Chemical synthesis of dna

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
1
Content
 Introduction : Chemical Synthesis of DNA
 Gene Machine
 Oligonucleotide synthesis
 Evolution of Chemical Synthesis
 Phosphodiester synthesis
 Phosphotriester synthesis
 Phosphite Triester Synthesis
Phosphoramidite method : chemical synthesis of DNA
steps involved :
 Detrylation
 Activation and coupling
 Capping
 Oxidation
 Recovery of final product
 Assembly of the product
 Significance of the phosphoramidite method
 Application
 Conclusion
 Reference
2
Introduction : Chemical Synthesis of
DNA
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.
3
Gene Machine OR DNA
Synthesizer
An automated
machine which
synthesizes the
desired gene
chemically from
the free
nucleotides is
known as the
“gene machine”.
The gene machine
contains
Ten
containe
r
Synthesi
zer
column
Valves
Spectro
photom
eter
Program
med
compute
r
DNA
synthesizers
are programmed
to introduce, in
the correct
order, specified
nucleotides and
the reagents
required for the
coupling of each
consecutive
nucleotide to the
growing chain.
4
5
Old version of DNA Synthesizer
6
7
Oligonucleotide Synthesis : In
General
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.
8
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
out .
• 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 .
9
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.
10
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 –
nucleosidic linkages.
This led to the discovery of phosphite triester
methodology .
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.
11
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 ………
12
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
deoxyribose.
The spacer unit is
attached to a solid
support, CPG bead.
13
Structure of phoshoramidite
Phosphoramidites are
available for each of
the four bases (A, C, G,
and T) that are used for
the chemical synthesis
of a DNA strand.
A diisopropylamine
group is attached to the
3′ phosphite group of
the nucleoside.
A β-cyanoethyl group
protects the 3′
phosphite group, and a
DMT group is bound to
the 5′ hydroxyl group of
the deoxyribose sugar.
14
PHOSPHORAMIDITE METHOD
Detrylation Activation
and coupling
Capping Oxidation
Steps involved
in this method
15
16
17
TCA and DMT are washed out and the amount of DMT released is estimated by
Spectrophotometer.
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.
Detritylation
18
19
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 .
20
Capping
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
capping .
21
Oxidation
Iodine mixture is pumped into the synthesizer column for
strengthening the phosphotriester bond between two
nucleotides. This is called oxidation.
22
Recovery of the Final Product
• .
23
Assembly of the DNA
24
Significance of Phosphoramidite
Method
Very high
yield
Ready
adaptability
Relatively
high purity
The phosphoramidite method is so
successful and it is currently
widely used :
25
Applications
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
the gene.
A set of mixed (degenerated) probes is often
used to screen a genomic library.
It is used in Genome sequencing ,
SNP genotyping , PCR .
26
Conclusion
It offers a highly effective technique to elucidate gene
functions and analyze protein – nucleic acid interactions.
It helped in modern biological techniques and biotech
applications .
Now it is possible to synthesize complicated genes
with reduced costs and shorter turnover time.
27
28Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 28
www.acade
mic.oup.c
om www.Slides
hare.net
www.ncbi.
nlm.nih.go
v
Molecular
biotechnology :
Principles and
applications of
recombinant
DNA :-textbook
by Bernard R.
Glick and Jack J.
Pasternak 4th
edition
THANK YOU
29
1 von 29

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Chemical synthesis of dna

  • 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 1
  • 2. Content  Introduction : Chemical Synthesis of DNA  Gene Machine  Oligonucleotide synthesis  Evolution of Chemical Synthesis  Phosphodiester synthesis  Phosphotriester synthesis  Phosphite Triester Synthesis Phosphoramidite method : chemical synthesis of DNA steps involved :  Detrylation  Activation and coupling  Capping  Oxidation  Recovery of final product  Assembly of the product  Significance of the phosphoramidite method  Application  Conclusion  Reference 2
  • 3. Introduction : Chemical Synthesis of DNA 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. 3
  • 4. Gene Machine OR DNA Synthesizer An automated machine which synthesizes the desired gene chemically from the free nucleotides is known as the “gene machine”. The gene machine contains Ten containe r Synthesi zer column Valves Spectro photom eter Program med compute r DNA synthesizers are programmed to introduce, in the correct order, specified nucleotides and the reagents required for the coupling of each consecutive nucleotide to the growing chain. 4
  • 5. 5
  • 6. Old version of DNA Synthesizer 6
  • 7. 7
  • 8. Oligonucleotide Synthesis : In General 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. 8
  • 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 out . • 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 . 9
  • 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. 10
  • 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 – nucleosidic linkages. This led to the discovery of phosphite triester methodology . 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. 11
  • 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 ……… 12
  • 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 deoxyribose. The spacer unit is attached to a solid support, CPG bead. 13
  • 14. Structure of phoshoramidite Phosphoramidites are available for each of the four bases (A, C, G, and T) that are used for the chemical synthesis of a DNA strand. A diisopropylamine group is attached to the 3′ phosphite group of the nucleoside. A β-cyanoethyl group protects the 3′ phosphite group, and a DMT group is bound to the 5′ hydroxyl group of the deoxyribose sugar. 14
  • 15. PHOSPHORAMIDITE METHOD Detrylation Activation and coupling Capping Oxidation Steps involved in this method 15
  • 16. 16
  • 17. 17
  • 18. TCA and DMT are washed out and the amount of DMT released is estimated by Spectrophotometer. 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. Detritylation 18
  • 19. 19
  • 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 . 20
  • 21. Capping 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 capping . 21
  • 22. Oxidation Iodine mixture is pumped into the synthesizer column for strengthening the phosphotriester bond between two nucleotides. This is called oxidation. 22
  • 23. Recovery of the Final Product • . 23
  • 24. Assembly of the DNA 24
  • 25. Significance of Phosphoramidite Method Very high yield Ready adaptability Relatively high purity The phosphoramidite method is so successful and it is currently widely used : 25
  • 26. Applications 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 the gene. A set of mixed (degenerated) probes is often used to screen a genomic library. It is used in Genome sequencing , SNP genotyping , PCR . 26
  • 27. Conclusion It offers a highly effective technique to elucidate gene functions and analyze protein – nucleic acid interactions. It helped in modern biological techniques and biotech applications . Now it is possible to synthesize complicated genes with reduced costs and shorter turnover time. 27
  • 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 www.acade mic.oup.c om www.Slides hare.net www.ncbi. nlm.nih.go v Molecular biotechnology : Principles and applications of recombinant DNA :-textbook by Bernard R. Glick and Jack J. Pasternak 4th edition