Concept explainers
Oligonucleotide Synthesis
In Section
we noted that synthetic oligonucleotides of defined sequence were commercially
available for use as primers for PCR and as probes for cloning DNA. Here we will examine how these oligonucleotides are prepared.
The method bears many similarities to the Merrifield solid-phase synthesis of peptides. A starter unit is attached to a solid support, nucleosides are attached one-by-one until the sequence is complete, whereupon the target oligonucleotide is removed from the support and purified. Like solid-phase peptide synthesis, the preparation of oligonucleotides relies heavily on protecting groups and bond-forming methods.
The starter units are nucleosides in which
Thymidine lacks an
These
The
(DMT) ether.
The nucleoside that is to serve as the
controlled-pore glass (CPG) bead by ester formation between its unprotected
The stage is now set for adding the second nucleoside. The four blocked nucleosides prepared
earlier are converted to their corresponding
Each phosphoramidite is coupled to the anchored nucleoside by a reaction in which the free
The product of the coupling is a phosphite; it has the general formula
The
Once all the nucleosides are in place and the last DMT is removed, treatment with aqueous
ammonia removes the acyl and cyanoethyl groups and cleaves the oligonucleotide from the CPG
support.
Consider the conjugate bases of structures
B.
C.
different.
D.
give the same conjugate base on deprotonation; the conjugate base of
different.
Section
Many important compounds contain two or more nucleotides joined together by
a phosphodiester linkage. The best known are those in which the phosphodiester joins the
Oligonucleotides contain about
phosphodiester links; polynucleotides can contain thousands of nucleotides.
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ORGANIC CHEMISTRY (LOOSELEAF)-PACKAGE
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