Chemical Synthesis of a Gene: Phosphodiester Approach

Chemical Synthesis of a Gene: Phosphodiester Approach Chemical synthesis of a gene is the process of synthesizing an artificially designed gene into a physical DNA sequence by chemical methods. The amino acid sequence of the protein encoded by a gene enables the deduction of base sequence of the concerned gene. From the amino acid sequence of the protein and using a set of optimal codons, the nucleotide sequence of the gene can be back translated. However, the degeneracy of genetic code may present some problems, but a functional sequence of the gene can nonetheless be worked out and can be optimized for codon usage as well as for base composition. In principle, a DNA synthesizer can be used to synthesize the DNA sequence chemically and this can be cloned in the usual manner. But this is not so simple. A synthesizer will add bases sequentially one at a time to the growing oligonucleotide chain through a series of chemical reactions and washing steps. Synthesis of oligonucleotides 30-50 bases long is very reliable, longer sequences can be synthesized but the practical limit is not more than 100 bases. One way to solve this is to synthesize short fragments and join them chemically or enzymatically to create the longer fragment. However, the synthesizer makes single-stranded DNA, so the complementary strand has to be synthesized again to create a double-stranded DNA. It involves a lot of work but is achievable. Early studies. The synthesis of nucleic acids in the laboratory started about thirty years ago. Early synthetic efforts used phosphodiester approach which enabled the synthesis of short oligonucleotides of 10-20 nucleotides. This approach was based on the selection of the proper condensing agents for phosphodiester bond formation and at the same time suitable protective groups were employed for the bases and the ribose moiety. These oligonucleotides were then assembled into longer DNA fragments with the help of kinase and DNA ligase. From the known primary structure of a ribonucleic acid, tyrosine tRNA, Dr H Khorana and his colleagues deduced the DNA sequence and synthesized successfully a DNA segment containing 200 bp coding for the structural gene for tyrosine tRNA. However, the low yields in the condensation step, the long reaction times, and especially the time-consuming purification of intermediates led to believe that chemical gene synthesis is unlikely to become a standard lab oratory method. Since then, the procedure for oligonucleotide synthesis has been improved by several workers and they provide different approaches for synthesis as well as protection of bases and sugar moieties. There are three distinct methods: (1) phosphodiester approach, (2) phosphotriester or phosphate triester approach and (3) phosphite triester or phosphoramidite approach. Phosphodiester approach This method involves the formation of an ester linkage between an activated phosphate group of one nucleotide with the hydroxyl group of another nucleoside, thus forming the natural phosphodiester bridge between the 5-OH of one nucleoside unit and the 3-OH of the next. Here, 3-O-acetylnucleoside-5-O-phosphate (a) is activated by N,N-dicyclo- hexylcarbodiimide (DCC) or p-toluenesulphonylchloride(PTS/PTsCl) and subjected to react with a 5-O-protected nucleoside (b) to give a protected dinucleoside monophosphate or phosphodiester (c). Activation of phosphate moiety is essential for easier formation of the phosphodiester linkage and this is mediated by DCC or PTsCl. Now, to increase the chain length, one has to remove the 3-O-acetyl group by base catalysed hydrolysis. Further chain elongation is carried out by repeating the process. The major drawback of the phosphodiester method is the formation of pyrophosphate oligomers and oligonucleotides branched at the internucleosidic phosphate. Phosphotriester approach In this method, oligonucleotide branch formation is avoided by protecting the phosphate group with an ethylcyano group. A nucleotide containing 5-OH protected and phosphate protected by MMT and 2-cyanoethyl group respectively (compound a) is activated with 2,4,6-Triisopropylbenzenesulfonyl chloride (TPSCl) and subjected to reaction with a 3-O-protected nucleoside (b). This generates a dinucleoside monophosphate or phosphotriester (c) in which phosphate group is protected by 2-cyanoethyl group. The basic difference between phosphodiester and phosphotriester method is that, in phosphodiester method, the phosphate group is protected by two phosphoester linkage but in phosphotriester method the phosphate group is protected by one extra phosphoester linkage with 2-cyanoethyl group. In phosphotriester method, the formation of oligonucleotide branch at the internucleosidic phosphate is avoided. Phosphite triester or phosphoramidite approach The phosphite triester or phosphoramidite approach for oligonucleotide synthesis was based upon the use of phosphoramidite monomers and the use of tetrazole catalysis. In phosphite triester method, the starting compound is N-6-benzoyldeoxyadenosinephosphoramidite (if adenine is the first base) where the phosphorous atom is in the +3 oxidation state. So unlike the other methods, the formation of oligonucleotides branch is not possible in this process. In this approach, the oligonucleotide is synthesized by a series of reactions described below. Protection of base and sugar In this step, the free -NH2 group of the bases are protected by benzoylation or acylation depending upon the nature of bases. The 5-hydroxyl group is also protected by dimethoxytrityl group (DMT), which protects only primary hydroxyl group but not secondary. The reactions are illustrated in CSG_Fig 3., the blocked bases are shown in the inset. Formation of phosphite triester or phosphoramidite In this step phosphite triester is synthesized by a series of reactions. First, 2-cyanoethanol on reaction with phosphorus trichloride produces an intermediate compound which on further reaction with di-isopropylamine (two-equivalent) and 5-OH protected nucleoside (one-equivalent) produces phosphite triester (CSG_Fig 4). This phosphoramidite will be repeatedly used during the oligonucleotide synthesis process described below. The synthesis procedure The synthesis is carried out in several steps described below: Step 1: The deblocking step The first base, which is attached to the solid support, is at first inactive because all the active sites have been blocked or protected. The free -NH2 groups in the bases remains protected by benzoylation or acylation depending upon the bases and the -OH group is protected by dimethoxytrityl group (DMT). To add the next base, the DMT group protecting the 5-hydroxyl group must be removed (deblocking). This step is also called detritylation. This is done by adding either dichloroacetic acid (DCA) or trichloroacetic acid (TCA) in dichloromethane (DCM), to the reaction column. The 5-hydroxyl group is now the only reactive group on the base monomer. This ensures that the addition of the next base will only bind to that site. The reaction column is then washed to remove any extra acid and by-products. Step 2: Base condensation The step2 is basically a condensation step. Now prior to addition of the well protected nucleotide to the column, it is essential to activate the phosphate group, so that the nucleophilic attack on phosphorous atom takes place easily. This is best done by adding tetrazole to the nucleotide in dichloromethane medium. In presence of tetrazole, diisopropylamine group of the nucleotide becomes positively charged and hence its departure would be easier after nucleophilic attack of 5-hydroxyl group of the previous nucleotide which is attached with resin column. After the reaction, the column was washed to remove extra tetrazole, unbound nucleotide and byproduct (diisopropylamine). Step 3: Capping In case of unreacted nucleoside attached with resin, the 5-hydroxyl group is unprotected this may react later with the addition of different nucleotides. If left unprotected, it will lead to the formation of a mixture of oligonucleotides. The 5-hydroxyl group is therefore blocked by adding acetic anhydride and N-methylimidazole (capping). After capping, the reaction column is thoroughly washed to remove extra acetic anhydride and N-methylimidazole. Step 4: Oxidation This step is basically an oxidation step. In this step, the phosphite linkage is oxidized to give more stable phosphate linkage. The oxidation is best done by adding a mixture of dilute aqueous iodine solution, pyridine (Py) and tetrahydorfuran (THF) to the reaction column. The steps one through four, i.e., deblocking, base condensation, capping and oxidation, are repeated until all desired bases have been added to the column. This cycle is completed once for each additional base. Step 5 Detachment of oligonucleotide from solid support After all bases have been added the oligonucletide must be cleaved from the solid support and deprotected before it can be effectively used. For detachment of oligonucleotides form resin, the column is treated with 28% ammonium hydroxide solution (NH4OH), and at the same time the ethylcyano group on the phosphate group is removed. Step 6: Purification and isolation of oligonucleotide In this step, NH4OH is evaporated from the ammonium hydroxide solution of oligonucleotides to get crude product. The crude product is a mixture of oligonucleotide, cleaved protective groups and oligonucleotides with internal deletions. Now this crude product is subjected to boiling in a sealed tube with NH4OH at 55 °C. The main purpose of this reaction is to remove the base protecting group. After evaporation of NH4OH, the crude product is subjected to desalting followed by Polyacrylamide Gel Electrophoresis, to purify the oligonucleotides. Desalting is used mainly to remove the ammonium ion. This is done by ethanol precipitation, size-exclusion chromatography, or reverse-phase chromatography. Oligonucleotides are synthesized by the stepwise addition of nucleoside-3à ¢Ã¢â€š ¬Ã‚ ²-phosphoramidite monomers to solid-phase supports in an automated DNA synthesizer. In solid-phase synthesis, 3-terminal hydroxy group of the first added nucleoside is attached to the solid surface by covalent interaction. The solid support is contained in columns whose dimensions depend on the scale of synthesis. The two most frequently used solid phase materials are Control Pore Glass (CPG) and macroporous polystyrene (MPPS). CPG is commonly defined by its pore size, for example pore sizes of 500Ã… are used to allow the oligonucleotides preparation of about 50 -mer. To improve the performance of native CPG some modification is required. This is done by treating the material with (3-aminopropyl)triethoxysilane) to give Aminopropyl CPG. The amino group then serves as the anchoring point for the first added oligonucleoside. MPPS is synthesized by polymerization of divinylbenzene, styrene, and 4-chloromethylstyrene in the presence of a porogeneous agent. It is a low-swellable, highly cross-linked polystyrene and suitable for oligonucleotide synthesis. The macroporous chloromethyl MPPS obtained is often converted to aminomethyl MPPS to improve the efficiency of the support. Annealing of oligonucleotides For chemically synthesize a gene, the next step will be to assemble the oligonucleotides to form a complete gene. This is achieved by enzymatic methods which include polymerase cycling and ligase reactions. Some of the strategies are discussed below. Assembling oligonucleotides by single-step PCR. For synthesis of a gene, the oligonucleotides (about 30-60 nt long) are synthesized chemically so that each oligonucleotide has a 6-9 nt overlap with its neighboring oligonucleotide. These are then assembled in a single-step PCR. In this method, oligonucleotides are first ligated and then the product, the entire gene, is PCR amplified using the outmost oligonucleotides as primers. This method was first used to synthesize a 924-bp gene coding for an isozyme of horseradish peroxidase. Another method was developed by WPC Stemmer which did not use any ligase for joining the oligonucleotide products. It however, relied on Taq DNA polymerase (PCR cycling) for joining the individual oligonucleotides. Assembling oligonucleotides by two-step PCR. The method involves two steps. (i) Synthesis of individual fragments of the DNA of interest: ten to twelve 60mer oligonucleotides with 20 bp overlap are mixed and a PCR reaction is carried out with high-fidelity DNA polymerase Pfu to produce DNA fragments that are 500 bp in length. (ii) Synthesis of the entire sequence of the DNA of interest: five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest, with the two outermost oligonucleotides as primers. Several modifications of the above procedure have been presented. One such method called PAS (PCR-based accurate synthesis) involves (i) synthesis of oligonucleotides to cover the entire DNA sequence (ii) PCR to synthesize DNA fragments (iii) second PCR for assembly of the products of the first PCR and (iv) cloning of the synthetic DNA and then verification by DNA sequencing. Besides, other methods in use for gene synthesis are successive extension PCR, simplified gene synthesis (PCR based), synthons and ligation by selection, to name a few. Review questions and problems What is the advantage of phosphatetriester method over phosphatediester method? What is the advantage of phosphitetriester method over phosphatetriester and  phosphatediester method? What is the main advantage to use DMTCl for protecting the 5-hydroxyl group? How could you attach the first nucleoside to the solid support? What is the utility of capping step in the oligonucleotides synthesis? Why capping is done by aceticanhydride? What is the function of iodine in the oxidation step of oligonucleotides synthesis? How could you protect only the free -NH2 group of the bases of a nucleoside? What is the reagent used for the removal of 2-cyanoethyl group from the  synthesized oligonucleotides? What is the byproduct produced from the base-condensation step of oligonucleotides  synthesis in phosphite triester method? How could you deprotect the bases of oligonucleotides? What is the function of tetrazole in the base condensation step of oligonucleotide synthesis? What is the basic principle for synthesizing a gene from the corresponding oligonucleotides by (a) PCR-based one-step DNA synthesis, (b) PCR-based two-step DNA synthesis?

Macbeth- General Information, Rough Essay

then is revealed as being weak and easily manipulated. He then descends into become a murderous madman. one realizes Macbeth’s transformation into one of drama’s most infamous villains coincides with a profound transformation of his conscience—to a point where he has none at all. Throughout the play Macbeth makes a journey from following a moral ethic, implementing a flawed ethic, and arriving to a point where he had none at all. When comparing Banquo and Macbeth, after they meet the weird sisters, one see’s that Banquo adheres to warrior ethics where Macbeth moves away from it.At the start of the play Macbeth was idolised by everyone and thought to have been a man of very great power whom could be defeated by nobody: â€Å"All is too weak for brave Macbeth – well he deserves that name† 1. 2. 15. It is stated in this quote by the Captain that at the start Macbeth was known as a brave heroic man, which his enemies were too weak to overthrow. B y the end of the play his status falls from a man of great magnificence to one that has barely managed to keep his own sanity.Thus our first description of Macbeth is that of a brave, loyal soldier defending his King and country He appears to be a strong military leader ‘brave Macbeth- well he deserves that name’’, is then called ‘noble Macbeth' and given the traitor’s title, Thane of Cawdor: ‘’with this former title greet Macbeth’’. The Captain tells the King killed the traitor Macdonald in a very horrible and gory manner ‘’unseamed him from the nave to th’chops’’ Therefore, we are led to believe that Macbeth is a good, loyal, courageous, and determined man.Things From the moment they are introduced to the play the witches are seen as a negative effect on Macbeth, creating chaos by prophesysing to Macbeth that he is going to become Thane of Cawdor ‘’All hail, Macbeth! Hail to thee, Thane of Cawdor! ’’ and that he is going to become king ‘’All hail, Macbeth! That shall be King hereafter’’and getting him to act. He knows he is already Thane of Glamis, but does not know that Duncan has promoted him to Thane of Cawdor. Macbeth is surprised by the promise of kingship.Banquo's prophecy is even more fantastic: he will be the father of kings but not king, and will be greater and happier than Macbeth. That is the moment when Macbeth wants to know more. The witches basically planted the seed of evil in Macbeth’s mind that later on grew to dominate his every action. However, it was Macbeth’s ambition that decided to take action on these prophesies, therefore it was he that decided the final outcome. When Ross and Angus enter to proclaim Macbeth's promotion, he is very surprised:‘’The Thane of Cawdor lives, why do you dress me in borrowed robes? ’ Moreover, Macbeth believes that this is the f ulfilment of the witches prophecies, However, there is no clear reason why Macbeth would become king, especially since the present King is so loved and admired. In the next few lines it becomes apparent that Macbeth not only has thought about being king, but he also believes what the witches told him is true: Glamis, and Thane of Cawdor: The greatest is behind Two truths are told, As happy prologues to the swelling act Of the imperial theme. (1. 3. 115-116, 126-128)This is the first time we see him realising that he might have to do something, as killing Duncan, in order to get to the throne. He debates the good and the bad side of the prophecies : ‘’If good, why do I yield to that suggestion whose horrid image doth unfix my hair’’ we are shown that Macbeth not only loves his King and country,’’our duties are to your throne and state, children and servants’’ but also himself. It still remains to be seen what action he will take. Macbeth's change has begun. **Soon enough, we are taken to Macbeth’s home, wherewe meet his wife, Lady Macbeth. Lady Macbeth has just received a letter from her husband in whichhe tells her everything that has happened. Because the witches got him very interested in their prophecies, he has had them investigated and has ’’ learned by the perfectest report that they have more in them than mortal knowledge. ’’ It is clear that after calling the witches ‘imperfect speakers' (1. 3. 68), Macbeth has now changed his mind. He also interprets the prophecies and tells his wife a slightly changed version.He addresses her ‘’my dearest partner in greatness’’ and seems to be sincere. Lady Macbeth, however, is determined that her husband becomes king. she says that Macbeth lacks the qualities necessary to assassinate Duncan without remorse or regret: ‘’yet do I fear thy nature, it is too full o’th’milk of human-kindness to catch the nearest way’’ . she hen prays for supernatural help to take away all of her feminine qualities and basically any traits of conscience: ‘’ unsex me here, and fill me from the crown to the toe top full of direst cruelty! ’, ‘’stop up th’access and passage to remorse’’, ‘’that my keen knife see not the wound it makes’’. After Macbeth arrives, Lady Macbeth is telling him to ‘’look like the innocent flower, but be the serpent under’t’’ and to ‘Leave all the rest to me' This implicates her in the murdering of Duncan and shows us that she is taking the responsibility. She is essentially taking over. After the King arrives at the castle and prepares to sleep peacefully, Macbeth is still debating how he can achieve the crown without getting caught.He doesn’t want to do it personally; he wants to get it over with:’ ’ If it were done, when’tis done, then ‘twere done well it were done quickly’ and doesn’t really want to kill his cousin and King; he has a conscience: ‘’he’s here in double trust’. On the other side, he’s obsessed with becoming king and his thoughts keep flowing in the same direction: ‘’I have no spur to prick the sides of my intent, but only vaulting ambitions, which o’er-leaps itself and falls on the other’’. We see duality when he says: ‘’we will proceed no longer in this business’’ and tries to procrastinate the murder.His wife then plays games with his mind and basically psychologically bullies and pressures him into killing Duncan by telling him that he is less than a man if he does not carry out the murder:’’ when you durst do it, then you were a man; and, to be more than what you were, you would be so much more the man’â€℠¢, and that she, being a woman has more strength of purpose than he does: ‘’i would, while it was smiling in my face, have plucked my nipple from his boneless gums, and dashed the brains out, had i sworn as you have done to this’. As soon as Macbeth has kills Duncan, he seems to start to lose his ind. He starts hallucinating: ‘’is this a dagger which i see before me, the handle toward my hand?Come, let me clutch thee. I have not, and yet I see thee still’’. He is unable to think clearly and is very paranoid. He is ready to eliminate anything that stands in his waybecause oft his ambitions for himself and the fear of being discovered. He just doesn't know what to do with himself while trying to keep the crown. Even though he is committed to Duncan, he Because Macbeth is afraid of the witches’ prophecy that he will lose the crown: ‘’To be thus in nothing, but to be safely thus.Our fears in Banquo stick deep’â₠¬â„¢ and Banquo will become king, he sends people to kill Banquo and Fleance. He hires three murderers in order to make sure that they won’t be able to escape. They ambush Banquo on his way to a royal feast, but Fleance escapes into the night. Macbeth is now the prisoner insolent and nagging doubts and fears,: ‘’But now I am cabined, confined, bound in to saucy doubts and fears’’, and is now paranoid because Fleace has escaped: ‘’There the grown serpent lies; the worm that’s fled hath nature that in time will venom breed’’.

Reactive Policing Essay

Reactive patrol is police responding to specific requests from individuals or groups in a community that provides â€Å"immediate† response to calls. Reactive patrol provides help to ensure that calls are responded to in an efficient and timely manner. Reactive patrol also involves the follow-up investigations required to get additional information to prosecute or otherwise help with assistance of the community. Reactive patrol is different from proactive control in the sense that reactive patrol is employed when a crime is or has already been committed and/or reported. Proactive policing is based on the concept of preventing crime, and making an appearance in the community to ensure they know there is assistance there for their own protection and safety. Proactive policing also provides a sort of deterrent for potential crimes and criminals due to the fact they know there is law enforcement active and interested in what is going on in the community. If police agencies only adopted one or the other, proactive, or reactive policing, the law enforcement system would become unbalanced. If proactive policing is the only type being employed, preventative measures might go so far that they begin to infringe on individual rights. If only reactive policing were used, there would be a rise in crime, because people would know there would only be a chance of them getting caught after the act had already been committed.

Historical And Contemporary Situation Of Torres Strait...

I aim to focus on the historical and contemporary situation of Torres Strait Islander and how the Edward Koiki Mabo (Eddie Mabo) decision has affected them. History Background Edward Koiki Mabo (Eddie Mabo) is the Australian Man born in 1936 from Torres Strait Islander campaigning for Indigenous land rights. His decision for land rights was the legal decision. In 1981, Mabo gave first speech at the land rights conference at the James Cook University explaining the traditional land ownership and inheritance system that his community followed on Mer Island. The lawyer in the conference noted the significance of Mabo’s speech and gave a suggestion there should be a test case to claim land rights through the court system. The case was heard for ten years, progressing from the Queensland Supreme Court to the high court of Australia. The choice of high court judgment in the Mabo case came about a clearing the myth of tera nullins and setting up a legitimate structure for native title by indigenous Australian. People recognize that Indigenous Australian have a prior claim to land taken by the British Crown since 1770. They were only land such as vacant cr own land, national parks and possibly some leased land, where the lease is subjected to the Aboriginal and Torres Strait Islander people. It is important because it was the turning point for the recognition of Aboriginal and People right which acknowledge their unique connection with land. The judgments of the High Court inserted