The design of oligonucleotides depends on the specific use of the oligonucleotide. However, the user is often free to choose the sequence within an area, e.g., when designing primers and hybridization probes.
Try to Avoid the Following
Since depurination will decrease the quality and yield of the oligonucleotide, many purines, especially adenosines, close to the 3’ end of the oligonucleotide (and especially an Adenosine at the 3' end) should be avoided. The longer the oligonucleotide, the more serious the problem. If the 3'-nucleotide lacks a base group, no base pairing to the template will occur and enzyme-directed primer elongation is inhibited.
Guanosine is prone to side reactions during synthesis, which may lead to mutations in cloning experiments. Therefore, try to avoid many guanosines in the oligonucleotide, especially stretches of guanosines. Positive clones should always be sequenced.
Furthermore, guanosine stretches have a strong tendency to self-associate, form triple helices, which may complicate purification and use of the oligonucleotide, e.g. guanosine stretches located near the 5’ end of the oligonucleotide will impede enzymatic kination.
Avoid oligonucleotides with partial or complete self-complementary sequences, except when specifically needed. These will form secondary structures and/or aggregates, which will be in equilibrium with the denatured oligonucleotide. In cloning experiments, secondary structures can lead to deletion or duplications of sequences. In primer extension experiments, the effective primer concentration is lowered and also primer-dimer molecules can form.
Since increasing length will decrease fraction of full-length product and increase purification problems, avoid making oligonucleotides longer than necessary.
It is recommended to limit the number of isomers in a mixed oligonucleotide synthesis to 16 since some species may be lost during purification (gel, HPLC or FPLC). Alternatively, deoxyInosine or a universal base (uB) can be incorporated instead. dI and uB have the capacity to base pair to all four bases, dG, dA, dC and dT.
For help designing your probes and primers please use this free RealTimeDesign™ software found on the LGC Biosearch Technologies website. You can submit your sequenced probes or primers through our Webshop.