Crime and Punishment | Character Analysis

umbrage and Punishment Character AnalysisIn the refreshing Crime and Punishment, the author Fyodor Dostoevsky prep ars a character in Raskolnikov who is plagued with dueling personalities. One side of Raskolnikov is caring and sensitive, often sharing what s movet(p) m integrityy he has with oppo sends, while the other side is dark and in contrasting, eventu alto pull backhery draging him to commit the murders of a pawnbroker and her sister. These personalities nominate an inner-conflict that progressively grows worse and drives Raskolnikov insane until he is forced to avow his act.Dostoevsky introduces the conflict within Raskolnikov archean in the first chapter. Raskolnikov is contemplating more than or less mysterious thing that is non announced to the endorser and simply referred to as that. In his mind, he continuously goes back and forth e realwhere whether he is truly thinking of doing that or if it is erect a sight going near in his betoken. As he is finis hing his thoughts, he attempts to shake off whatever that is by telling himself, It is not that serious at every last(predicate). Its simply a fantasy to am affair myself a toy, scarcely after a short moment he ends with, Yes, peradventure it is a plaything, (Dostoevsky, pg. 2). In this quotation the maybe exclusivelyows a reader to assume that Raskolnikov may, in fact, be considering this thing. The chat Raskolnikov holds with his self, as he goes back and forth over this issue, shows that he his mind is conflicted. This thought so primal in the novel lets us see Raskolnikov is already thinking to himself entirely is not even current of his own thoughts. Such indecision over what it now an unmenti atomic number 53d matter dress outs the t single for this inner-conflict that he struggles with by discover the novel and that nearly consumes him.When the truth of what that is comes out, readers grasp the extremity of the inner-dual of Raskolnikov. That, the murder of an inn ocent woman, is several(prenominal)thing truly unimagin able-bodied by, what society considers, a rational person. Raskolnikov is endlessly at odds with whether or not this is reasonable an idea formed in his head or if he is imparting to commit such an act. In a dream he sees the brutal torture of a horse, which leads to its death. after the dream Raskolnikov wonders how he could even contemplate murder and verbalise the thought of it construction, My God Anyway I couldnt bring myself to do it I couldnt do it, I couldnt do it (Dostoevsky, pg. 54). Such adamant renouncement of the act is suddenly and easily altered as he strolls upon a conversation where he discovers that Alyona Ivanova, the woman he is thinking of murdering, will be home alone at a certain fourth dimension. Raskolnikov sees this as fate and completely forgetting almost his dream once again alters his expectation and decides to commit the crime. The plot thickens and the clash continues inside the mind of Ra skolnikov, as what was once just a plaything grows into a reality.Throughout the novel the reader is able to see the tremendous amount of unsupported pridefulness Raskolnikov holds for himself. Although he lives in what seems to be a slum in St. Petersburg, does not pay his rent, and he has deep dropped out of school, he believes that he is better than those approximately him. In spite of the level of pride he possesses he is allay content to leave his house dressed as though he has no regard for his appearance. With that said, in explaining an phrase that he wrote, he says,I simply hinted that an extraordinary man has the slumpthat is not an official right, but an inner right to decide in his own conscience to come aboutcertain obstacles, and and in case it is essential for the practical fulfilment of his idea,(Dostoevsky, pg. 226)whatever that idea may be. He feels that he is so superior to others that he even has the right to take the life of other human beingness, thou gh he adds, some ages, perhaps, of benefit to the whole of humanity, (Dostoevsky, pg. 226). He attempts to validate his point by saying that it is more reasonable for a superior being to break the law when the act benefits all of society. This allows the reader to see that his mind is clear mergeed up. He believes that he is superior, though he does nothing to upgrade it. His sole claim to fame is this article that was published in a new-fashionedspaper and that does not contain his sig spirit, only his initials.One of the most signifi discharget views of Raskolnikovs dueling personalities washbowl be seen through his interaction with others. The moral-psychological traits of his character incorpo sum up this antinomy amid consciousnessive kindness, sympathy, and pity (Unkown). Theses ar the words of a law student who explains that when acting on instinct Raskolnikov is kind, sympathetic and has pity for others. While he can often be extremely compassionate for those around him his admitted feeling of superiority over others leads him to the mistreatment of them as well as his delusional ideal mentioned in his article On Crime. The compassion he shows is frequently counteracted, because after performing an act of kindness Raskolnikov is often moot at himself for doing the deed. What a stupid thing Ive done, they postulate Sonia and I want it myself, (Dostoevsky, pg. 23) Raskolnikov says this after he leaves some money, which he does not bring in much of, for a friend and his poor family. This shows his feeling of regret for doing what is clearly a good deed. Later, he shows regret after protecting a drunken unfledged girlfriend who is being pursued by an older man. The fact that Raskolnikov is not able to make what he sees as quality decisions in spur of the moment situations can be attributed to the confusion at play in his mind and the insanity it is causing. His personality is always at odds and Dostoevsky uses the continual shifting in his cha racters head to show this. Raskolnikovs idea that he is superior is disproved not by his actions but by the regret he has for making decisions that seem to be rational.In the weeks following Raskolnikovs crime his mental condition deteriorates even more. After many days of sick sleep he is surrounded by many of the commonwealth he knows and some he does not know. Raskolnikov finds that he actually feels he is wrong for what he did. The conflict in his head shifts from the decision of whether or not to take the life of the pawnbroker, to whether or not he should confess to his crime. However, Raskolnikovs beliefs be undermined by the guilt and illness he experiences after the murder, creating a split in which he desires to alleviate his guilt and also desires to affirm himself as extraordinary, (TheDoctor). This new split in his mind grows out of the original conflict. His guilt is something that is not completely shown, but it is present. Raskolnikov believes, even as a convict i n a Siberian labor camp, that his crime would have been something under die hardable had not the pawnbrokers innocent sister walked in and seen him. Raskolnikovs guilt grows due to those he is closest to. Sonia encourages Raskolnikov to confess his crime because it is the only way he could be redeemed. Even with the one he loves most telling him to confess, Raskolnikovs pride is still present and he possesses a pack to prove that he is truly a superior being. With all these desires going on in his head Raskolnikov does not know how to swallow his pride and accept that the only way he can exist is through repentance.In his insanity Raskolnikov is driven to do many fanatical things as he abandons his sister, mother, and best friend, and nearly lets the truth, that he committed the murders, slip out. He is once again alienated from everyone, draw out Sonia. The relationship between Raskolnikov and Sonia grows strong and he learns to trust her. In the books concluding scene Raskolnik ov finds himself in a state of near delirium at the police transmit, and he confesses his crime, (Unknown). This quotation explains Raskolnikovs mental condition as the story is coming near to a close. Raskolnikov is so taken by his insanity that he wonders through the city in an attempt to unrestrictedly confess. He is so horrified by the crowd that he cannot bring himself to do the confession publicly. His mind could b bely function as he walked into the police station where he would finally declare his guilt and own up to his crime. When he hears of the suicide of someone he knows he becomes so conf utilise that he walks out of the police station. His mind is now so debile that he can be sidetracked easily. He reenters the station when he sees Sonia, the one who has finally convinced him to confess. It seems as though she has to actually control his mind in the final scenes in come out for him to have replete strength to confess.Crime and Punishment, as a novel, contains man y ideas of the author Fyodor Dostoevsky. Nearly everything in the book revolves around his character Raskolnikov. Raskolnikovs inner-struggle accounts for a majority of the novel. From his love for his family, to his devotion to his friends, and even his willingness to risk his life for strangers, he can be considered good. Due to his selfishness and pride, which he allows to alienate him from society, he may be considered just the opposite. When these dual personalities mix with the ideals he has created during his extended periods of separation from others, it figurerates a man whose mind is torn into many counsels. Raskolnikov is unable to hire his own direction and allows chance to control his actions. He is close to going completely insane and he probably would have if not for the relationship formed with Sonia in which she takes the place of chance and guides Raskolnikov in the direction of redemption.Impact of picture The Kennedy Nixon DebatesImpact of Television The Ke nnedy Nixon DebatesWhy were the Kennedy-Nixon debates in 1960 so important for the political charm of American television receiver?IntroductionThe debates between John Kennedy and Richard Nixon in the last days of the 1960 presidential shift have become both famous (in the American publics imagination) and influential (in determining the nature of subsequent political campaigns, not only in America but in other westbound democracies as well). These debates were watched by more than 70 million viewers in America (and millions more listened on the communicate). However, beforehand the start of the campaign, it was by no means clear that Kennedy would win. At the beginning of 1960, President Eisenhower was still a popular candidate, and would have won had he been allowed by the constitution to stand for a trine term. Lyndon Johnson had a regionalised support based in the South, Kennedy seemed far too young and inexperienced, and Vice-President Nixon did not have the cartel of t he electorate.1 These television debates (the first time that a presidential debate had been televised) were therefore crucial in lovable the election for John Kennedy, and for securing the defeat of Nixon, though the television (and the photograph created from being on television) had never before played such a pivotal role in an election campaign. How did this come about?Television as crucial to Kennedys campaignKennedy formalised his declaration for the presidency on 2nd January 1960, which some professional politicians felt was too early to begin a presidential campaign. Kennedy realised that he had a problem, but he was forced both by his own inexperience (he needed more time to prove himself) and by the hostility he faced from senior members of the Democrat party. In secernate to stand any change of being nominated, Kennedy needed to make his campaign open and public, and importantly, to use television to create a good public check, to attract the public, and to excite the voters by presenting himself as the candidate best fit to take America forward into this promising new era, embodied by hope and optimism.2 Kennedy also had to sweep over several problems that would have had a hugely negative impact on his public image he suffered from chronic poor health and he was promiscuous, despite his prominent and public marriage to Jacqueline Bouvier in 1953 at a society wedding in Newport, Rhode Island, in front of a crowd of 3,000. It is notable that Kennedy managed to go for this aspect of his personal life quiet only because the media paid no attention the private lives of those in office, but this was in 1960, and before Kennedy was about to remake politicians as television celebrities. Kennedy managed both to overcome these detractors to his image, and to create a positive image despite others criticisms of his ability to hold office, by appealing straight to voters, via the new mass media technology of television and television marketing this was a remaking of American politics.3The medium is the kernelIt is the nature of the medium of television, with its combination of visual and audio clues, and its instant, mass communication, that a great many people can come to an instant judgement of a candidate based not only on what he says, but also on how he says it. The first presidential debate was key all triplet subsequent debates were based upon the Kennedys initial television succeeder. It is interesting to compare the television with the radio debate for an example of how influential the television debate was, and for how it would secure the primacy of the medium of television over radio, something which has not yet been changed in the modern age of political electioneering. Radio listeners rated Nixon the victor in these debates, while television viewers believed that Kennedy had won. If there was such a clear difference between views on different media, thusly(prenominal) it must be the case that the nature of the me dia is the explanation. On television, viewers could see a Kennedy who was well-dressed, handsome and tell (in other words, a political celebrity) against a poorly presented and badly dressed Nixon. Neither candidate had in actual fact any great difference between them in price of their political policies Kennedy was just incredibly flourishing at emphasising his dynamism, youth, vigour and optimism, in other words, a triumph of style over political substance. The voter turn-out of the election shows just how good Kennedy was at stimulation the interest of the electorate via his good image-presentation. The turn-out (in terms of percentage of adults of voting age casting votes) was the highest it had ever been in the history of American politics, especially among African American voters, whose vote Kennedy had managed to secure by his high-profile knowledge with the civil rights movement.4President as celebrity the pore on imageThe television debates greatly advanced the popula r image of Kennedy. For example, many people had thought that Kennedy was too young (he was the first American president to take office born in the twentieth century, at age 43)5 and too inexperienced to take office. This would have been a serious criticism of the would-be(prenominal) president, and in fact, this was only dispelled by his appearance on television, something that Kennedy himself later admitted We wouldnt have had a appeal without that gadget.6 He came across as cool, calm and collected, with poise and knowledge enough to endure the responsibleness of holding presidential office.7 It was these presidential debates in 1960 that really propelled Kennedy to power, on the back of the positive public image he had generated. In fact, so successful was the careful management of presidential appearance on television, that the image of Kennedy as youthful, dynamic, optimistic and able to seize the moment was never lost after his election to office. Instead, Kennedy went on to embody the nascent optimism and expectancy of early 1960s America (and the image of Kennedy as glorious and expansive was established all the more firmly by his assassination on 22nd November, 1963).8 This was a time when great social plans and movements could be won (the civil rights movement), a time when new frontiers were being expand and explored (the space race) and a time when many dreamt of increased affluence and economic prosperity.9The lesson learntNo one more than failed presidential candidate Nixon was aware of how important creating a positive image on television (and radio) was spirit back on all four of them, television debates there can be no question but that Kennedy had gained more from the debates than I.10 It is clear that these television debates were crucial for securing electoral success Nixon failed to win the presidential campaign in 1960, and he attributed this directly to the way his image was managed (especially on television) I recognized the basic mistakes I had do. I had concentrated too much on substance and not enough on appearance.11 Nixon never made that mistake again. In 1968, when he again ran for president, he made sure that his appearances on television were well-nigh controlled and timed, in order to appear cool, calm, and collected in other words, just as Kennedy had so successfully appeared in 1960. It worked. Nixon became president in 1968, though he often tried to deny that he was one of the first presidents to realise that image (most easily communicated via the mass media of TV) was crucial to electoral and thence political success I dont worry about polls. I dont worry about imagesI never have.12 This, of course, was not true. Nixon expended a great deal of energy into maintaining a good public image,13 something which no president had done to quite the extent before, and something which has also set a precedent for all subsequent western political campaigns they have learnt Nixons 1960 presidential lesson never ignore the importance of show well on television.ConclusionBy looking at the Kennedy-Nixon debates of 1960, we can see that they were important because they set the precedent of television having an enormous political influence, in terms of setting up and disseminating an instant and successful public image. The lesson of the importance and influence of television is one that no candidate for office has ever been able to ignore, either in America or wherever television has a wide bedcover hold on mass-media communications.BibliographyBarnouw, E., The Image Empire (New York, Oxford University Press, 1972)Havel, J.T., US Presidential Candidates and the Elections A Biographical and Historical Guide (New York, Simon Schuster Macmillan, 1996)Nixon, R.M., Six Crises (London, W.H.Allen, 1962)Paper, L.J., The look for and the Performance The Leadership of John. F.Kennedy (New York, Crown Publishers Inc., 1975)Rorabaugh, W.J., Kennedy and the Promise of the Sixties (Cambridge, Cam bridge University Press, 2002)1Footnotes1 Rorabaugh, 2002, 6.2 ibid., 2002, 7.3 ibid., 2002, 13.4 Rorabaugh, 2002, 17.5 Havel, 1996, 318.6 Barnouw, 1972, 169.7 Paper, 1975, 300-301.8 Havel, 1996, 318.9 Rorabaugh, 2002, x.10 Nixon, 1962, 384.11 ibid., 36612 From a rare interview with Nixon conducted by Barbara Walters in March 1971 on the Today programme on NBC television.13 For example, the highly stage-managed presidential visit to mainland China in 1972, in which Nixon was concerned to impress upon the electorates mind the image of a presidential diplomat, bring home the bacon in establishing good relations with a country who had been an outspoken critic of America for over 20 years. He did this, of course, via a carefully controlled media campaign, which often seemed ilk a campaign for some new consumer item.Genotyping ApoE Variants For archeozoic Diagnosis of ARCGenotyping ApoE Variants For Early Diagnosis of ARCNeven AbushabanGenotyping ApoE renderingsPredictor of rare mali gnant neoplastic disease in young adultsAccording to Yamashiro (2017), The rare ApoE related cancer (ARC) occurs in mostly in young adults with 80% of all cases being in people between the ages of 20-30 years old. ARC is unbiased to gender and there is demo that it is a hereditary infirmity linked to the inheritance of eight variants of the gene Apolipoprotein E (ApoE), that are spread out through the gene, including two that are in intronic eras. These alleles seem to be recessive with some of them that when they are recessive homozygotes being strong predictors of ARC. It was also sometimes found that when there is heterozygosity of two recessive ApoE alleles, one being ApoE4, can act as recessive homozygotes. However, ApoE4 has the weakest association with cancer at less than 2%, but when another ApoE allele is present the ApoE4 allele pairs as a normal ApoE gene (Yamashiro, 2017a).The ApoE gene encodes for the apolipoprotein E which combines with fats in the body to form lipo proteins (US National Library of Medicine, 2017). The ApoE lipoproteins are responsible for maintaining normal cholesterol levels in the bloodstream and the brain by transporting cholesterol and other fats in the bloodstream and assisting deposition of amyloids and the modify of deposits from the parenchyma of the brain (Garg Roth, 2015). The allele ApoE4 has the weakest link to ARC, less than 2% and when it pairs with another recessive ApoE allele they seem to be equivalent to a normal ApoE gene (Yamashiro, 2017a). Some variants of the ApoE gene increase the risk of transgressing totality disease, Alzheimers diseases (AD), and ARC. Compared to the other alleles ApoE 4 increases the risk for AD and it was also found to be a risk factor cerebral amyloid angiopathy, dementia, and multiple sclerosis (Zhong, et al., 2016).Tests for Genotyping ApoE VariantsEarly receiveion of ApoE alleles that are high-risk factors for ARC is infallible for the most effective treatment of the canc er. It is essential to use rapid and cost-effective block outs to genotype all 8 recessive alleles of ApoE to determine carrier status of the recessive alleles and homozygosity of the recessive alleles that will most likely lead to ARC development. There are several discharge rules for ApoE genotyping and this case study will focus on RT-PCR, Oligonucleotide Microarrays, and Next coevals Sequencing.Real-time PCR (RT-PCR)Conventional polymerase chain reception (PCR) which was conceptualized by Kary Mullis in 1983 and it has the ability to hit the roof special nucleic acid eons exponentially in a short amount of time. PCR refinement is achieved through multiple cycles of denaturation, annealing, and extension in a thermocycler that controls the temperature for distributively cycle (Kuslick, Chul, Yamashiro, 2008). In the denaturation step the chemical reaction mix (which contains the desoxyribonucleic acid template that will be amplified, a pair of primers, Taq polymerase, the four building blocks of the deoxyribonucleic acid which are the deoxyribonucleotide triphosphates (dATP, dCTP, dGTP,and dTTP), a salt with Mg2+ (a divalent cation), and a buffer) is heated for some time to cause the double-stranded deoxyribonucleic acid to dissociate in preparation for interbreeding of the primers onto the desoxyribonucleic acid template. During the annealing step, the temperature is brought down in order for the primers hybridize on severally(prenominal)(prenominal) deoxyribonucleic acid strand. Finally, in the extension step, the thermostable deoxyribonucleic acid polymerase, the Taq polymerase synthesizes the DNA strands with the primers to make new complementary DNA strands (Kuslick et al, 2008). The amplicons, which are the amplified double stranded deoxyribonucleic acids, are visualized as bands by agarose gel electrophoresis. Conventional PCR is useful for DNA amplification however, it has a time-consuming procedure and data analysis of the result s as each marker postulate to be investigated separately by PCR, thus taking a long time to get to a final diagnosis (Irshad et al, 2016).Real-time PCR (RT-PCR) is a PCR technique that allows for visualization of the DNA while it is amplifying by the accompaniment of a fluorescent primer/ try out to the reaction mix and running the reaction under unseeable light with a video camera recording each cycle, and translating the data into an amplification curve (Valasek Repa, 2005). RT-PCR has the readiness of genotyping all eight ApoE gene variants through SNP (single nucleotide polymorphisms) genotyping using TaqMan technology by amplifying each variant in a separate tube using a forward and reverse primer specific for each target sequence of the alleles. TaqMan is an RT-PCR system from Roche and utilizes a primer/probe with a reporter spot and a quencher tinge attached, for visualization and follows a similar protocol to that of conventional PCR. The difference is, however, when the Taq polymerase is extending the DNA it frictions the probe and a 5-3 exonuclease activity will cleave the probe which in turn untethers the reporter dye past from the quencher dye releasing the signal from the reporter dye and the signal is then timed by the equipment that the reaction is being conducted in (Zhong, et al., 2016). Some advantages of SNP TaqMan RT-PCR are that it is a closed reaction system which reduces the risk of pollution of amplicons, has very little labor in the protocol, and takes less than a day to get to a final diagnosis. A disadvantage is that requires an RT-PCR machine that can read data at real time quite than using a fluorescence reader utilise for conventional PCR (Geyer, Reisbig, Hanson, 2012).Non-technical Parameters. When designed optimally the primers for RT-PCR can be very accurate with high specificity and sensitivity of the results. There are many companies other than Roche, like Thermo Fisher scientific that offer a variety of TaqMan baulk formats for real-time PCR such as singles, 96-well plates, 384 microfluidic cards, and openArray plates (Thermo Fisher Scientific, 2017). The cost per assay for TaqMan can be from around $3 and goes up to $350 (Science Exchange, 2017a).Oligonucleotide MicroarraysDNA microarray technology was originally designed to measure the RNA transcriptional levels of genes in a genome. With this technology, it is now possible gene expression patterns for studying diseases, disease progression, detect single nucleotide polymorphisms (SNPs), and identification for drug targeting. Microarrays use single stranded DNA sequences as probes just like in PCR to form complementary hybrids with the target DNA sequences to measure the expression of multiple genes. Thousands of DNA probes for the target sequences are bound, synthesized, or spotted to a silicon chip wafer similar to those used for estimator microchips.There are two main types of DNA microarray chips methodologies and it depends on the type of probes that are to be spotted (Trevno, Faclciani, Barrera-Saldaa, 2007). One type was developed by Affymetrix that is adapted from the manufacturing of semiconductors and synthesizes short single-stranded oligonucleotides, about 22 nucleotides in length, in situ onto the wafer (Trevno et al, 2007) (Yamashiro, 2017c). The second type uses reverse transcription of messenger RNA (mRNA) to get complementary DNA (cDNA) for the cloning of the double-stranded DNA gene sequence, and then amplification of the open reading frames using PCR. The cDNA are the probes bounds to the wafer. A limitation of the cDNA method is that there is an uneven melting temperature due to the differences in the CG- content of the large open reading frames or cDNA sequence probes. There is also non-specific hybridization from overlapped genes, related sequences, and variations in splicing. The oligonucleotide method is designed in such a way that overcomes that of the cDNA probes, by designing the ol igonucleotide probes to be complementary to the target sequence and redundantly detect the target segments (Pastinen, et al., 2000) (Trevno, 2007).The extracted nucleic acid sequences are designate with fluorescent dyes and are hybridized onto the DNA array through incubation and afterward, non-specific hybrids are washed off. The fluorescent dyes are detected through a laser in a confocal scanner that excites them and then produces a digital image of the microarray. Special software is used to analyze the image that assigns a final reading of a value that is relative to concentration in each spot of the probe of the target sequence being measured. There are some microarray methods that are competitive two-dye assays that uses two types of fluorophore dyes, one for the target sequence and the other for the reference sample (Trevno, 2007). The microarray reading assigns a ratio of the two dyes equal to the amount of the target sequence to the reference sample. This method is suitabl e to for measuring a small number of genes (Trevno, 2007).Frequently the oligonucleotide microarray method is used for large scale multiplex genotyping of multiple alleles, mutations, and single nucleotide polymorphisms (SNP), and it would be the method of picking for genotyping the ApoE alleles (Pastinen, et al., 2000). chromosomal microarray is a type of oligonucleotide microarray, that is commonly used in clinical laboratories as a genetic test for analyses of genomic copy number, SNP, karyotyping for visualization and analyses of chromosomal rearrangements like gains and losses (Miller, et al., 2010). Each ApoE variant sequence would be identify using two to three oligonucleotides for the sense and antisense strands. The array would have data points for the sense and antisense primers for analyses in order to reduce the occurrence of false positives. A genotyping software would then identify the variant sequences in each patient tested (Schrijver, 2005).A downside of microarray s is that for some genetic carrier screening, such as Cystic Fibrosis carriers, a second tier of testing is often required to prove carrier status. The second tier is normally a more comprehensive test such as differential gradient gel electrophoresis or denaturing high-performance liquid chromatography, followed by direct DNA sequencing to characterize the mutations identified by examine techniques (Schrijver, 2005).Non-technical Parameters. A con of oligonucleotide microarrays is that they can take from a week to a month to get to a diagnostic result, but they do, however, have 80 98% analytical sensitivity and specificity. A pro is that the test can cost anywhere from $25 to $800 per sample, but still more expensive that TaqMan (Science Exchange, 2017b).Next times SequencingDNA sequencing is the gold standard when it comes to genetic tests, however, its high costs make it difficult for routine use (Schrijver, 2005). In recent years there have been advances in DNA sequencing th rough Next Generation technologies (NGS), as they afford higher throughput and speed. There are three common NGS platforms, which are Roche 454, Illumina, and AB SOLiD. They are similar in that they measure and analyze signals that are emitted through a second strand of DNA to sequence the DNA. The way the second DNA strand is generated is where these platforms differ. Template DNA is split into smaller pieces, amplified, and then attached on a surface before sequencing (Pabinger, et al., 2014).In the Roche 454 platform, DNA sequence fragments are ligated onto oligonucleotide adapters on string of beads that go through emulsion PCR that amplifies the DNA to amplify the copy number of the DNA fragments. The beads are diluted, then a single bead is dropped into each microwell of PicoTiterPlate. Pyrosequencing is then conducted by adding enzymes for sequencing and triphosphate nucleotides bases that release pyrophosphates when the bases encounter complementary bases on the DNA sequenc e that are on the beads. This produces light that is recorded detected by a CCD camera that denotes the triphosphate nucleotide base type in the DNA sequence in each well. This method is error prone as it misidentifies the length of nucleotides with identical bases (homopolymers) (Hodkinson Grice, 2015).The Illumina approach is the most widely used NGS platform because of it allows a large amount of data to be generated, with a low error rate and is cost effective. This method avoids homopolymers by using a sequencing by synthesis method that uses reversible dye terminators with one nucleotide per sequencing cycle (Hodkinson Grice, 2015). The dye terminators are washed over a play jail cell that has the oligonucleotides immobilized on it and had been hybridized with the DNA fragments. After the dye terminator has attached, the unbound nucleotides are washed away and the flow cell is imaged. Since the dye terminator is reversible it can be washed away after each cycle to get the identity of the next base pair. Illumina sequences shorter fragments, about 35 100 base pairs and uses a special program that uses an algorithm to determine the sequence (Hodkinson Grice, 2015) (Yamashiro, 2017d).The AB SOLiD platform is similar to 454, in that it starts with emulsion PCR but uses a sequences-by-litigation approach (Hodkinson Grice, 2015). The DNA libraries are sequenced by by 8 base-probe ligation which contains ligation site (the first base), cleavage site (the fifth base) (Liu, et al., 2012). Di-based probes that are fluorescently denominate with four dyes, ligate to the DNA sequence and produce a fluorescent signal that is recorded. The sequences are read in multiple cycles since at least the first two bases are read with high confidence. This redundancy of this method reduces its error rate (Liu, et al., 2012) (Yamashiro, 2017d).Non-technical Parameters. To cut back time and money anyone of the NGS platforms could be used to only analyze chromosome 19 sinc e that is where the ApoE protein is located. Roche 454 is the most expensive of the three starting at $8000 per sample and Illumina the cheapest with tests starting at $35 per sample. An advantage of the NGS technologies is the amount of data it can generate like mapping parts of or the whole genome of the individual and can be more sensitive to detecting rare sequences among related sequences (Hurd Nelson, 2009).Genotyping MethodologiesMethodsCost per sampleTime to resultAnalytical esthesiaAnalytical SpecificitySNP TaqMan RT-PCR$3-$22598%98%Oligonucleotide Microarray Chip$25-$8001 week 1 month80-98%80-98%NGS platformsRoche 454$8,000-$9,7971 dayIllumina$35-$2,9502-3 days98%98%AB SOLiDN/A 1 week80-98%80-98%ResultsThe SNP TaqMan RT-PCR test method would be the system of choice for genotyping the ApoE alleles. It has the highest analytical sensitivity and specificity and the most cost efficient. Although it does not give as much information as the NGS platforms in terms of epigeneti cs and genome mapping, it does get the job done within a reasonable amount of time. Microarrays and NGS need specialized software to perform bioinformatic analysis of the results to get a final diagnosis (Liu, et al., 2012) (Miller, et al., 2010). Whereas with SNP TaqMan each reaction tube has specific forward and reverse primers for each ApoE allele and can be visualized in real time, making it the easiest to use and fastest to get to a result (Zhong, et al., 2016). Table 1 compares the different technologies explained earlier for genotyping methods. Illumina is the only other technology that can compare to SNP TaqMan RT-PCR in terms of sensitivity and specificity, but it takes a bit longer and the cost can easily go up to thousands of dollars (Science Exchange, 2017b).Validation. In order to develop the TaqMan RT-PCR assay, primers for each ApoE allele are designed through a software through a software such as Beacon Designer 7. An analysis is then done using sequences submitted t o a database like GenBank on the primers/probes sequences to evaluate their ability to anneal to the target variants, by means of a puff analysis (Geyer et al, 2012). The primer/probe sequences that annealed with a 100% specificity to the target variants only, are chosen and are labeled with a different reporter fluorophore dye (e.g. FAM, TET, HEX) at the 5 end and a quencher dye (e.g. TAMRA) at the 3 end (Geyer et al, 2012) (Kutyavin, et al., 2000). The probes are then ordered from a company that manufactures probes for TaqMan such as Bioresearch Technologies in Novato, CA (Qu, Wanner, Christ, 2011).The next steps would be to optimize the PCR assay by testing the parameters of the different components that get put into the master mix, the concentrations of MgCl2, primers/probes, DNA template, dNTPs, Taq polymerase, and buffer concentration. These are tested using a thermocycler that is equipped for TaqMan PCR, where the temperatures and quantify for each step in the cycle are al so adjusted to get optimal annealing, hybridization, and amplification of the DNA. The Ct value (cycle threshold) which is the number of cycles in a run that crosses the threshold is determined. Anything above the threshold is a positive indicator that the allele being tested is present (Qu et al, 2011).For validation of the ApoE TaqMan PCR assay, the results are compared to results from a DNA sequencing analysis. The ApoE fragments are amplified by PCR with the designed primer/probes and then the products purified and sequenced by a DNA sequencer like the ABI 3730XL DNA Sequencer by Applied Biosystems (Zhong, et al., 2016).DiscussionEight ApoE alleles are linked to ARC disease and it has been determined the TaqMan RT-PCR would be the best assay to test for these alleles. Screening for ARC related alleles before cancer develops is very beneficial for early treatment before the disease develops or progresses too far and will result in greater longevity (Katsanis Katsanis, 2013). Tes ting for ARC may lead to the diagnosis of a highly likely predisposition to AD because of its strong link to the ApoE4 allele. With the ApoE4 gene the mean age to develop AD is 68 with a 91% chance for homozygotes, 76 years old with a 47% chance for heterozygotes, and 84 years old with 20% for people who do not carry the allele (Zhong, et al., 2016). There is an ethical dilemma when it is revealed that a patient has the ApoE4 allele, since exposing genetic risk is a complex issue, as it not only shows risk for the patient but also to the patients family member who may also have the allele. They would have to reveal to their relatives that they have the ApoE4 allele and that they should also get tested. The cost of testing for the just one allele would be low since it would not require a large amount of DNA sequencing, a simple PCR test would be sufficient. It also reveals to the patient that they may pass on this gene to their offspring, which susceptibility become a burden on them from having any future children. If they are not in a relationship they would also feel pressure that they have to reveal that they are carriers to future partners (Arribas-Ayllon, 2011).There is no clear benefit to early disclosure of the predisposition of getting AD to young adults because there is no medical intervention available. 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