Download The solid- phase synthesis of nucleic acids

The solid- phase synthesis of
nucleic acids
Prepared by Mary Naluguza
Definition
Solid-phase synthesis of nucleic acids is an
automated method in which molecules are
bound on a bead and synthesized step-by-step
in a reactant solution.
This idea was first developed by Bruce Merrifield
who synthesized polypeptides and earned him the
Nobel Prize in 1984.
Principle
DNA strands can be synthesised by the
sequential addition of activated monomers to
the growing chain that is linked to an insoluble
(solid) support.
The chain is synthesized in 3' to 5' direction.
Activated monomers are the protonated
deoxribonucleoside 3’-phospharamidites.
Activated monomer
Description of protocol
Step 1 (coupling)
The 3’- phosphorus atom of this incoming unit becomes
joined to the 5’- oxygen atom of the growing chain to form a
phosphite trimester atom of the activated monomer.
The 5’- OH group of the activated monomer is unreactive
because it is blocked by a dimethoxytriyl (DMT) protecting
group, and the 3’- phosphoryl group is rendered unreactive by
attachment of the β- cyanoethyl (βCE) group. Likewise, the
amino group on the purine and pyrimidine bases are blocked.
Coupling is carried out under anhydrous conditions because
water reacts with phosphoramidites.
Solid- phase synthesis of DNA chain by the phosphite triester
method
Description of protocol continued……..
Step 2 (oxidation)
The phosphite triester (in which P is trivalent) is oxidised by
iodine to form a phosphotriester (in which P is pentavalent).
Step 3 (deprotection)
The DMT protecting group on 5’-OH group of the growing
chain is removed by the addition of dichloroacetic acid, which
leaves other protecting groups intact.
The DNA chain is now elongated by one unit and ready for
another cycle of addition. (i.e. ready to react with another
incoming activated monomer)
Removal of protecting group
-At the end of the synthesis, NH3 is added to
remove all the protecting groups and release
the oligonucleotides from the solid support.
-The sample can then be purified by highpressure liquid chromatography or
electrophoresis on polyacryamide gels.
-Each cycle takes only about 10 times and
usually elongates more than 99% of the
chains.
Applications
• Synthesis of new tailor- made genes. New proteins
with novel properties can now be produced in
abundance by expression of synthetic genes.
• Synthesis of interferons that have antiviral activity
and ribonuclease that is catalytically active.
• PCR amplifications
• Manipulation of gene fragments
• Gene fragment and total gene synthesis
• Primers for radioactive labelling
• Hybridisation: “fishing” in libraries with probes
Conclusion
This method has the advantage of washing away all
the soluble impurities without loosing any product.
This is because the growing nucleotides is attached
to an insoluble support. Thus, this method has the
ability of rapidly synthesising DNA chains and this has
opened many experimental avenues.