General Design Guidelines

The following guidelines should be taken into account when designing modified oligonucleotides.

 

1. Sequence Length - SYNTHEGEN can synthesize oligonucleotides from 5 to 110 bases in length. Most sequences range from 18 to 30 bases with the average being 24 bases. Remember that the longer the oligonucleotide, the less the percentage of full length product in the crude synthesis. This results in lower yields after purification.

    

2. Sequence Composition - Make sure your sequence is free of hairpins and self complementary regions. Also, more than six of the same consecutive bases (ie. GGGGGGG) can be problematic and reduce final yields.

    

3. Modification Placement - Whenever possible, place modifications at the 5' end. Automated DNA synthesis occurs in the 3' to 5' direction. Each nucleotide addition is 98-99% efficient resulting in 1-2% of the oligonucleotide being truncated and capped at each position. Placing the modification at the 5' end ensures that only the full length oligonucleotide is modified. Furthermore, because most modifications are more hydrophobic than unmodified oligonucleotide, the full-length modified oligonucleotide binds more tightly to the reverse phase media during HPLC purification. This enhances the separation between the full-length, modified oligonucleotide sequences and the truncated, unmodified olignucleotide sequences.

    

4. Synthesis Scale - The term "synthesis scale" refers to the amount of derivatized solid support used. The final quantity of product delivered will depend on sequence length, sequence secondary structure, type of modification used, placement of modification, number of modifications per oligonucleotide and purification methods used.

    

5. Purification Method - Choose a purification method on the basis of the level of purity required for your specific application. Remember that in general, the higher the purity, the lower the final yield.