ABI3900 Synthesizer Columns with CUTAG CPG Frits

Proligo® presents the next generation support for reliable, cost-effective oligo synthesis without cross-contamination.

A novel 0.2 μmol ready-to-use Universal ABI 3900 synthesizer column for the synthesis of top quality oligonucleotides has been developed. This new column contains a universal CUTAG loaded CPG 1000 Å frit instead of a polystyrene or CPG support filling, and is therefore powder free. The CPG frit has several advantages over conventional powder filling, which include:

  • Prevention of cross-contamination from airborne support particles
  • Enhanced flow control resulting in improved synthesis consistency
  • Reduction of reagent consumption and lower cost

Structure

synthesis frit containing polyethylene-CPG material

Figure 1. The oligonucleotide synthesis frit consists of a polyethylene-CPG composite material. The CPG is loaded with CUTAG universal anchor, which is related to the Ionis UnyLinker™.1


1 The original UnyLinker™ contains an N-phenyl instead of the N-methyl group, which avoids the release of toxic aniline during the deprotection process. UnyLinker™ is protected by US patent 7,202,264.

 

Product Application

This novel column is designed for ABI 3900 synthesizers. It includes the column body and the CUTAG CPG 1000 Å frit. For the synthesis of DNA oligonucleotides there are only a few minor changes compared to the use of nucleoside loaded supports.

Due to the application of the CUTAG universal support, these changes are related to the downstream processing of the synthesized oligonucleotides.

  • The columns can be fixed in the banks as for the original ABI columns (including tapping with the hammer)
  • The instrument pressure should be set to 5 psi, the porosity of the frits is adjusted to avoid fast run through or overflow
  • The original ABI 3900 0.2 μmol synthesis protocol can be employed
  • After completion of the solid-phase synthesis the columns are removed from the banks using the column removal tool and placed in a manifold, or processed according to the customer’s setup for downstream processing

Adjustment of reaction conditions for deprotection
In contrast to nucleoside-loaded supports, the CUTAG support requires dephosphorylation in addition to cleavage and deprotection. If dephosphorylation is incomplete, a modified sequence can be formed as a by-product, whereby the (partial) anchor group remains on the oligonucleotide. To prevent this, we recommend the following deprotection conditions:

  Temperature Time
Ammonium hydroxide 80 °C
55 °C
2 h
8 h
Ammonium hydroxide / 40% methylamine 1:1 (AMA) 80 °C
55 °C
30 min
2 h

Other conditions might be also suitable but should be verified by analysis of the oligonucleotide product by LC-MS.

Synthesizer Platforms

The novel Proligo® ABI 3900 synthesizer columns are applicable for a number of synthesizer platforms.

  • ABI 3900 High Throughput DNA Synthesizer
  • Biolytic ABI 3900 and Dr. Oligo
  • OligoMaker ApS
  • Bioautomation

Besides the physical properties of the CPG frit, such as the porosity that determines the flow characteristics, the column body design is also critical.

Example of ABI3900 Synthesizer Columns containing a CUTAG CPG Frit

Figure 2. Example of the new ABI 3900 Synthesizer Column with CUTAG CPG Frit

Where You Can Really Benefit

The novel Proligo® ABI 3900 synthesizer column contains a CPG-polymer composite as the solid support. The single piece composite material, or CPG-frit, replaces the three part content of a conventional column, which consists of a bottom and top filter and the CPG or polystyrene powder in between. This modification has several advantages:

  • The column does not contain any support powder so loss of support caused by misplaced filters or cross-contamination from airborne particles is eliminated. Particularly useful when applying gas-phase deprotection.
  • The composite material does not swell in any of the common solid-phase synthesis solutions, resulting in low reagent consumption and more reliable synthesis results.
  • Due to the porous properties of the composite material the solvents and reagent solutions are absorbed like a sponge and equally distributed into the pores of the CPG, leading to more effective usage of reagent volumes.
  • Instead of four different nucleoside loaded support columns, just one universal support column is required; the state of the art CUTAG anchor is related to the Iosys UnyLinker™ support which enables reliable cleavage and dephosphorylation under mild conditions.
AX-HPLC chromatogram of crude dT80 synthesized on a Proligo® 0.2 µmol, ABI 3900 synthesizer column

Figure 3. AX-HPLC chromatogram of crude dT80 synthesized on a Proligo® 0.2 µmol, ABI 3900 synthesizer column.

Materials

     
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