Homogenous method for the detection of a polynucleotide, with e.g. cleavable lanthanide chelate label

• US 7,371,544 B2
• Filed: 02/13/2001
• Issued: 05/13/2008
• Est. Priority Date: 02/16/2000
• Status: Expired due to Fees

• Current Assignee: Jussi Nurmi
• Original Assignee: Jussi Nurmi
• Inventors: Nurmi, Jussi
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): BAUGHMAN, MOLLY E
• Application Number:
  US10/203,516
• Publication Number:
  US 20040029119A1
• Time in Patent Office: 2,646 Days
• Field of Search: 435/6, 435/18, 435/91.1, 436/800, 536/24.3, 935/77, 935/78
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides

Abstract

The present invention relates to a homogenous method for detecting at least one target polynucleotide in a sample comprising the steps of

• • contacting a sample comprising said polynucleotide or polynucleotide forming nucleic acids or derivatives thereof, with labelled oligonucleotide probe under conditions where said oligonucleotide probe selectively hybridizes to said target polynucleotide, wherein the signal of the label increases or decreases upon the hybridization between the oligonucleotide probe and the target polynucleotide,
  • exciting the label, and
  • monitoring the signal intensity of said label to indicate the presence or absence in a sample of said target polynucleotide.
    According to the invention, said label is measured by time resolved fluorometry.

Claims

1. A homogenous method for detecting at least one target polynucleotide in a sample comprising the steps of

contacting a sample, comprising said polynucleotide with an oligonucleotide probe labeled with a label under conditions where said oligonucleotide probe selectively hybridizes to said target polynucleotide, wherein the signal of the label itself increases or decreases upon the hybridization of the oligonucleotide probe and the target polynucleotide, and the label consists of a fluorescent lanthanide chelate,

exciting the label, and

without removing unhybridized probes, monitoring the signal intensity of said label by time resolved fluorometry to indicate the presence or absence in a sample of said target polynucleotide.

2. The method according to claim 1, wherein the sample is contacted with additional oligonucleotide probes having different base sequences, said probes being labeled with different labels so that all labels have distinguishable signal wavelengths, said additional oligonucleotide probes thus being able to hybridize to different target polynucleotides in the sample.

3. The method according claim 1, wherein the method is carried out in the real-time monitoring of nucleic acid synthesis reactions by measuring the signal intensity of said label at regular intervals during a nucleic acid synthesis reaction and comparing the signal intensities of a reaction containing a sample of nucleic acids and of a negative control reaction that does not contain said target polynucleotide, a difference in the signal intensities of these two indicating the presence of said target polynucleotide in said sample;

and detecting the point of time when the signal intensity of a reaction containing said target polynucleotide clearly starts to differ from the signal intensity of said negative control reaction, the detected point of time indicating the initial amount of said target polynucleotide in said sample.

4. The method according to claim 1, wherein the method is carried out in the end-point detection of nucleic acid synthesis reaction products by measuring the signal intensity of said label after said nucleic acid synthesis reaction and comparing the signal intensity of a reaction containing said sample of nucleic acids and the signal intensity of a negative control reaction that does not contain said target polynucleotide, a difference in the signal intensities of these reactions indicating the presence of said target polynucleotide in said sample of nucleic acids.

5. The method according to claim 1, where said oligonucleotide probe is made of DNA, RNA, peptide nucleic acid (PNA), or a chimera thereof.

6. The method according to claim 1, wherein said target polynucleotide is a single stranded nucleic acid, DNA or RNA, or a double stranded stretch of DNA or RNA.

7. The method according to claim 1, wherein said label is attached to the 5′

terminal nucleotide of said oligonucleotide probe.

8. The method according to claim 1, wherein said label is attached to the 3′

terminal nucleotide of said oligonucleotide probe.

9. The method according to claim 1, wherein said label is attached to the 5′

and 3′

terminal nucleotides of said oligonucleotide probe.

10. The method according to claim 1, wherein said label is attached to any nucleotide of said oligonucleotide probe between 5′

and 3′

terminal nucleotides.

12. The method of claim 1, wherein the fluorescent lanthanide chelate is terbium or europium chelates of a derivative of 2,2′

,2″

,2′

″

-{[6,6′

-(Pyrazole-1″

,3″

-diyl)bis(pyridine)-2,2′

-diyl]bis(methylenenitrilo)}tetrakis (acetic acid) or 2,2′

;

6′

,2″

-terpyridine.

11. A method of determining the presence of a target nucleic acid sequence in a sample, comprising the steps of

providing a mixture of

a sample of interest, and

a nucleic acid probe comprising a nucleic acid sequence selectively binding said target nucleic acid sequence and one or more labels wherein the one or more labels consist of fluorescent lanthanide chelates,

wherein each of said fluorescent lanthanide chelate is capable of changing the intensity of its own fluorescence emission upon hybridization of said probe to said target nucleic acid sequence,

subjecting the mixture to conditions under which the probe selectively hybridizes to the target nucleic acid,

and

without removing unhybridized probes from the mixture, determining whether each fluorescent lanthanide chelate changes the intensity of its own fluorescence emission by measuring fluorescence emission from the mixture by time resolved fluorometry,

thereby determining the presence of said target nucleic acid sequence in the sample if the intensity does change over time.

13. The method of claim 11, wherein the fluorescent lanthanide chelate is terbium or europium chelates of a derivative of 2,2′

,2″

,2′

″

-{[6,6′

-(Pyrazole-1″

,3″

-diyl)bis(pyridine)-2,2′

-diyl]bis(methylenenitrilo)}tetrakis (acetic acid) or 2,2′

;

6′

,2″

-terpyridine.