did you graduate? Abby: My name is Abby! I grew up pretty much halfway in between Washington DC and Baltimore, MD, and I have lived in Maryland all my life. I went to the University of Maryland, College Park for my undergraduate degree, and received a Bachelor of Science in Animal Sciences with a minor in Spanish Language and Cultures. I graduated in 2016, and headed straight to medical school a few months later! Accepted: Where are you currently attending medical school? What year are you? Abby: I am currently a second year medical student at the University of Maryland School of Medicine, in Baltimore City. Accepted: When did you figure out you wanted to be a physician? Abby: My path into medicine was a little bit interesting  Ã¢â‚¬â€œ I originally wanted to go to veterinary school. During my senior year of high school, I took anatomy and really enjoyed it, which kind of confirmed my suspicions that something in the general medical field may be the right choice for me. This encouraged me to build on my background of always being curious about science, my love of learning, and also of animals, and to explore veterinary medicine with a major in Animal Sciences, which is what I did. However, once I started getting the necessary experience for veterinary school, I realized I wasnt passionate about it. I figured this out somewhat late for some  Ã¢â‚¬â€œ in my sophomore year of college. I really missed and craved more meaningful interactions with people, as well teaching, which I think medicine contains a wonderful aspect of. Working directly with the individuals you are treating allows for a unique dynamic of education, exploration, leadership, build ing relationships, and so many more things that I love. So, I kind of fell into medicine after deciding to shadow my old pediatrician, and deciding that I enjoyed those types of interactions so much better than my experiences in veterinary medicine. Looking back, it makes sense. I remember watching Discovery Health on TV as an elementary schooler, always being curious about the body and science, loving school, loving to read and learn, considering myself a leader, so on and so forth. In the end, I think it was the right decision for me! Accepted: How has medical school been so far for you? Is there anything that has surprised you? Is there anything that you wish you could change? Abby: Medical school has been great so far! Its really quite the ride. Its been the most challenging thing Ive done yet – its rigorous, its difficult. It doesnt really stop, and its taxing, especially now as a second year, getting into more pathology, having a little bit of constant anxiety about Step 1 at the back of my mind. I think it surprised me just HOW MUCH I was going to study in medical school. I knew going into it that I was going to be studying a lot, but you never really know until youre in the thick of it. I dont know what I expected – I didnt really know that weekends before exams would be spent studying literally 10+ hours a day, and that when a friend is in town close to an exam, you do have to tell them sorry, but you cant go out tonight because you have to study. Its a lot of give and take, and you need to find that balance. In terms of what I wish I could change – Im not really sure! There are things I like about medical school, and things that I dont. I think the biggest thing I wish I could change is just the fact that, like I said, life pretty much stops once an exam is approaching. Its a lot of stress, its difficult to stay grounded, and its easy to feel like youre missing out on the rest of life, as youre seeing people moving on and doing other things around you as youre studying for hours on end, feeling kind of stagnant. But, it will be worth it in the long run! Accepted: Looking back at the application process, how did it go for you? Did you experience any hiccups along the way? Abby: I was extremely fortunate in my application process. I applied once, was lucky enough to be accepted to several schools, and am very happy where Ive ended up. I just had one little hiccup, which was a me problem– I will never forget this. My personal statement went right up to the character limit– so much so, that the period at the very end of the statement was cut off. And I realized it after I already submitted! I flipped out a little bit. I thought, theyre going to think Im so careless, submitting this statement and not ending the last sentence with a period, my chances are done for. So, when my interviews started rolling in, I was SO relieved. If I didnt notice a missing period, I guess nobody else did either. Accepted: How did you prepare for the MCAT? Did you feel prepared on test day? Abby:  In terms of how I prepared for the MCAT, I personally did not take a course, I didnt really have the time or the money to invest in it. I purchased a set of MCAT books and attempted to self-study from those. I took a January exam, so I was trying to study during a semester packed with some difficult classes, like nutrition and biochemistry, while working and being a TA. Looking back, I think I would have tried to take it over a summer break, or maybe when I had some more time to myself to just dedicate to studying. I did end up having about three weeks over winter break just for studying. I ended up at the library most days, and starting about 8 weeks out from the test, I would go to a building on campus every Sunday morning at 8 am (with my best friend, who took it on the same day as I did, and now goes to medical school with me!) and we would do a full-length practice test. It wasnt fun to give up every Sunday morning, but I think that I learn best from practicing, so this was something extremely helpful that I highly recommend! Take the time to do practice exams  Ã¢â‚¬â€œ simulate the testing environment, the breaks, everything. Get comfortable with taking practice exams, and then thoroughly go over your answers to see what you got wrong, and why. You want to analyze your testing strategy to see what youre doing right and wrong, and improve from there. Accepted: Readers can find out more about you either on your blog, or your Instagram account! What made you want to start sharing your journey with others? Abby: I would love for readers to learn more about me! My blog is medstudentsaga.wordpress.com, and my Instagram is @medstudentsaga. Thinking back to why I started, I think I was really inspired by other accounts, which I started looking at once I was accepted to medical school, to kind of get a feel for what it might be like and see the experiences other students were sharing. Ive always loved taking photos, so I enjoyed the concept of documenting the huge journey that is medical school on a platform where I could be creative, easily access it myself, but also share it with others! I have really loved being able to mentor and guide other students along the way over the years, so I think I saw it as an outlet for that as well. Im always super happy to get other students reaching out to me with questions or for advice. Ive even been able to edit a few personal statements and essays (time permitting!), and its just an overall really fun way to share my experience as a medical student, while seeing what other people are experiencing, and inspiring and pushing each other to do our best. I think Ive found such an amazing community, especially on Instagram, of other medical students, physicians, and other professionals that I really did not expect, and Im so glad that I found what I did! Im looking forward to continuing sharing my journey with you all. Accepted: Lastly, can you share your top three tips for surviving your first year of medical school? Abby: 1. KEEP UP. Some of you may have heard the pancake analogy, and I really do think its a great way to describe medical school. The material, the work, the hours – it never stops. So every day, you need to eat your pancakes (do your work) from that day, so to speak. You can choose not to do it one day – I can choose to take an afternoon off and not review a lecture or take notes from the day. But those pancakes arent going away, so that makes double the pancakes for me to eat tomorrow. You can pick and choose how youd like to complete your work and study, but in order to prevent getting completely bogged down, I suggest trying to keep up with those pancakes. There will be days where you really dont want to, and thats okay. But, just find good ways to motivate yourself and take some time off if you really need it. 2. Take time for yourself. We need to stay human in medical school. Stick to your hobbies! And always remember that while medicine may define our careers, medicine does not define us. I heard a great quote from a physician the other day – she said I am a person before I am a doctor. Medicine may be your dream career, but you are a human being at the end of the day. What do you do to take care of yourself, to have fun, to enjoy your life outside of medicine? Find those things, those people, and cling to them. 3. Dont panic because you dont know what you want to do, or because you change your mind. This is still a piece of advice I am struggling with and need to remind myself of daily. The decision to declare a specialty approaches closer, and I need to remind myself that its okay I dont know what I want to do! While time goes fast, there really is a lot of it to explore – take the time to shadow, talk to physicians, do some reading and research. I came into medical school thinking I might want to do family medicine, Ive toyed around now with the idea of med-peds, pediatrics, maybe OBGYN, and at the end of the day, I still have no clue. And thats okay! Everyone figures it out at some point, and it really is never too late to make a change. Ive met several physicians who have switched residencies to specialize in something different, so while its difficult and takes time, its possible and worth it to do what you love. You can continue following Abbys story on Instagram (@medstudentsaga) and on her blog A Medical Student Saga. Thank you Abby for sharing your story with us, we wish you much success! For one-on-one guidance on your med school applications, check out our catalog of  med school admissions services. Do you want to be featured in Accepted’s blog? If you want to share your med school journey with the world (or at least with our readers), email us at  bloggers@accepted.com. For 25 years, Accepted has helped applicants gain acceptance to their dream healthcare programs. Our outstanding team of admissions consultants features former admissions directors, admissions committee members, pre-health advisors, postbac program directors, and doctors. Our staff has guided applicants to acceptance at allopathic (MD) and osteopathic (DO) medical schools, residencies and fellowships, dental school, veterinarian school, and physician assistant programs at top schools such as Harvard, Stanford, Penn, UCSF, Johns Hopkins, Columbia, and many more.  Want an admissions expert  to help you get Accepted? Click here to get in touch! Related Resources: †¢ Navigate the Med School Application Maze, a free guide †¢ Meaningful Experiences For Medical School Applicants †¢ Med School Kicks Off: Ten Tips to Get You Through the Season

Personal Narrative Friends And Family Extroversion

Friends and Family Extroversion: I utilize the traits of extroversion with my friends and family by being an extremely social person. I am very easy for people to get to know. I am not closed-off and detached like introverts. I am a very open-minded person, and am willing to express my feeling, thoughts and desires without hesitation. I am an extremely approachable person, I am the first one at a social gathering to go and introduce myself to others. I am also a huge component of small talking when first meeting someone, it is a way that can help you start to build a relationship with others. In the end I do not have to come best friends with the person, but being able to network myself, and build acquaintances will help me not only in my personal life but in my professional career as well. Finally, as an extrovert I use social setting as a means of energy for myself. I am always up to going to social gathering, and group outings and the more people the better. My mom used to work fo r the Erie County Health Department, and part of her job would be going to health fairs, and other events. She travelled all over Erie and Niagara Counties, when I was younger I was always dragged with her. Every year we went to the Erie County Fair, and as an 9-10 year old I had no other option then introducing myself and talking to people. I think this is where I got my ability to openly go up and talk to people. Friends and Family Trustworthiness: The way I utilize trustworthiness inShow MoreRelatedCareer Choices Are A Reflection On An Individual’S Personality1325 Words   |  6 Pageswith others and rewarded for sociability and humanitarianism. Nevertheless, enterprising environments are focused on leadership; they are rewarded for displays of leadership and self-confidence. Finally, conventional environments emphasize attaining personal and organizational goals through predictable and specific standards. 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The Debate Over Same Sex Marriage - 1555 Words

For years on end, same-sex marriage has been a topic of controversy. It has always been an on-going argument; there is the for-side and there is the against-side. There are many arguments for each side but which side has the most convincing argument? Maybe some of these arguments can be debunked. There needs to be a discussion about the holes in arguments and which side is the most convincing. The history of same-sex marriage was anti-climatic for a long time. It seemed that for too many years it was frowned upon, banned, and ultimately seen as disgraceful. â€Å"On May 18, 1970, two University of Minnesota students, Richard John Jack Baker and James Michael McConnell applied to Hennepin County District Court clerk Gerald Nelson for a†¦show more content†¦Those opposed to it came up with far-fetched arguments and went to extensive lengths to rid the USA of same-sex marriage. Their extensive lengths being abuse against the LGBT+ community, rallying at public hearings, and continuing to treat gay people as animals. Those opposed to same-sex marriage have come up with many arguments that do not really have any thought behind them. One of their arguments is, â€Å"Same-sex marriage is against my religion.† Religion is to be respected but how many people have not read the Bible front to back. The Bible has spoken against many other things, but these things are excused every day. â€Å"The Bible says that you can execute people for cheating on their spouses [...] The Bible also says that you can keep slaves† (10 Reasons). People have decided to pick and choose which parts of the Bible they want to follow. This argument has been continuously used over the years. Religion has often been used as a scapegoat for abuse against the LGBT+ community. Religion should be hope, support, and faith†¦ not inhumanity, violence, and discrimination. Another argument often used is, â€Å"Marriage has always been between man and woman.† This is the common â€Å"we-cannot-break-tradition† argument. Over the years, we have broken tradition many times. One example would be discrimination against black people. It was considered â€Å"tradition† for white people to own black slaves†¦ do we still follow this tradition? No, because it

Philosophical Transactions of Royal Society

Question: Discuss about the Philosophical Transactions of Royal Society. Answer: Introduction The ceratotrichia, collagenous protein present inside the fins of shark are the main constituent that serves as the delicacy in the Shark fin soup. There is an increased activity of shark fishing that drives the soup market which is a very unsustainable. In light of the declining shark population the Convention of International Trade in Endangered Species (CITES) have listed eight species of shark which are Cetorhinus maximus, Lamna nasus, Sphyrna lewini, Carcharhinus longimanus, Carcharodon carcharias and S. zygaena as well as S mokarran (Shivji et al. 2005). It is to note that any kind of international trade involving the listed species should be accompanied with special permit that certifies that the trade is not detrimental to the population of the shark species. Therefore, there is a common need for both the exporting and the importing countries to understand and validate which species of shark are they trading. Though shark species can be determined by looking at morphological features, processed samples are difficult to be identified. Using DNA barcoding methods can help in such situations. The PCR amplification of cytochrome c oxidase I gene is a standard barcoding technique that is used. However, this process may be limited by extensive processing of the samples with chemicals that can damage the DNA making it literally impossible to obtain intact DNA and sequences for amplification to aid in genetic identification. DNA barcoding is a technique that aids in identification of species through sections of DNA from a specified standard region of the genome. It is given the term barcoding as every species has a unique sequence that is conserved throughout the species and is not different among members of the same species. Therefore establishing a specified sequence as a DNA barcode will eventually help in species identification with ease. The most often used gene region is the 648 base pair cytochrome C oxidase 1 gene (Moritz Cicero 2004). The COX1 gene has been extensively used to establish species identification among many oranisms such as birds (Hebert et al. 2004), butterflies (Wiemers Fiedler 2007), fishes (Weigt et al. 2012) etc. the region is extensively used for species barcoding because it is long enough to be sequenced and yet get a good result as well as small enough to be easily amplified using a PCR. However, the COX1 gene is not so useful in identification of plant species (Chase et a l. 2005). In order to establish a barcode the obtained specimen is lysed and DNA is isolated. The isolated DNA is then amplified for the COX1 gene and sequenced. The sequence is then called a barcode that is deposited in the barcode of Life Data Systems (BOLD) database. Thus the BOLD database is a repository of DNA barcodes and can be used to identify unknown species using DNA sequence from the COX1 gene (Ratnasingham Hebert 2007). Similarly, though not a barcode repository the sequence database NCBI GenBank stores sequences so far identified of organisms. It is an extensive repository of sequences and all sequences known till date is updated in the database. Therefore searching the database for similarity in the obtained sequence of a specimen will also help in establishing the proximity of phylogenetic relationship between two organism. The two approach BOLD and GenBank search using BLAST (basic Local Alignment and Search Tool) will help in proper identification of unknown sample based on t heir DNA sequence. There are other methods that employs other segments of DNA (Pinhal et al. 2008)other than the cytochrome oxidase gene present in the mitochondria (Naylor et al. 2012). However, COX 1 gene remains the most exploited DNA sequence. The present technique aims to identify shark specimen based on their COX 1 gene obtained by PCR amplification of DNA obtained from tissue samples. Genomic DNA isolation DNA from the shark fin samples were isolated after pulverization of the hard tissues using a kitchen blender to yield single cells. The shark tissues collected were stored at 70% ethanol before pulverization. 2 mm3 of the pulverized cells were cells were lysed with proteinase K (20g/mL) and 580 L of TNES (50mM Tris, 400mM NaCl, 100mM EDTA, 0.5% SDS) buffer followed by incubation at 55oC for 3 hours in a waterbath. After incubation the tube is transferred to room temperature and 170 L of 5mM NaCl was added followed by vortexing for 15 seconds. The sample was then centrifuged at 14000 rpm for 10 minutes to remove cell debris. The supernatant is recovered to which 770 L of ice cold 100% ethanol was added. The mixture was inverted 5 times to allow precipitation of DNA followed by centrifugation at 14000 rpm for 5 minutes. The pellet was collected followed by washing with 70% ethanol to remove residual salt followed by centrifugation at 14000 rpm for 5 minutes. The supernatant was removed and pellet was dried at 55oC for 10 minutes followed by resuspension in 30 L of sterile water. The quality of DNA was determined by measuring absorbance at 260 and 280 nm and taking the ratio of the two. The amount is determined by measuring absorbance at 260 nm. PCR reaction and sequencing DNA isolated from the shark tissue samples were subjected to PCR amplification using cytochrome oxidase I gene forward primer 5 TCA ACC AAC CAC AAA GAC ATT GGC AC 3 and reverse primer 5TAA ACT TCA GGG TGA CCA AAA AAT CA3 was used. 500ng of the template was taken in a PCR tube with 100pM of primers and mixed with premixed BYBR green mastermix (KAPA SYBR FAST qPCR Master Mix 2X, KAPABiosystems). Final volume of 20L was used for the PCR set up. PCR was conducted at 95oC denaturation, 60oC annealing for 30 seconds and amplification at 72 oC for 30 Seconds. The cycle was repeated for 30 cycles followed by a final extension step of 72oC for 5 minutes. The product was then analyzed on an agarose gel to validate the size of the product. The PCR product was then subjected to DNA sequencing. The sequence obtained was subjected to alignment using NCBI BLAST and Clustal W on Bioedit software. The sequence alignment at NCBI BLAST was also used for establishment of species identity and drawing a phylogenetic tree to identify the species of the shark to which the sample belong to. In BLAST alignment the most similar sequence to the query sequence was considered as the species. Further the species was confirmed using the barcode of life website BOLDSYSTEMS. During alignment using ClustalW segments spanning 150 bp without any missing nucleotides in all the six sequence provided were used for in depth analysis of similarity and difference in the nucleotides. Around 20g of DNA was recovered from the tissue sample provided which was estimated by measuring absorbance at 260nm using a UV spectrophotometer. 500ng of DNA was used as a template for PCR amplification with the 100pM of forward and reverse primer as described in materials and methods. The PCR yielded around 650 bp product which was the expected size of the product (Figure 1). The amplified product showed a good amplification but was also seen with some non-specific amplified product that needs to be cleaned up before setting up a sequencing experiment. The PCR product obtained was excised from the agarose gel and purified using a spin column to obtain the specific PCR product and to negate the non-specific amplification that was observed along with the PCR amplicon. The sequence yielded on analysis of the PCR product (Figure 2) was subjected to alignment using BLAST and Clustal W. The sequence alignment of the DNA sequence obtained from the PCR product yielded a very similar hit on BLAST analysis. The shark species was identified as Carcharhinus obscurus. The phylogenetic tree was also established that showed their evolutionary relationship with other shark species (Figure 3). The sequence that was obtained after PCR amplification when aligned with already established sequences of species revealed a 100% match with the shark species Carcharhinus obscurus and other subspecies of the same. We also found a close phylogenetic relationship of the identified species with Carcharhinus leucas. The barcode assay that we conducted using the COX 1 gene led to successful amplification of the target with the expected product size. The primers used may be used as a common or universal primer for identification of other shark species as it will amplify the COX 1 genes of most species. However, a distinction between species can only be made after realizing the actual difference in the sequence. Therefore, the PCR amplification is not the only step that can identify the species but obtaining a sequence of the PCR product is a must. The sequence that was obtained after PCR amplification when aligned with already established sequences of species revealed a 100% match with the shark species Carcharhinus obscurus. Other shark species that were closely related to the identified species were sequences identified as Carcarhinus obscurus 2, 3 and 4. These may be subspecies of the identified Carcharhinus obscurus species. The identified species is closely related to Carcharhinus leucas and prionace glauca as seen from the phylogenetic report. The identified sequence was also similar to Carcharhinus sps. Indicating a close phylogenetic relationship. GenBank and BOLD sequence alignment is helpful in identification of species that are listed in CITES. We observed a high sequence similarity between CITES listed species and the closest relative was as similar in sequence as a difference of only 2 bp as observed earlier (Fields et al. 2015). A more thorough identification strategy may be formulated to discriminate between closely related Carcharhinus obscurus species. A combination of BOLD and BLAST identification is recommended for such work. The data thus obtained will help in establishing species of sharks that are being commercially exploited and will help conserve endangered species from over exploitation (Baum, Kehler Myers 2005). The combination of BOLD and BLAST strategy is highly efficient but may need visual inspection of short nucleotide sequences. Using the barcoding assay many applications can be formulated which are: law enforcement agencies can be confident of the identification technique and use it as a legal tool to present evidence of species identification, the method can be employed to both processed and unprocessed samples. The sequence can also be used to establish stock records of shark species and may be helpful in understanding origin of the stock and migration pattern (Ward, Hanner Hebert 2009; Ward et al. 2005). The protocol may also be useful to quantify types of species that are being extensively marketed and used as soup ingredients. Nevertheless, there are shortcomings that need to be addressed as we have tried the amplification with a sample that is already available with us. We may try to collect sample from various sources in both processed and unprocessed forms to validate our result and show its applicability in different sample forms. However, we are confident that the protocol will eventually help in identification of endangered species that has been overexploited and help in enforcing conservation strategies to save declining population of some species of sharks. References: Baum, J, Kehler, D Myers, R 2005, 'Robust estimates of decline for pelagic shark populations in the northwest Atlantic and Gulf of Mexico', FISHERIES-BETHESDA-, vol. 30, no. 10, p. 27. Chase, MW, Salamin, N, Wilkinson, M, Dunwell, JM, Kesanakurthi, RP, Haidar, N Savolainen, V 2005, 'Land plants and DNA barcodes: short-term and long-term goals', Philosophical Transactions of the Royal Society of London B: Biological Sciences, vol. 360, no. 1462, pp. 1889-95. Fields, AT, Abercrombie, DL, Eng, R, Feldheim, K Chapman, DD 2015, 'A novel mini-DNA barcoding assay to identify processed fins from internationally protected shark species', PloS one, vol. 10, no. 2, p. e0114844. Hebert, PD, Stoeckle, MY, Zemlak, TS Francis, CM 2004, 'Identification of birds through DNA barcodes', PLoS Biol, vol. 2, no. 10, p. e312. Moritz, C Cicero, C 2004, 'DNA barcoding: promise and pitfalls', PLoS Biol, vol. 2, no. 10, p. e354. Naylor, GJ, Caira, JN, Jensen, K, Rosana, K, White, WT Last, P 2012, 'A DNA sequencebased approach to the identification of shark and ray species and its implications for global elasmobranch diversity and parasitology', Bulletin of the American Museum of Natural History, pp. 1-262. Pinhal, D, Gadig, OB, Wasko, AP, Oliveira, C, Ron, E, Foresti, F Martins, C 2008, 'Discrimination of shark species by simple PCR of 5S rDNA repeats', Genetics and Molecular Biology, vol. 31, no. 1, pp. 361-5. Ratnasingham, S Hebert, PD 2007, 'BOLD: The Barcode of Life Data System (https://www. barcodinglife. org)', Molecular ecology notes, vol. 7, no. 3, pp. 355-64. Shivji, MS, Chapman, DD, Pikitch, EK Raymond, PW 2005, 'Genetic profiling reveals illegal international trade in fins of the great white shark, Carcharodon carcharias', Conservation genetics, vol. 6, no. 6, pp. 1035-9. Ward, RD, Hanner, R Hebert, PD 2009, 'The campaign to DNA barcode all fishes, FISH?BOL', Journal of fish biology, vol. 74, no. 2, pp. 329-56. Ward, RD, Zemlak, TS, Innes, BH, Last, PR Hebert, PD 2005, 'DNA barcoding Australia's fish species', Philosophical Transactions of the Royal Society of London B: Biological Sciences, vol. 360, no. 1462, pp. 1847-57. Weigt, LA, Driskell, AC, Baldwin, CC Ormos, A 2012, 'DNA barcoding fishes', DNA Barcodes: Methods and Protocols, pp. 109-26. Wiemers, M Fiedler, K 2007, 'Does the DNA barcoding gap exist?a case study in blue butterflies (Lepidoptera: Lycaenidae)', Frontiers in zoology, vol. 4, no. 1, p. 8.

Learn How to Assertiveness and Appropriate Confirmation - Learn the Right Way to Act When Someone Says Something Wrong

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What You Need to Know About Essay Topics about Social Media

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