Optimally fluorescent oligonucleotides

• US 6,072,043 A
• Filed: 06/04/1996
• Issued: 06/06/2000
• Est. Priority Date: 06/04/1996
• Status: Expired due to Fees

• Current Assignee: Polyprobe, Inc.
• Original Assignee: Polyprobe, Inc.
• Inventors: Nilsen, Thor W.
• Primary Examiner(s): Marschel, Ardin H.
• Assistant Examiner(s): Riley, Jezia
• Application Number:
  US08/657,961
• Time in Patent Office: 1,463 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: 536/22.1
• 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 fluorescent oligonucleotides wherein fluorescent dye-conjugated nucleotide triphosphates are incorporated into a nucleic acid sequence in a defined repetitive manner which allows for the optimal specific fluorescence of the oligonucleotide. The oligonucleotides of the present invention are useful in the assay of a wide variety of nucleic acid sequences, specifically wherever highly fluorescent nucleic acid probes are desired.

Claims

1. An oligonucleotide comprising a nucleotide repeat region having the formula:

space="preserve" listing-type="equation">(N.sub.m N.sup.f)

wherein N represents any nucleotide which is not conjugated to a fluorescent dye moiety, N^f represents a nucleotide conjugated to a fluorescent dye moiety, and m is an integer from 1 to 11, and wherein for each said repeat N^f and the fluorescent moiety are the same, and further wherein m and said fluorescent dye moiety are selected such that said oligonucleotide exhibits maximum specific fluorescence.

2. The oligonucleotide of claim 1, further comprising a radiolabled nucleotide sequence.

3. The oligonucleotide of claim 2, wherein said radiolabel is ³² P.

4. The oligonucleotide of claim 1, which is single-stranded.

5. The oligonucleotide of claim 1, which is double-stranded.

6. The oligonucleotide of claim 1, which contains from about 20 to about 1000 total nucleotides.

7. The oligonucleotide of claim 1, linked to a probe that hybridizes with a nucleic acid of interest.

8. The oligonucleotide of claim 1, in combination with a DNA matrix.

9. An oligonucleotide containing a single fluorescent dye moiety and prepared by a method comprising:

(a) preparing a primer;

(b) preparing a template oligonucleotide, said 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 which cannot form a base pair with a fluorescent dye-conjugated nucleotide triphosphate, and N^t is a nucleotide which can form 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 polymerase, unlabeled nucleotide triphosphates and a predetermined concentration of said fluorescent dye-conjugated nucleotide triphosphate, wherein said polymerase and m are chosen to cause said optimally fluorescent oligonucleotide to exhibit maximum specific fluorescence;

(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.

10. The oligonucleotide of claim 9, wherein said method further comprises step (f) attaching said oligonucleotide to a probe that hybridizes with a nucleic acid of interest.

11. The oligonucleotide of claim 10, wherein said attaching comprises ligation.

12. The oligonucleotide of claim 10, wherein said attaching comprises randomer extension.

13. The oligonucleotide of claim 10, wherein said attaching comprises cloning.

14. The oligonucleotide of claim 9, wherein said (e) isolating comprises precipitation, size fractionation, gel electrophoresis or antigen-specific binding.