Synthesis Cycle

  1. Deblocking

    The first nucleotide attached to the solid support is deprotected by removing the DMT-protecting group. This produces a free 5’ hydroxyl group to react with the next nucleotide.
  2. Coupling

    The second nucleotide is activated then added to the reaction and is covalently attached (i.e. coupled) to the previous nucleotide.
  3. Capping

    Any of the first nucleotide that failed to react is capped so that it will no longer participate at any subsequent step.
  4. Oxidation

    The bond between the first nucleotide and the successfully coupled second nucleotide is oxidized to stabilize the growing chain.
  5. Deblocking

    The 5’ DMT group is removed from the second nucleotide to prepare it for further cycles.

    At the end of the oligo synthesis, the crude product is cleaved from the solid support (CPG or polystyrene beads) and purified using one of a variety of methods.

Use of amino-modified oligos

In the first case the label is conjugated to an amino-modified oligonucleotide (3’, 5’ or on a dT) using its amino-reactive version (N-hydroxysuccinimide (NHS) ester in most cases).

Use of a maleimide-modified label

The second possibility (originally also used for synthesis of molecular beacons) is the addition of a maleimide-modified label to a thiol-modified oligonucleotide.

View the Modification list