Synthesis of oligonucleotides by phosphoramidite method
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The document discusses the synthesis of oligonucleotides, detailing the chemical processes and reagents involved, including the importance of phosphoramidite chemistry and the role of various protective groups. It highlights the methods of cleavage, capping, and monitoring of coupling efficiency, emphasizing the advantages and disadvantages of automated oligonucleotide synthesis. Additionally, applications such as cloning, site-directed mutagenesis, and DNA sequencing are mentioned, along with references for further reading.
Synthesis of oligonucleotides by phosphoramidite method
- 1. SYNTHESİS OF OLİGONUCLEOTİDES 198612 FREYA CARDOZO
- 2. Nucleoside Oligonucleotides Nucleotide Structure of Nitrogenous bases Oligos= few Short stretches of single stranded DNA or RNA molecules Sugar + Nitrogenous base Nucleoside + Phosphate
- 5. 5’--> Dimethoxytrityl (DMT) 3’--> Diisopropylamine Nitrogenous bases (Except T)-->Benzoyl group 3’--> 2-Cyanoethane Phosphoramidite
- 6. Bruce Merrifield in 1960s Nobel prize in Chemistry in 1984 Discovery
- 7. MAJOR STEPS IN SYNTHESIS Unreacted 3’ OH Acetic anhydride Unstable- Phosphite triester bond to Phosphate triester Stable- bond İodine + Pyridine Removal of DMT Activation of 3’ Trichloroacetic acid Previous to next nucleotide Tetrazole DETRITYLATION CAPPING COUPLING OXIDATION
- 8. 1 Linking 1st NUCLEOSIDE TO THE COLUMN ● Controlled pore glass (CPG) - Rigid & non-swelling → deep pores - Prefered for short oligos - <40 → blocks the pores and reduces diffusion ● Polystyrene - Highly cross-linked polystyrene beads - Good moisture & exclusion properties - Small scale (e.g. 40 nmol)
- 10. 3 ACTIVATION & COUPLING Tetrazole
- 11. 3 COUPLİNG
- 12. 4 CAPPİNG The 5’ end using acetic anhydride and dimethyl aminopyridine
- 13. Deletion mutation:- Sequence of the correct oligonucleotide (top); and a failure sequence (bottom) containing a deletion mutation, corresponding to the deletion of the thymine base at position 6 Why do we need to cap the unreacted linked nucleosides?
- 14. 5 OXIDATION Phosphite triester (unstable) → Phosphate triester (pentavalent) İodine + Pyridine
- 15. linking Detrity lation Activa tion Capping oxida tion Repeat Acetonitrile Argon Trichloroacetate (TCA) Wash with Acetonitrile→ Ar Iodine Pyridine Tetrazole Remove- Argon Cleavage Deprotection Acetic anhydride Dimethyl aminopyridine
- 17. Cleavage of DNA bound to bead Conc. aqueous NH3 Beta cyano group & benzoyl at the bases Conc. aqueous NH3 DMT at the 5’ end of the last base Pyridine Phosphorylation of 3’ terminus T4 polynucleotide kinase / chemical procedure POST SYNTHESIS MODIFICATIONS
- 18. OVERVIEW
- 19. Coupling Efficiency Determination Cleavage of trityl group → resultant orange solution Absorbance at 495 nm is measured in a UV/visible spectrophotometer.
- 20. Eg. Efficiency= 99% at each cycle 20-unit oligonucleotide 19 coupling reactions 0.9919 × 100= 83% of the product will be 20 nucleotides long. For 60-mer is synthesized with 99% efficiency at each cycle ??
- 22. OLIGONUCLEOTIDE CHARACTERIZATION Electrospray Mass Spectrometry (ESMS) Deconvoluted ES MS of crude oligonucleotide 5'-DMT-T20 (calculated mass 6324.26 Da).
- 23. CLONİNG SITE DIRECTED MUTAGENESİS DNA SEQUENCING SCREENING GENE LIBRARIES USES OF OLIGONUCLEOTIDES 01
- 24. Easily automated Impurities & excess reagents are washed away - x purification after each step . Economical Advantages Excess reagents- Quick completion
- 25. Poor yield- Increasing chain length Monitoring coupling efficiency Disadvantages
- 26. REFERENCES Glick, Bernard R. Molecular biotechnology : principles and applications of recombinant DNA / Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten. — 4th ed https://www.atdbio.com/content/17/Solid-phase-oligonucleotide-synthesis https://www.sigmaaldrich.com/technical-documents/articles/biology/dna-oligonucleotide -synthesis.html https://www.mt.com/us/en/home/applications/L1_AutoChem_Applications/L2_ReactionAn alysis/oligonucleotide-synthesis.html?GLO_YT_Autochem_OTH_Youtube_Autochem#over viewaf http://technologyinscience.blogspot.com/2013/10/oligonucleotide-synthesis.html#.YIDc-e gzZPa https://www.youtube.com/watch?v=GmR2UWJbGzs https://forms.gle/bsN1JgejeLNBm8mq5