Synthesizing the Desired DNA/ Gene, has many applications in the
Biochemical and Biotechnological experiments.
I am here going to focus on the,
“Advanced and Contemporary method of chemical
synthesis of DNA” .
In 1947, HAR GOBIND KHORANA, for the first
time synthesized his gene of interest ( which is,
alanine t-RNA of yeast)
The Evolution of chemical
Many years ago, several laboratories, focussed on developing new
procedures for polynucleotides synthesis, so that they would be
readily accessible for biological and biochemical experiments.
In 1981, this goal has been achieved as methods were available
that could be used for rapid preparation of deoxyoligonucleoties.
And those procedures were based on the earlier works of
R.LETSINGER, which are generally known today as the
Significance of the,
The Phosphoramidite method is so successful and it is
currently widely used because of,
I. READY ADAPTABILITY - to automated DNA Synthesis machines
II. VERY HIGH YIELD and
III. RELATIVELY HIGH PURITY - Of polynucleotides having 100 or more
“Gene Machine” – The 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 the,
“Oligonucleotide synthesis” – In General
“Oligonucleotide synthesis”, is the chemical synthesis of relatively short
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'
In chemical synthesis, a DNA strand is synthesized from free nucleotides
without the aid of TEMPLATE STRAND, and DNA POLYMERASE.
Deducing the Nucleotide sequence
Isolation and Purification of Target Protein from the
The Nucleotide Sequence is deduced from the Amino
Acid sequence of Target Protein.
This is the “Nucleotide sequence of the Gene” for
Before chemical synthesis, the deduced nucleotide sequence
is divided into many small units of 60 – 80 base sizes.
The computer is programmed to make nucleotides sequence
of one of the units.
The Reagents and Nucleotides are filled in the containers.
To prevent the side reactions the amino group of Nitrogenous
base, Demethoxytrityl (DMT), Methyl group, and Di-Iso
propylamine group attaches to the Nucleotide. And this
Chemically protected Nucleotide is called the
Hence this method is called “Phosphoramidite
The First Nucleotide is linked with the Spacer Molecule, which when pumped
into synthesizer column, binds with the glass beads, to form the ‘starting
complex’, which acts as solid support for chemical synthesis.
Unreacted Nucleotides are washed out by Acetonitrile and then Acetonitrile
itself is washed out by Argon.
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 Detritylation.
TCA and DMT are washed out , and the amount of DMT released is
estimated by Spectrophotometer
STEP 5 Activation and Coupling Reaction
As programmed, the second nucleotide is
pumped into synthesizer column, and
simultaneously TETRAZOLE is pumped
into it. The Tetrazole activates the
Phosphoramidite to form a covalent bond
between its 3’ P-group, and 5’ OH-group of
This is called Activation and Coupling!
STEP 6 Capping Process…
Phosphoramidite and Tetrazole
are 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 reactions.
This is called Capping!
STEP 7 Oxidation…
Iodine mixture is pumped
into the synthesizer column
for strengthening the
between the two Nucleotides.
This is the Oxidation
Recovery of the Final Product
The steps 2 – 7 are repeated many times till the final nucleotide gets added to the growing
In every cycle, a new nucleotide is added to the DNA strand. At the end of the reaction,
many single stranded DNAs with a unique sequences are found attached with the glass
The DNA at the final nucleotide is removed by treating the column with TCA
The ssDNAs are eluted from the column using a buffer solution.
Methyl group at phophodiester bonds is removed from the DNA. Amino protecting groups
are removed chemically.
The final DNA solution contains a mixture of single stranded DNAs. They differ in their
length. The full length DNA strands are separated from others by electrophoresis.
In this way many different ssDNA units are made separately and joined together to make
complete duplex DNA (Insert DNA).
Assembly of a Synthetic Gene
All the ssDNA ‘s are mixed together
and treated with T4 DNA Ligase.
The Complementary strands
hybridized with each other to form
duplex DNA and the overlapping
sequences bind the adjacent strands
by hydrogen bonding.
As a result Nicks are there in duplex
DNA. The Nicks are sealed by T4
Final Product is the “Full Length
Desired DNA” , which is amplified and
used in Gene Manipulation.
The New 4 to 8 columns oligo synthesizer,
the smallest one in the world, presented for the first time at
ANALYTICA CHINA SHANGHAI in Sept. 2010!
The smallest in the world…..
Even though the other DNA Chemistries have also been
explored and improved significantly during the past decade,
like “Phosphate - Triester” approach, proposed by A. TODD,
THE OVERALL YIELD,
SPEED OF SYNTHESIS, AND
QUALITY OF DNA,
From these procedures have not yet matched the
I Conclude hereby Stating that,
the “Phosphoramidite Methodology”,
Continues to be the most preferred procedures for the
‘Chemical Synthesis of DNA’.
19th, November, 2010