Some sequencing strategies as well as PCR probes require the 3'- terminus of an oligonucleotide to be blocked from allowing polymerase extension. This may be achieved by modifying the 3'-terminus with a phosphate group, a phosphate ester, or using an inverted 3'-3' linkage. However, side reactions during deprotection of the oligonucleotide or enzymatic impurities may free the 3'-hydroxyl group to a small extent. So far, the 3'-propyl phosphate formed using 3'-Spacer C3 oligo modification has proved to be the simplest and most effective non-nucleosidic blocker of the 3'-terminus. We have no definitive data on the propyl group to support the assertion that it protects oligos from exonuclease digestion and does not permit polymerase extension. Our conclusion is based by analogy to the propylamino modification. This modification protects oligos from exonuclease digestion but permits polymerase extension to a small extent since the modifier is eliminated to a level of about 10% from the 3' terminus, leaving the 3'-hydroxyl group available. HPLC experiments have shown that there is no detectable elimination of the propyl group from oligos made from the spacer C3 modification
2',3'-Dideoxynucleosides
The surest way to guarantee blocking the 3'-terminus is using a 2',3'-dideoxynucleoside support. Unfortunately, only ddC are amenable to attachment to the support through the exocyclic amino group.
3'-Deoxynucleosides
In situations where it is necessary to have a selection of all four bases available, it is possible to use the 3'-deoxynucleoside supports as 3'-terminators. Although the 2'-hydroxyl group is still present in the final oligonucleotides, it is not a substrate for at least the routinely used polymerases. All four 3'-deoxynucleoside supports will shortly be available, along with their phosphoramidite counterparts.
- 3'-Phosphate CPG
- 3'-Spacer C3 CPG
- 2',3'-ddC-CPG
- 3'-dA-CPG
- 3'-dC-CPG
- 3'-dG-CPG
- 3'-dT-CPG
REFERENCE(S): (1) J.G. Zendegui, K.M. Vasquez, J.H. Tinsley, D.J. Kessler, and M.E. Hogan, Nucleic Acids Research, 1992, 20, 307-314.