DNA Synthesis

DNA Synthesis via Phosphoramidites

We manufacture synthetic oligonucleotides using the phosphoramidite method. First, a nucleoside is attached to a solid support, controlled pore glass (CPG), through the 3’ hydroxyl. Oligonucleotide synthesis is done by sequential addition to the 5’ end of the growing oligo. The high coupling efficiency (typically >99%) permits the manufacture of longmers in excess of 100 bases. Solid phase synthesis allows excess reagents to be washed away and avoids polymerization that would occur in a solution phase reaction. 

1. Detritylation: The dimethoxytrityl (DMT) protecting group is removed by treatment with acid to form a 5’ reactive hydroxyl.
2. Coupling: The 5’ reactive hydroxyl couples to a dexoynucleoside phosphoramidite (activated with tetrazole) to link the two nucleosides.
3. Capping: Unreacted 5’ hydroxyls are capped by acetylation to prevent extension in subsequent coupling reaction. 
4. Oxidation: An iodine solution is used to oxidize the phosphite triester group resulting from the coupling step to a phosphotriester.

This cycle is repeated until the desired sequence is synthesized. This stepwise procedure is well-suited to automation.

DNA Synthesis

The history at LGC Biosearch Technologies in custom oligonucleotide synthesis can be traced back to the early 1980’s during the prenatal stages of the synthetic DNA market. The company’s first entity, originally known as ‘Biosearch’ was one of the first manufacturers of solid phase DNA synthesizers. Today, Biosearch Technologies manufactures the reagents for oligonucleotide synthesis (dyes, amidites, and CPGs), and uses these building blocks to synthesize custom oligonucleotides and fluorogenic probes.  We are our own primary supplier for synthesis reagents!

We manufacture synthetic oligonucleotides using the phosphoramidite method. First, a nucleoside is attached to a solid support, controlled pore glass (CPG), through the 3’ hydroxyl. Oligonucleotide synthesis is done by sequential addition to the 5’ end of the growing oligo. The high coupling efficiency (typically >99%) permits the manufacture of longmers in excess of 100 bases. Solid phase synthesis allows excess reagents to be washed away and avoids polymerization that would occur in a solution phase reaction. 

1. Detritylation: The dimethoxytrityl (DMT) protecting group is removed by treatment with acid to form a 5’ reactive hydroxyl.
2. Coupling: The 5’ reactive hydroxyl couples to a dexoynucleoside phosphoramidite (activated with tetrazole) to link the two nucleosides.
3. Capping: Unreacted 5’ hydroxyls are capped by acetylation to prevent extension in subsequent coupling reaction. 
4. Oxidation: An iodine solution is used to oxidize the phosphite triester group resulting from the coupling step to a phosphotriester.

This cycle is repeated until the desired sequence is synthesized. This stepwise procedure is well-suited to automation.

Placement of Modifications

Because synthesis is done in the 3’ to 5’ direction, truncated sequences will have 3’ modifications. Therefore, it is best to place nonfluorescent modifications on the 3’ end and reporter dyes on the 5’end to avoid fluorescent impurities.

Dyes, quenchers and other modifications should be added as CPGs or phosphoramidites if possible. This is less expensive, easier to automate, increases the amount incorporated and facilitates purification.