Linkers play a vital role in oligo synthesis, specifically in two main applications:

Solid Phase Attachment: During oligonucleotide synthesis, a linker molecule covalently attaches the growing oligonucleotide chain to a solid support. This solid support is typically a controlled-pore glass (CPG) bead. The linker offers a stable bond to the solid phase, allowing for the stepwise addition of nucleotides to create the desired oligonucleotide sequence. After synthesis is complete, the linker is cleaved under specific conditions, releasing the purified oligonucleotide from the solid support.

Bioconjugation: Linkers can also be incorporated at either the 5′ or 3′ terminus of the oligonucleotide to facilitate conjugation with various biomolecules. These biomolecules can include fluorophores, quenchers, antibodies, nanoparticles, or other functional groups. Bioconjugation allows researchers to attach functionalities to the oligonucleotide, enabling diverse applications such as:
• Fluorescence in situ hybridization (FISH) for cellular imaging
• Detection of specific nucleic acid sequences
• Immunoassays
• Drug delivery

There are various types of linkers available for oligo synthesis, each with unique properties and functionalities. Some common linker types include:

• Cleavable Linkers: These linkers are designed to be cleaved from the solid support under specific conditions, such as exposure to acid or base. This allows for the release of the purified oligonucleotide.
• Non-Cleavable Linkers: These linkers form a permanent bond with the solid support. While the oligonucleotide remains tethered to the solid phase, non-cleavable linkers are useful for applications like microarrays where the oligos stay attached to the surface.
• Biotinylated Linkers: These linkers contain biotin, a small molecule that can bind to streptavidin with high affinity. This property allows for easy purification of the oligonucleotide using streptavidin-coated magnetic beads.
• Fluorescent Linkers: These linkers incorporate a fluorescent moiety, enabling the direct detection of the labeled oligonucleotide.

The choice of linker in oligo synthesis depends on the desired application and the downstream processes involved.