1. Pt. RAVISHANKAR SHUKLA UNIVERSITY, Raipur
S.o.S in Biotechnology
TOPIC : - Chemical synthesis of DNA
GUIDED BY :
Dr. Afaque Quraishi
SUBMITTED BY – P . Sujata
M.Sc. 3rd Sem
Introduction : Chemical Synthesis of DNA
Evolution of Chemical Synthesis
Phosphite Triester Synthesis
Phosphoramidite method : chemical synthesis of DNA
steps involved :
Activation and coupling
Recovery of final product
Assembly of the product
Significance of the phosphoramidite method
3. Introduction : Chemical Synthesis of
Chemical synthesis of nucleic acids
(DNA/RNA) refers to synthesis of short
nucleic acid fragments with defined
chemical structure and sequence.
Chemically synthesized single-stranded
DNA oligonucleotides are used for
assembling whole genes, amplifying
specific DNA sequences, introducing
mutations into cloned genes, screening
gene libraries, sequencing DNA, and
facilitating gene cloning.
4. Gene Machine OR DNA
known as the
The gene machine
to introduce, in
required for the
coupling of each
nucleotide to the
8. Oligonucleotide Synthesis : In
Oligonucleotide synthesis is the chemical synthesis
of relatively small fragments of nucleic acids with
defined chemical structure.
Whereas enzymes synthesize DNA and RNA in a 5’
to 3’ direction. Chemical oligonucleotide synthesis is
carried out in the opposite 3’ to 5’ direction.
In chemical synthesis , a DNA strand is synthesized
from free nucleotide without the aid of TEMPLATE
STRAND and DNA POLYMERASE.
9. Evolution of Chemical Synthesis
Early work before Phosphoramidite method :
• Phosphodiester synthesis :
• In the 1950’s , khorana and coworkers developed a phosphodiester method where 3 O’- Acetylnucleoside -5’
O- phosphate 2 was activated N,N ‘ dicyclohexylcarbiimide or 4- tolunesulfonylchloride and a 5’ O-
nucleoside was reacted with the activated species to give a protected dinucleoside monophosphate .
• Upon the removal of 3-O’ acetyl group using base catalyzed hydrolysis , further chain elongation was carried
• This methodology , sets of tri and tetradeoxyribonucleotides were synthesized and enzymatically converted
to longer oligonucleotides , which allowed elucidation of the genetic code .
• LIMITATION :
• The major limitation of the phosphodiester method is oligonucleotides branched at the internucleosidic
phosphate. The lack of convenient protection stratergy made the synthesis slower and less selective
chemistry to achieve the ultimated goal of the study .
10. Phosphotriester synthesis
In the 1960’s groups led by R. Letsinger and C.
Rese developed a phosphotriester approach.
In the method the phosphate moeity in the building
block and in the product with 2- cyanoethyl group.
This protected the formation of oligonucleotides
branched at the internucleosidic phosphate.
Limitation : The higher selectivity of the method allowed
the use of more efficient coupling agents and catalysts,
which dramatically reduced the length of the synthesis.
11. Phosphite Triester Synthesis
In the 1970’s substantially more reactive P(III)
derivatives of nucleosides, 3-O-chlorophosphites ,
were successfully used for the formation of inter –
This led to the discovery of phosphite triester
The group led by M. Caruthers look the
advantage of less aggressive and more selective
1H – tetrazolidophosphites and implemented the
method solid phase.
The use of 2- cyanoethyl phosphite- protecting
group in place of less userfriendly methyl group
led to the nucleoside phosphoramidite currently
used in oligonucleoside synthesis.
12. Phosphoramidite method : chemical
synthesis of DNA
Hence this method is called “phosphoramidite method “.
To prevent the side reactions the amino group of nitrogenous base , Demethoxytrityl (DMT) Methyl group and Di –
Isopropylamine group attaches to the nucleotide , and this Chemically protected Nucleotide is called the phosphoramidite.
The Reagents and nucleotides are filled in containers .
The computer is programmed to make nucleotide sequence of one of the units.
The nucleotide sequence is isolated and purified, the deduced nucleotide sequence is divided into many small units of 60-
80 base sizes.
BEFORE CHEMICAL SYNTHESIS ………
13. Starting complex for the chemical
synthesis of DNA strand.
The initial nucleoside has a
protective DMT group
attached to the 5’.
A spacer molecule
attached to the 3’
hydroxyl group of the
The spacer unit is
attached to a solid
support, CPG bead.
14. Structure of phoshoramidite
available for each of
the four bases (A, C, G,
and T) that are used for
the chemical synthesis
of a DNA strand.
group is attached to the
3′ phosphite group of
A β-cyanoethyl group
protects the 3′
phosphite group, and a
DMT group is bound to
the 5′ hydroxyl group of
the deoxyribose sugar.
18. TCA and DMT are washed out and the amount of DMT released is estimated by
As a result starting complex alone exists in the column . TCA is pumped into the
column to release the DMT group, from nucleotide which provides free 5’ OH group
for chain elongation. This is called Detrylation.
Unreacted nucleotides are washed out by acetonitrile. And acetonitrile itself is
washed away with argon .
The first nucleotide is linked with spacer molecule which when pumped into
the synthesizer column, binds with the glass beads to form the starting
complex , which acts as solid support for chemical synthesis.
20. Activation and coupling
The second nucleotide is pumped into the synthesizer
column and simultaneously TETRAZOLE is pumped
into it . The TETRAZOLE activates the
phosphoramidite to form a covalent bond between 3’
P group and 5’ OH group of previous nucleotide .
This is called Activation and coupling .
Unincorporated phosphoramitide and tetrazole is washed out by
acetonitrile and argon .
Acetic anhydride and dimethyl amino pyridine which when pumped into
the column add an acetyl group to the 5’ OH group of previous
nucleotide which in turn prevents the further reaction. This is called
Iodine mixture is pumped into the synthesizer column for
strengthening the phosphotriester bond between two
nucleotides. This is called oxidation.
The applications are including large-scale production of
proteins, testing protein function after changing specific
codons, and creating nucleotide sequences that encode
proteins with novel properties .
These can be used to screen a genomic library for
A set of mixed (degenerated) probes is often
used to screen a genomic library.
It is used in Genome sequencing ,
SNP genotyping , PCR .
It offers a highly effective technique to elucidate gene
functions and analyze protein – nucleic acid interactions.
It helped in modern biological techniques and biotech
Now it is possible to synthesize complicated genes
with reduced costs and shorter turnover time.
28. 28Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 28
by Bernard R.
Glick and Jack J.