Temporary Protecting Groups,Oligonucleotide Support,Synthesis Cycle,Coupling,Oxidation,After the Synthesis,Dichloracetic Acid,Nucleoside

Scholarships Dictionary Visa Details Study Abroad Exam Results Online Test Jobs

	Home
Isolation, Sequencing and Synthesis of Genes Isolation, Sequencing and Synthesis of Genes - Introduction Isolation of Genes Isolation of Genes Coding for Specific Proteins Isolation of Genes with Known or Unknown Products having Tissue Specific Expression Isolation of Genes with Known or Unknown Products Using DNA or RNA Probes Use of Heterologus Probes Use of Synthetic Probes Isolation of Genes Coding Use of Transposable Elements Transposon Tagging T-DNA-Insertion Mutagenesis for Isolation of Plant Genes Promoter Enhancer and Gene Trap for Isolation of Genes Differential Screeing and Differential Display Technique for Isolation of Genes Subtractive Hybridizatioon for Gene Isolation Map Based Cloning for Gene Isolation Isolation of Novel Genes by Asis Data at JNU New Delhi

Home >> Biotechnology and Genomics >> Isolation, Sequencing and Synthesis of Genes >> Temporary protecting groups

Your Ad Here	Temporary protecting groups These protecting groups need to be removed after every step during the synthesis. The 4, 4’ dimethoxytrityl group (DMTr) is used to protect the hydroxyl group at position 5’ of the incoming nucleotide. It is removed in every step using mild acid solutions (1% trichloracetic acid = TCA or 3% dichloracetic acid = DCA). Oligonucleotide support The support used in oligonucleotide synthesis consists of tow main parts : (i) the first nucleoside unit, from which with synthesis stars; (ii) the solid phase (an organic support to which the first nucleoside is linked by means of a spacer arm;) The solid phase technique helps in rapid and efficient washing of the growing oligonucleotide between the steps, to remove excess reactants.
A solid support should have the following features: (i) should be insoluble in all solvents and reagents, (ii) should not swell under the conditions used for synthesis, (iii) should be inert towards chemicals used, (iv) should be macroporous (to give rapid access to reagents) (v) should have large surface area, (vi) should be rigid (to withstand solvent pressure applied during synthesis), (vii) should be capable of bearing functional groups (which can react with deoxyribonucleotides in a reproducible manner). A solid support should have the following features : (i) should be insoluble in all solvents and reagents, (ii) should not swell under be inert towards chemicals used, (iv) should be macroporous (to give rapid access to reagents), (v) should have large surface area, (vi) should be rigid (to withstand solvent be capable of bearing functional groups (which can react with deoxyribonucleotides in a reproducible manner).
The main four types of solid support include : (i) silica, (ii) glass beads (CPG), (iii) plastic (e.g polystyrene) and (iv) cellulose paper. Of these four, glass needs to be derivatised to enable coupling of the first deoxyribonucleoside to it. An amino group is introduced for this purpose at the end of a spacer arm. A nucleoside attached to a solid support	Your Ad Here
The synthesis cycle The synthesis of an oligonucleotide starts with the first deoxyribonucletide (in its protected form) linked to the solid phase through its 3’ end. All the four nucleotides are added as protected amides. The reaction cycle then consists of the following four steps: (i) Deprotection (detritylation): the solid support is washed with dichloroethane and the 5’ hydroxyl group of deoxyribonucleotide is deprotected removing DMTr group (1.5 to 3 minutes of acid treatment); the reagents are removed by a wash with dichloroethane and anhydrous conditions for coupling reaction are achieved by a wash with dichloroethane and anhydrous conditions for coupling reaction are achieved by an acetonitrile wash; (ii) Coupling : 3’-5’ phosphodiester linkage is achieved using 3’ phosphate group of the incoming protected deoxyribonucleotide (a phosphoramidite or phosphite triester); this is achieved by phosphite triester synthesis, in which phosphoramidite is firs converted to a tetrazolide (using tetrazole as a weak acid);excess reagents are washed by acetonitrile.
	(iii) Capping: Any unreacted 5’ hydroxyl groups anchored at the solid phase are protected by acetyl group using acetic anhydride (AC₂O+ N-methylimidazole + 2,4,6-collidine in (acetonitrile). This allows only the correct chains to elongate in subsequent coupling reaction; (iv) Oxidation: Before adding the next nucleotide, the phosphate triester is oxidized to the more stable 5-valent oxidation state, by treatment with a mixture of iodine, collidine and water in acetonitrile. After the synthesis. Following two steps are used to obtain the final product : (i) removal of permanent protecting groups and cleavage of oligonucleotide from the solid phase, (ii) analysis and/or purification of the product by chromatography or electrophoresis.

Sever versions pf gene machines are now available. These machines, under the control of microprocessor, synthesize specific short sequence of single stranded DNA automatically.The desired sequence is entered on a keyboard and the microprocessor automatically opens the valve of the containers of successive nucleotides, reagents and solvent need at each step, into a synthesizer column, which is packed with tiny silica beads. These beads provide support on which DNA molecules are assembled.

	Home
Sequencing of a Gene or a DNA Fragment Maxam and Gilbert's Chemical Degradation Method Sanger's Dideoxynucleotide Synthetic Method Automatic DNA Sequencers Slab Gel Versus Capillary Sequencers Slab Gel Sequencing Systems Capillary Gel Sequencing Systems Direct DNA Sequencing Using PCR Ligation Mediated PCR LMPCR DNA Sequencing Through Transcription DNA Sequencing by Hybridization Using Microarrays on DNA Chips DNA Sequencing by DE-MALDI-TOF Mass Spectrometry Synthesis of Genes Synthesis of Gene for Yeast Alanyl tRNA Synthesis of Oligonucleotides Synthesis of Three Duplex Fragments Synthesis of Gene from Three Duplex Fragments Synthesis of Gene for a True Precursor tRNA Total Synthesis of a Human Leukocyte Interferon Gene Gene Synthesis Machines Protection Chemistry and Amidites Permanent Protecting Groups Temporary Protecting Groups Synthesis of Gene Using PCR Synthesis of Genes From mRNA