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US 6,046,038 AGranted
Optimally labeled oligonucleotides
AssigneePoly Probe, Inc.
InventorThor W. Nilsen
Life of this patent
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Abstract
A method for the preparation of optimally labeled oligonucleotides wherein label-conjugated nucleotide triphosphates are incorporated into a nucleic acid sequence in a defined repetitive manner which allows for the optimal specific detectability of the oligonucleotide. The oligonucleotides of the present invention are useful in the assay of a wide variety of nucleic acid sequences, specifically wherever labeled nucleic acid probes are desired.
Claims16
A method of synthesizing an oligonucleotide containing a single fluorescent dye moiety, comprising:
- (a) preparing a primer;
- (b) preparing a template oligonucleotide containing a nucleotide sequence complementary to said primer and a nucleotide repeat region downstream from said complementary nucleotide sequence, said nucleotide repeat region having the formula:
- (N.sub.m N.sup.t)
- wherein m is an integer from 1 to 11, N is a nucleotide that does not form a base pair with a fluorescent dye-conjugated nucleotide triphosphate, and N.sup.t is a nucleotide that forms a base pair with the fluorescent dye-conjugated nucleotide triphosphate, wherein for each repeat, N.sup.t is the same;
- (c) annealing the template and the primer in a reaction medium comprising a first polymerase, unlabeled nucleotide triphosphates and a predetermined concentration of fluorescent dye-conjugated nucleotide triphosphate;
- (d) initiating synthesis of a complementary strand on the template which defines said optimally fluorescent oligonucleotide;
- and
- (e) isolating said oligonucleotide from the reaction medium, wherein m, said fluorescent dye moiety, and said polymerase are selected such that said oligonucleotide exhibits maximum specific fluorescence.
5 more elements
A method for preparing an optimally fluorescent probe that hybridizes with a target sequence of interest, comprising:
- (a) preparing a primer;
- (b) preparing a template oligonucleotide containing, from 5' to 3', a nucleotide sequence complementary to said primer, a nucleotide repeat region having the formula:
- (N.sub.m N.sup.t)
- wherein m is an integer from 1 to 11, N is a nucleotide that does not form a base pair with a fluorescent dye-conjugated nucleotide triphosphate, and N.sup.t is a nucleotide that forms a base pair with the fluorescent dye-conjugated nucleotide triphosphate, wherein for each repeat, N.sup.t is the same, and an overhang region comprising 6-200 nucleotides;
- (c) annealing the template and the primer in a reaction medium comprising a first polymerase, unlabeled nucleotide triphosphates and a predetermined concentration of fluorescent dye-conjugated nucleotide triphosphate;
- (d) initiating synthesis of a complementary strand on the template which defines said optimally fluorescent oligonucleotide having a 3'end complementary to said overhang region;
- (e) isolating said oligonucleotide from the reaction medium, wherein m, said fluorescent dye moiety, and said polymerase are selected such that said oligonucleotide exhibits maximum specific fluorescence
- (f) denaturing a target sequence, thus producing a denatured target sequence;
- and
- (g) adding an excess of said optimally fluorescent nucleotide, nucleotide triphosphates and a second polymerase in a reaction medium, whereby said 3' region of said optimally labeled oligonucleotide anneals to said target and is extended so as to produce an optimally fluorescent probe.
7 more elements
A method of preparing an optimally fluorescent probe that hybridizes with a target nucleic acid sequence of interest, comprising:
- (a) preparing a recombinant nucleic acid molecule comprising from 5' to 3' and in operable association, a promoter, a template oligonucleotide containing, from 5' to 3', a nucleotide repeat region having the formula:
- (N.sub.m N.sup.t)
- wherein m is an integer from 1 to 11, N is a nucleotide that does not form a base pair with a fluorescent dye-conjugated nucleotide triphosphate, and N.sup.t is a nucleotide that forms a base pair with the fluorescent dye-conjugated nucleotide triphosphate, wherein for each repeat, N.sup.t is the same, and a target sequence of interest;
- and
- (b) providing a reaction medium comprising said recombinant DNA molecule, a polymerase, nucleotide triphosphates conjugated to a fluorescent dye and non-labeled nucleotide triphosphates, to allow synthesis of said probe that is complementary to said template and said target sequence, wherein said polymerase, said fluorescent dye and m are chosen to cause said probe to exhibit maximum specific fluorescence.
2 more elements
Dependencies are inferred from the claim text.