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Cartridge-based high-throughput purification of oligonucleotides for reliable oligonucleotide arrays
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
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2006 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 356, no 1, 132-141 p.Article in journal (Refereed) Published
Abstract [en]

A novel, cartridge-based procedure for the efficient and irreversible detritylation of oligonucleotides is reported. This method, combined with a process for the elimination of depurinated fragments produces, in a highly parallel fashion, oligonucleotides with better purity than those traditionally obtained using reversed-phase high-performance liquid chromotography purification. Our combined detritylation and purification methodology compares favorably with commercial cartridge-based purification systems. The benefits of working with pure oligonucleotides, with regard to higher signal and better signal linearity, are shown in array-based hybridization experiments.

Place, publisher, year, edition, pages
2006. Vol. 356, no 1, 132-141 p.
Keyword [en]
oligonucleotide, synthesis, purification, cartridge, microarray
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-94934DOI: 10.1016/j.ab.2006.05.008ISI: 000240170700017PubMedID: 16769031OAI: oai:DiVA.org:uu-94934DiVA: diva2:168960
Available from: 2006-10-02 Created: 2006-10-02 Last updated: 2011-06-20Bibliographically approved
In thesis
1. Novel Methods for Synthesis of High Quality Oligonucleotides
Open this publication in new window or tab >>Novel Methods for Synthesis of High Quality Oligonucleotides
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The first part of the work describes a procedure of oligonucleotide purification using a reversed-phase cartridge. The developed method employs a very efficient yet mild oligonucleotide detritylation on the cartridge support allowing fast purification of oligonucleotides regardless of their 5´-modification. Thiol- and amino-modified oligonuc-leotides were detritylated and purified with the same high efficiency as non-modified oligonucleotides. The method enables fast, parallel and automated purification of many oligonucleotide probes that was not possible before. In combination with the method of removal of tritylated failure fragments oligonucleotides were produced with purity superior to that of oligonucleotides purified using RP HPLC.

In the second part of the present study a method of solid-phase RNA synthesis using 2´-tert-butyldithiomethyl (2´-O-DTM) is discussed. The stability of the DTM group during oligonucleotide assembly and deprotection in ammonia, together with its ability for rapid deprotection under mild conditions, allowed the synthesis of RNA with the quality similar to that of synthetic DNA oligonucleotides. The advantage of the 2´-O-DTM group is that it is completely orthogonal to all protecting groups used for the traditional solid-phase DNA synthesis. Therefore, the synthesis can be performed using a standard DNA synthesis procedure – no changes are needed for the product assembly. RNA oligonucleotides synthesized with retained 5´-terminal trityl group can be subjected to a cartridge-based purification using the procedure described in the first part of the study. The phosphoramidite synthesis was optimized for a large scale preparation and gives versatility for introduction of other alkyldithiomethyl groups according to the preference to their certain properties.

The third part of the thesis describes the synthesis of a dithiomethyl linker and its utility for reversible conjugation of oligonucleotides. A dithiomethyl group, cleavable under mild conditions, was introduced onto 3´-OH of tritylated nucleosides via 3´-O-methylthiomethyl derivatives. The influence of different alkyl substituents on the disulfide bond stability was investigated, and stable analogues were employed in oligosyntheses. Two applications were developed using the present linker: 1) purification of oligonucleotides linked to the solid support; and 2) cartridge-based purification of tritylated oligonucleotides having an additional hydrophobic group on their 3´- terminus.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 62 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 179
Organic chemistry, oligonucleotide, solid-phase synthesis, RNA synthesis, phosphoramidite, abasic sites, depurination, 2'-O-DTM, protecting group, cartridge, conjugate, dithiomethyl linker, base-stable linker, oligonucleotide purification, nucleoside, Organisk kemi
urn:nbn:se:uu:diva-7172 (URN)91-554-6672-9 (ISBN)
Public defence
2006-10-23, Rudbecksalen, Rudbeck Laboratory, Dag Hammarskjölds väg, 20, Uppsala, 09:15
Available from: 2006-10-02 Created: 2006-10-02Bibliographically approved

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