Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1991-03-20
1995-07-25
Robinson, Douglas W.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536 253, 536 2531, 536 243, C07H 2104
Patent
active
054363276
DESCRIPTION:
BRIEF SUMMARY
There are several potential applications for oligonucleotides bound to solid supports. They could be used to test for the presence of mutations in complex DNAs--for example for disease loci in Humans. They could be used to select specific nucleic acids from the complex mixtures; for example specific mRNAs from a whole cell population. They will be useful in the invention described in International Application PCT/GB89/00460 filed 2 May 1989.
Several papers describe methods for attaching nucleic acids to solid matrices (1-6). These methods suffer from two problems: they require complex and often inefficient steps; the nucleotide is linked through the bases as well as the ends. Linkage through the bases interferes with subsequent use of the bound polynucleotide in hybridization reactions.
Methods for synthesizing oligonucleotides on solid supports are well established (7-14). The linkage between the oligonucleotide and the support is labile to the the final reagent used to remove blocking groups in the bases, and so this step in the process also removes the oligonucleotide from the solid support. Oligonucleotides would remain tethered to the support of a stable link were used. Sproat and Brown (1985) have shown that a urethane link is more stable than the usual succinate link, however, it requires a complex synthesis, and is not completely stable to the final deprotection step.
Crea and Horn (1980) used a ribonucleotide, linked through the 5'-hydroxyl group to cellulose, to initiate oligonucleotide synthesis. The link between the first and second residues of the resulting chain is labile to the final deprotection step.
Arnold and Berg (1985) describe a polymeric support with a covalently bonded primer for oligonucleotide synthesis, wherein the primer is cleaved by selective oxidation without oxidizing other bonds of the oligonucleotide.
It is an object of this invention to provide a new link which is easy to synthesize and completely stable to standard deprotection steps.
In one aspect the invention provides a method of making a derivatized support suitable for oligonucleotide synthesis, which method comprises attaching a nucleoside reagent to a support carrying hydroxy groups by a covalent phosphodiester link which is stable to conditions used for removing protective groups from oligonucleotide chains, characterized in that the hydroxyl groups are aliphatic hydroxyl groups in which the aliphatic moiety is --(C.sub.n H.sub.2n)-- where n is at least 3 or alkoxy or poly(alkoxy).
In another aspect the invention provides a method of preparing an oligonucleotide bound to a support by including protecting groups, and nucleoside becomes attached to the support by a covalent phosphodiester link which is stable to the conditions used in step c), characterized in that the hydroxyl groups are aliphatic hydroxyl groups in which the aliphatic moiety is --(C.sub.n H.sub.2n)-- where n is at least 3 or alkoxy or poly(alkoxy).
In a further aspect the invention provides a derivatized support suitable for oligonucleotide synthesis comprising a nucleoside linked to a support by means of a covalent phosphodiester link of the structure --O--PY--O--, where Y is a protected or unprotected oxygen atom characterized in that the link to the support is through an aliphatic moiety which is --(C.sub.n H.sub.2n)-- where n is at least 3 or alkoxy or poly(alkoxy).
In yet another aspect the invention provides a support-bound oligonucleotide wherein the oligonucleotide is bound to the support through a terminal phosphate group by a covalent phosphodiester link, characterized in that the link has the structure --O--PO.sub.2 --O--R-- where R is an aliphatic moiety which is --(C.sub.n H.sub.2n)-- where n is at least 3 or alkoxy or poly(alkoxy).
The nature of the support is not critical to the invention. It may be massive or particulate and may for example be of derivatized silica gel or Kieselguhr-polydimethyl-acrylamide, or controlled-pore glass, or a plain glass surface. What is essential is that it carry aliphatic hydroxyl groups, and these i
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Pierce Handbook and General Catalog. Pierce Chemical Co. 1979-1980, p. 361.
Sigma Chemical Co. Handbook 1989, p. 142.
Pon et al. BioTechniques 6(8):768-775 (1988).
Crea et al. Nucl. Acids Res. 8(10):2331-2348 (1980).
Gilham, P. T. J.A.C.S. 86:4982-4985 (1964).
Froehler et al. Nucl. Acids Res. 14(13):5399-5407.
Maskos Uwe
Southern Edwin M.
Isis Innovation Limited
Kunz Gary L.
Robinson Douglas W.
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