# US 6,046,038 A · Optimally labeled oligonucleotides

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- Number: US 6,046,038 A
- Document ID: US6046038A
- Status: Granted
- Kind: A
- Application number: 08/909,539
- Filed: 1997-08-12
- Granted: 2000-04-04
- Earliest claimed: 1996-06-04
- Assignees: Poly Probe, Inc.
- Inventors: Thor W. Nilsen
- Web page: https://patentgrep.com/patent/US6046038A

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/682: Signal amplification
- C12Q 1/6841: In situ hybridisation
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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.

## Claims

### Claim 1

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.

### Claim 2 (depends on 1)

The oligonucleotide of claim 1, wherein said method further comprises (f) attaching said oligonucleotide to a probe that hybridizes with a nucleic acid of interest.

### Claim 3 (depends on 2)

The oligonucleotide of claim 2, wherein said attaching comprises ligation.

### Claim 4 (depends on 2)

The oligonucleotide of claim 2, wherein said attaching comprises randomer extension.

### Claim 5 (depends on 2)

The oligonucleotide of claim 2, wherein said attaching comprises cloning.

### Claim 6 (depends on 1)

The oligonucleotide of claim 1, wherein said isolating comprises precipitation, size fractionation, gel electrophoresis or antigen-specific binding.

### Claim 7

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.

### Claim 8 (depends on 7)

The method of claim 7 wherein said template has a 3' end comprising a random sequence of 6 to 12 nucleotides.

### Claim 9

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.

### Claim 10 (depends on 9)

The method of claim 9 wherein said polymerase is an RNA polymerase.

### Claim 11 (depends on 10)

The method of claim 10 wherein said promoter is a T3 promoter.

### Claim 12 (depends on 10)

The method of claim 10 wherein said promoter a T7 promoter.

### Claim 13 (depends on 10)

The method of claim 10 wherein said promoter is a SP6 promoter.

### Claim 14 (depends on 9)

The method of claim 9 wherein said polymerase is a DNA polymerase, said conjugated and non-labeled nucleotide triphosphates are deoxyribonucleotide triphosphates and wherein said reaction medium further comprises a primer.

### Claim 15 (depends on 14)

The method of claim 14 wherein said template further comprises a nucleotide sequence complementary to a primer 5' to said nucleotide repeat region, and wherein said primer hybridizes with said nucleotide sequence complementary to said primer.

### Claim 16 (depends on 14)

The method of claim 14 wherein said optimally fluorescent labeled probe is generated by random priming.