Chemical Synthesis of a Gene: Phosphodiester Approach

Chemical Synthesis of a factor Phosphodiester ApproachChemical implication of a cistron is the process of synthesizing an by artificial means designed gene into a physical deoxyribonucleic blistery chronological instalment by chemical modes. The amino acid period of the protein encoded by a gene enables the deduction of swinish sequence of the concerned gene. From the amino acid sequence of the protein and using a set of optimal codons, the fundament sequence of the gene can be back translated. However, the degeneracy of genetic code may present some problems, besides a functional sequence of the gene can n singletheless be worked forbidden and can be optimized for codon riding habit as well as for alkali composition.In principle, a desoxyribonucleic acid synthetic thinkinger can be utilise to compound the deoxyribonucleic acid sequence chemically and this can be cl angio tensin converting enzymed in the usual manner. But this is not so simple. A synthesizer wi ll add root words sequentially tincture of iodine at a epoch to the growing oligo nucleotide chain through a series of chemical moveions and washing steps. Synthesis of oligonucleotides 30-50 bases long is very reliable, all-night sequences can be synthesized but the practical limit is not to a greater extent than 100 bases. genius way to solve this is to synthesize short fragments and wedlock 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 discount of nucleic acids in the research lab started about thirty years ago. Early synthetic efforts used phosphodiester address which enabled the tax write-off of short oligonucleotides of 10-20 nucleotides. This approach was based on the selection of the prissy condensing agents for phosphodiester bond relieve ones elfation and at the same time equal protective conclaves were employed for the bases and the ribose moiety. These oligonucleotides were therefore assembled into longer DNA fragments with the help of kinase and DNA ligase. From the known prime 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 compressing step, the long reply times, and oddly the time-consuming purification of intermediates led to believe that chemical gene synthesis is unlikely to become a standard laboratory mode.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 atomic number 18 tether distinct methods (1) phosphodiester approach, (2) phosphotriester or orthoph osphate triester approach and (3) phosphite triester or phosphoramidite approach.Phosphodiester approachThis method involves the formation of an ester gene linkage between an trip phosphate root word of one nucleotide with the hydroxyl group of an separate nucleoside, thus forming the natural phosphodiester link 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- protect nucleoside (b) to contrive a protected dinucleoside monophosphate or phosphodiester (c). Activation of phosphate moiety is meaty for easier formation of the phosphodiester linkage and this is mediated by DCC or PTsCl. at present, 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 phosphod iester method is the formation of pyrophosphate oligomers and oligonucleotides discriminateed at the internucleosidic phosphate.Phosphotriester approachIn 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 ( conflate a) is activated with 2,4,6-Triisopropylbenzenesulfonyl chloride (TPSCl) and subjected to response 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 twain phosphoester linkage but in phosphotriester method the phosphate group is protected by one special(a) phosphoester linkage with 2-cyanoethyl group. In phosphotriester method, the formation of o ligonucleotide branch at the internucleosidic phosphate is avoided.Phosphite triester or phosphoramidite approachThe 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 outgrowth base) where the phosphoric share is in the +3 oxidation state. So unlike the opposite methods, the formation of oligonucleotides branch is not possible in this process.In this approach, the oligonucleotide is synthesized by a series of receptions described below.Protection of base and sugarIn this step, the exhaust -NH2 group of the bases are protected by benzoylation or acylation depending upon the temper 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 occlude bases are shown in the inset.Formation of phosphite triester or phosphoramiditeIn 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 perennially used during the oligonucleotide synthesis process described below.The synthesis procedureThe synthesis is carried out in several steps described below grade 1 The deblocking stepThe first base, which is devoted 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 protec ting 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 methylene chloride (DCM), to the reaction column. The 5-hydroxyl group is now the only reactive group on the base monomer. This ensures that the auxiliary of the next base will only bind to that site. The reaction column is then washed to remove any extra acid and by-products. tint 2 Base condensationThe 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 trump done by adding tetrazole to the nucleotide in dichloromethane medium. In presence of tetrazole, diisopropylamine group of the nucleotide becomes positively charged and thusly its departure would be easier after nucleophilic attack of 5-hydroxyl group of the previous nucleotide which is affixed with resin column. afterward the reaction, the column was washed to remove extra tetrazole, unbound nucleotide and byproduct (diisopropylamine).Step 3 CappingIn contingency 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 form 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 OxidationThis 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 c ondensation, capping and oxidation, are repeated until all desired bases have been added to the column. This cycle is completed once for for each one additional base.Step 5 Detachment of oligonucleotide from solid supportAfter all bases have been added the oligonucletide must be cleaved from the solid support and deprotected earlier it can be effectively used. For detachment of oligonucleotides form resin, the column is handle with 28% ammonium hydroxide solution (NH4OH), and at the same time the ethylcyano group on the phosphate group is removed.Step 6 shade and isolation of oligonucleotideIn this step, NH4OH is evaporated from the ammonium hydroxide solution of oligonucleotides to get uncut 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 resistance with NH4OH at 55C. The main purpose of this reaction is to remove the base protecting g roup. After evaporation of NH4OH, the crude product is subjected to desalting followed by Polyacrylamide colloidal 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-3-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 500 are used to allow the oligonucleotides preparation of about 50 -mer. To improve the deed of nativ e 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 desirable for oligonucleotide synthesis. The macroporous chloromethyl MPPS obtained is often converted to aminomethyl MPPS to improve the efficiency of the support.Annealing of oligonucleotidesFor 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. few 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 chemic ally 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 full 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 real 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 dozen 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 fi ve to ten PCR products from the first step are combined and used as the guidebook 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) clone of the synthetic DNA and then verification by DNA sequencing.Besides, other methods in use for gene synthesis are successive mention PCR, simplified gene synthesis (PCR based), synthons and ligation by selection, to name a few. recapitulation questions and problemsWhat is the advantage of phosphatetriester method over phosphatediester method?What is the advantage of phosphitetriester method over phosphatetriester andphosphatediester method?What is the main advantage to us e 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 thesynthesized oligonucleotides?What is the byproduct produced from the base-condensation step of oligonucleotidessynthesis 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 dance DNA synthesis, (b) PCR-based two-step DNA synthesis?