Optimally labeled oligonucleotides

• US 6,046,038 A
• Filed: 08/12/1997
• Issued: 04/04/2000
• Est. Priority Date: 06/04/1996
• Status: Expired due to Term

• Current Assignee: CF Partners Acquisition III, LLC
• Original Assignee: Poly-Pro, Inc.
• Inventors: Nilsen, Thor W.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US08/909,539
• Time in Patent Office: 966 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/91.1
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2563/101 radioactivity, e.g. radioactive labels
• CPC Class Codes: C12Q 2563/107 fluorescence

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

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;

space="preserve" listing-type="equation">(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^t is a nucleotide that forms a base pair with the fluorescent dye-conjugated nucleotide triphosphate, wherein for each repeat, N^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.

2. 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.

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

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

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

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

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;

space="preserve" listing-type="equation">(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^t is a nucleotide that forms a base pair with the fluorescent dye-conjugated nucleotide triphosphate, wherein for each repeat, N^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.

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

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;

space="preserve" listing-type="equation">(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^t is a nucleotide that forms a base pair with the fluorescent dye-conjugated nucleotide triphosphate, wherein for each repeat, N^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.

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

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

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

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

14. 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.

15. 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.

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