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

• US 20040029119A1
• Filed: 08/12/2002
• Published: 02/12/2004
• Est. Priority Date: 02/16/2000
• Status: Active Grant

• Current Assignee: Jussi Nurmi
• Original Assignee: Jussi Nurmi
• Inventors: Nurmi, Jussi
• Granted Patent: US 7,371,544 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 or polynucleotide forming nucleic acids or derivatives thereof with an oligonucleotide probe labelled with one single kind of 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 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, characterized in that said label is measured by time resolved fluorometry.

2. The method according to claim 1, characterized in that the label is cleaved chemically from the oligonucleotide probe as a result of hybridization.

3. The method according to claim 1 or 2, characterized in that the label is a fluorescent lanthanide chelate.

4. The method according to claim 1, 2 or 3, characterized in that the sample is contacted with additional oligonucleotide probes having different base sequences, said probes being labelled 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.

5. The method according to any one of the claims 1 - 4, characterized in that 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.

6. The method according to any one of the claims 1 - 4, characterized in that 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.

7. The method according to any one of the claims 1 - 4, characterized in that said oligonucleotide probe is made of DNA , RNA or any nucleic acid derivative such as peptide nucleic acid (PNA) or where said oligonucleotide probe is a chimera of any two or more different kinds of nucleic acids or derivatives thereof.

8. The method according to any one of the claims 1 - 4, characterized in that said target polynucleotide is a single stranded nucleic acid, DNA or RNA or a derivative thereof, or a double stranded stretch of DNA or RNA or a derivative thereof or a hybrid of DNA and RNA or of DNA or RNA and a nucleic acid derivative such as PNA.

9. The method according to any one of the claims 1 - 8, characterized in that said method is an assay where said label is released from said oligonucleotide probe by the 5′

to 3′

exonuclease activity of a nucleic acid polymerase.

10. The method according to any one of the claims 1 - 8, characterized in that said method is an assay where said oligonucleotide probe comprises RNA bases and is cleaved by ribonuclease H when hybridized to a single stranded target DNA.

11. The method according to any one of the claims 1 - 8, characterized in that said label is attached to the 5′

terminal nucleotide of said oligonucleotide probe.

12. The method according to any one of the claims 1 - 8, characterized in that said label is attached to the 3′

terminal nucleotide of said oligonucleotide probe.

13. The method according to any one of the claims 1 - 8, characterized in that said label is attached to the 5′

and 3′

terminal nucleotides of said oligonucleotide probe.

14. The method according to any one of the claims 1 - 8, characterized in that said label is attached to any nucleotide of said oligonucleotide probe between 5′

and 3′

terminal nucleotides.