Nucleic acid probes and methods
AssigneeDuke University
InventorsMark W. Grinstaff, Amy E. Beilstein, Shoeb I. Khan
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Abstract
The present invention provides metal-containing purines, pyrimidines, nucleosides, nucleotides and oligonucleotides; including phosphoramidite and photolabile derivatives thereof, including methods of making and method of using same. The present invention provides a method for detection of nucleic acid sequences via electrochemical or photochemical means.
Claims40
A nucleoside, nucleotide, purine or pyrimidine derivative comprising a detectable marker which is a metal complex containing at least one of a M(diimine)xY+ complex, M(terpyridine)xY+ complex or a metallocene wherein M is a transition metal, said diimine is selected from a bipyridine, phenanthroline or terpyridine derivative;
- x is 1, 2, or 3 and y is 0, 1, 2, 3, or 4, said bipyridine, phenanthroline, terpyridine and metallocene being optionally substituted by any one of alkyl, alkene, alkyne, aryl, alkylaryl, and carboxyalkyl, amide, ester or ether.
An improved method of synthesizing a labeled oligonucleotide comprising:
- attaching a nucleoside of the oligonucleotide to be synthesized to a solid support through a labile linker arm;
- said nucleoside containing a removable protecting group attached to a 5′hyroxyl of a sugar group of said nucleoside;
- removing said protecting group;
- adding a further nucleoside containing a labile linker arm and a protecting group to said solid support under conditions where said further nucleoside is coupled to said 5′hydroxyl of said sugar group through said linker arm;
- optionally capping any uncoupled nucleoside;
- removing the protecting group of said further nucleoside;
- repeating the above with further nucleoside derivatives wherein at least one of said further nucleoside contains a detectable label, until a desired oligonucleotide is formed;
- and
- cleaving said oligonucleotide from said solid support;
- wherein the improvement comprises adding at least one dimethoxytrityl, phosphoramidite halodonucleoside derivative in place of one of said at least one nucleoside containing a detectable label;
- and
- adding a detectable label to said halodonucleoside derivative subsequent to formation of said oligonucleotide.
An improved method of oligonucleotide synthesis comprising combining phosphoramidite nucleoside derivatives in combination with a solid support wherein the improvement comprises adding at least one phosphoramidite halonucleoside during the synthesis and subsequently labeling said halonucleoside with a marker in a metal catalyzed reaction.
Dependencies are inferred from the claim text.