Oligonucleotides are chemically synthesis by the Phosphoramidite method. It involves four main steps- Detritylation, Coupling, Oxidation and Capping. Advantages and Disadvantages of the method are also discussed. Happy Learning!

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

3. ADENINE CYTOSİNE THYMİNE
GUANİNE

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)

9. 2 DETRITYLATION
TCA
Wash with
acetonitrile
Flush with
Ar

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

16. Ref: https://www.atdbio.com/content/17/Solid-phase-oligonucleotide-synthesis

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
??

21. Ref: https://www.sigmaaldrich.com/technical-documents/articles/biology/dna-oligonucleotide-synthesis.html

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