Abstract
A method is described as an alternative to the use of nucleoside pre-functionalized supports for DNA synthesis. The procedure should allow the generation of 3'-OH terminal moieties of any natural or modified DNA fragment using a single derivatized solid support material. The method utilizes 1-O-(4,4' dimethoxytrityl)-2- O-succinoyl-3-N-allyloxycarbonylpropane immobilized on amino-propyl CPG followed by subsequent coupling of unit phosphoramidites. Work up is accomplished by removal of the 3-N-allyloxycarbonyl group [Pd(0) at 50 degrees C for 15 min] followed by cleavage under very mild conditions (aqueous TEAA/NH3 buffer pH 10, room temperature) to release the desired product. The mechanism is believed to involve nucleophilic attack of the linker-derived amino group on the 3'-phosphate triester, followed by elimination of the desired product. DNA synthesis with the new support and with classical nucleotide synthesis supports have been performed, and the products shown to be identical. Further proof of product integrity was given by MALDI mass spectral studies and the efficacy of DNA primers made with the new support in PCR amplification.
Full Text
The Full Text of this article is available as a PDF (73.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Crea R., Horn T. Synthesis of oligonucleotides on cellulose by a phosphotriester method. Nucleic Acids Res. 1980 May 24;8(10):2331–2348. doi: 10.1093/nar/8.10.2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nelson P. S., Frye R. A., Liu E. Bifunctional oligonucleotide probes synthesized using a novel CPG support are able to detect single base pair mutations. Nucleic Acids Res. 1989 Sep 25;17(18):7187–7194. doi: 10.1093/nar/17.18.7187. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vu H., Joyce N., Rieger M., Walker D., Goldknopf I., Hill T. S., Jayaraman K., Mulvey D. Use of phthaloyl protecting group for the automated synthesis of 3'-[(hydroxypropyl)amino] and 3'-[(hydroxypropyl)triglycyl] oligonucleotide conjugates. Bioconjug Chem. 1995 Sep-Oct;6(5):599–607. doi: 10.1021/bc00035a015. [DOI] [PubMed] [Google Scholar]
- Wang Y. Y., Lyttle M. H., Borer P. N. Enzymatic and NMR analysis of oligoribonucleotides synthesized with 2'-tert-butyldimethylsilyl protected cyanoethylphosphoramidite monomers. Nucleic Acids Res. 1990 Jun 11;18(11):3347–3352. doi: 10.1093/nar/18.11.3347. [DOI] [PMC free article] [PubMed] [Google Scholar]