JP2003144197A - Method of detection for nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of detection for sequence of a nucleic acid especially useful for gene diagnosis such as detection of a mutant gene in genetic disease, diagnosis of cancer, and method of detection for gene relating to virus, and to provide an oligonucleotide used for the same. SOLUTION: This method detects the existence of the specific nucleic acid sequence by reacting a marker oligonucleotide and a specific nucleic acid marker to the specific nucleic acid sequence included in a sample and measuring interaction between the marker oligonucleotide and the nucleic acid marker.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting a specific target nucleic acid molecule present in a sample.

[0002]

Sequence-specific hybridization of labeled oligonucleotide probes has long been used as a means of detecting and identifying selected nucleotide sequences. Hybridization analysis of nucleic acids
Very small number of target nucleic acids (DNA or RNA) among many kinds of nucleic acids
This is a technique for detecting a protein with a probe, and a highly sensitive reporter (enzyme, fluorescent dye, radioisotope, etc.) has also been developed. Such methods of labeling probes, especially with fluorescent labels, have provided relatively sensitive non-radioactive means to aid in detection of probe hybridization. However, in a general hybridization method, an operation of removing unreacted probe and removing a non-specific signal is indispensable, and a great deal of labor is required. The recently developed detection method uses fluorescence resonance energy transfer (Fluorescence Resonance Energy Transfer) rather than direct detection of fluorescence intensity for detection of probe hybridization.
escence Resonance Energy Transfer (FRET) method is used. Fluorescence resonance energy transfer involves donor fluorophore and quencher dye (whether or not fluorophore).
And the absorption spectrum of one (quencher) overlaps with the emission spectrum of the other (donor),
Occurs when two dyes are in close proximity. Dyes with these properties are called donor / quencher dye pairs or energy transfer dye pairs. The excited state energy of the donor fluorophore is transferred to nearby quenchers by dipole interactions caused by resonant dipoles. As a result, quenching of the donor phosphor occurs. In some cases, when the quenching agent is also a fluorescent substance, its fluorescence intensity may be enhanced. Energy transfer efficiency is highly dependent on the distance between the donor and the quencher, and the formula for predicting these relationships is Forster (194
8. Ann. Phys. 2, 55-75). The distance between the donor and the quencher dye, which has an energy transfer efficiency of 50%, is
Called the Forster distance (Ro). Other mechanisms of fluorescence quenching include, for example, charge transfer quenching and collision quenching.

Energy transfer and other mechanisms based on quenching through the interaction of two dyes in close proximity are attractive ways to detect or identify nucleotide sequences, since they can be performed in a homogeneous manner. The homogeneous assay format is simpler than the conventional probe hybridization assay method, which is based on the detection of the fluorescence of one fluorophore label. This is because heterogeneous analysis generally requires the additional step of separating the hybridized label from the unhybridized free label. Typically F
RET and related methods are based on monitoring changes in the fluorescent properties of one or both dye labels when two complementary oligonucleotides are joined by hybridization. In this manner, changes in fluorescence properties are measured as changes in energy transfer or changes in fluorescence quenching, and are typically shown as an increase in fluorescence intensity of one dye. In this method, it is possible to detect the nucleotide sequence of interest without separating the oligonucleotide that has not hybridized and the oligonucleotide that has hybridized. Hybridization can occur between two separate complementary oligonucleotides, one labeled with the donor fluorophore and the other labeled with the quencher. Compared to single-stranded oligonucleotides, the double-stranded form has reduced donor fluorescence (increased quenching) and
/ Or increased energy transfer occurs. Some methods for FRET hybridization analysis are Nonisotopic
DNA Probe Techniques (1992. Academic Press, Inc., p
ags. 311-352). Alternatively, the donor and quencher can be combined into a single oligonucleotide so that there is a detectable difference in the fluorescence properties of one or both of the oligonucleotides when unhybridized and when hybridized to complementary sequences. Can be combined. In this format, typically, oligonucleotide hybridization increases donor fluorescence and decreases energy transfer / quenching. For example, when self-complementary oligonucleotides labeled at both ends form a hairpin structure, this brings the two fluorophores (ie, 5'end and 3'end) into close proximity, causing energy transfer and quenching. When the self-complementary oligonucleotide and the complementary sequence in the second oligonucleotide hybridize,
The hairpin structure is destroyed and the distance between the two dyes is increased, resulting in reduced quenching. The drawbacks of hairpin structures are that they are very stable, their conversion to unquenched hybrids is often slow, with only a slight bias, and generally poor performance. Tyagi and Kramer (1996. Nature Biotech. 14, 30
3-308) report a hairpin structure labeled as above containing a detection sequence in the loop between the self-complementary arms of the hairpin structure forming the stem. The base-paired stem must melt in order for the detection sequence to hybridize to the target and cause reduced quenching. The "double hairpin" probe and how to use it is described in B. Bag
well, et al. (1994. Nucl. Acids Res. 22, 2424-2425;
U.S. Pat. No. 5,607,834). These structures contain the target binding sequence within the hairpin structure, thus involving competitive hybridization between the target and the self-complementary sequence of the hairpin structure. Bagwell solves the problem of unfavorable hybridization kinetics by destabilizing the hairpin structure due to mismatches.

Homogeneous methods that utilize energy transfer or other mechanisms of fluorescence quenching to detect nucleic acid amplification have also been reported. LG Lee, et al. (1993. Nuc. Acids
Res. 21, 3761-3766) discloses a real-time detection method in which a double-labeled detection probe is cleaved specifically during PCR during target amplification. When the detector probe hybridizes downstream of the amplification primer, the 5'-3 'of Taq polymerase
Exonuclease activity digests the detection probe and separates the two fluorochromes that form an energy transfer pair.

A signal primer (often referred to as a detection probe) that hybridizes to a target sequence downstream of the hybridization site of an amplification primer has been reported for homogeneous detection of nucleic acid amplification (US Pat. No. 5, US Pat. No. 5,
5547,861). The signal primer is extended by the polymerase in the same manner as the extension of the amplification primer.
By extension of the amplification primer, depending on the amplification of the target,
The extension product of the signal primer is displaced, producing a double-stranded secondary amplification product that is detectable as an indicator of target amplification. Examples of homogeneous detection methods for use with single-stranded signal primers are US Patent No. 5,550,025 and US Patent
No. 5,593,867. More recently, FRET
The signal primers have been modified for detection of nucleic acid and target using the method. US Pat. No. 5,691,145 discloses a G quartet structure with a donor / quencher dye pair attached to the 5 ′ end of the target binding sequence of a single-stranded signal primer. When the complementary strands are synthesized during target amplification, the G quartet structure unfolds, widening the distance between the donor and quencher dye, resulting in a detectable increase in donor fluorescence. Recently, some single-stranded and partially double-stranded signal primers labeled with a donor / quencher dye pair have also been reported. For example, EP
0 878 554 discloses a signal primer having a donor / quencher dye pair flanking a single stranded restriction endonuclease recognition site. In the presence of the target, the restriction site becomes double-stranded and can be cleaved by the restriction endonuclease. Cleavage separates the dye pairs and reduces donor quenching. EP 0 881 302 reports a signal primer having an intermolecular base pairing structure. The donor dye of the donor / quencher dye pair bound to the intermolecular base-pairing structure is quenched when the structure is folded,
In the presence of the target, intermolecular base pairing structures and complementary sequences are synthesized. This unfolds the intermolecular base-pairing structure and separates the donor and quencher dye, causing a reduction in donor quenching. Nazarenko, et al. (US Patent No. 5,86
6,336) reported a similar method in which the amplification primer was formed by a hairpin structure with a donor / quencher dye pair.

Energy transfer and other methods of fluorescence quenching detection have also been applied to the detection of target sequences by hybridization of specific probes. Japanese Patent No.9301543
9B discloses a method for measuring a polynucleotide by hybridizing a single-stranded target with two labeled single-stranded polynucleotide probes that form an energy transfer pair. The double-stranded hybrid is cleaved between the labels by a restriction enzyme, and the fluorescence of one of the labels is measured. The disadvantage of this method is that the restriction site in the probe is
This is also the point that it must be present in the target sequence to be detected.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and provides a nucleic acid detection method that is more versatile and easier to operate than conventional nucleic acid detection techniques. Characterize.

[0008]

As a result of earnest research, the inventors of the present invention have carried out the above-mentioned conventional method in which two probes or a donor and a quencher labeled with a required restriction enzyme site, a donor or a quencher are labeled. As a homogeneous detection method that does not require a probe labeled with two types of the above, a labeled oligonucleotide specific to a specific nucleic acid sequence and a nucleic acid-specific label are allowed to act, and the interaction between the label of the oligonucleotide and the nucleic acid-specific label Found that the specific nucleic acid sequence can be detected,
The present invention has been completed.

That is, the present invention has the following configuration. [1] A labeled oligonucleotide specific to a specific nucleic acid sequence contained in a sample and a nucleic acid-specific label are allowed to act, and the specific nucleic acid sequence is detected by the interaction between the label of the oligonucleotide and the nucleic acid-specific label. Method. [2] The method according to [1], wherein the nucleic acid-specific label is double-stranded nucleic acid-specific. [3] The method according to [1] or [2], wherein the nucleic acid-specific label is a fluorescent dye. [4] The method according to [1] to [3], wherein the oligonucleotide label is a fluorescent dye. [5] The method according to [1] to [4], wherein the interaction is fluorescence resonance energy transfer. [6] The method according to [1] to [5], wherein a specific nucleic acid sequence contained in the sample is previously amplified. [7] The method according to [1] to [6], which comprises performing an amplification reaction using the labeled oligonucleotide and measuring during and / or after the amplification reaction. [8] A labeled oligonucleotide specific to a specific nucleic acid sequence contained in the sample is caused to act as a primer to carry out an extension reaction, and then a nucleic acid-specific label is allowed to act on the extension product to label the oligonucleotide. A method for detecting the specific nucleic acid sequence by the interaction of the nucleic acid-specific label. [9] The method according to [8], wherein the nucleic acid-specific label is double-stranded nucleic acid-specific. [10] The method according to [8] or [9], wherein the nucleic acid-specific label is a fluorescent dye. [11] The method according to [8] to [10], wherein the oligonucleotide label is a fluorescent dye. [12] The method according to [8] to [11], wherein the interaction is fluorescence resonance energy transfer. [13] The method according to [8] to [12], wherein the specific nucleic acid sequence contained in the sample is previously amplified. [14] The method according to [8] to [13], which comprises performing an amplification reaction using a labeled oligonucleotide primer and measuring during and / or after the amplification reaction. [15] In the method of detecting a specific nucleic acid sequence contained in a sample, the following steps are performed: 1) A labeled oligonucleotide specific to the nucleic acid sequence is caused to act as a primer to carry out an extension reaction. 2) After the extension product is denatured into a single strand, an oligonucleotide complementary to the extension product is allowed to act to perform an extension reaction to produce a double-stranded nucleic acid. 3) A double-stranded nucleic acid-specific fluorescent dye is allowed to act, and the interaction between the fluorescent dye and the label of the oligonucleotide is measured.
A method comprising: [16] The method according to [15], wherein the oligonucleotide label is a fluorescent dye. [17] The method according to [15] or [16], wherein the interaction is fluorescence resonance energy transfer. [18] The method according to [15] to [17], wherein the specific nucleic acid sequence contained in the sample is previously amplified. [19] Repeat steps 1 and 2 to carry out the amplification reaction,
The method according to [15] to [18], which comprises measuring during and / or after the amplification reaction. [20] A kit for detecting a specific target nucleic acid molecule present in a sample containing at least a labeled oligonucleotide, a polymerase, and a nucleic acid-specific label.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
A chromosome or a fragment thereof containing a specific nucleic acid sequence contained in a sample is a target nucleic acid carrying information of a target gene,
There is no particular limitation. Examples of the target nucleic acid include genes derived from viruses or pathogens, Alu sequences, exons and introns of genes encoding proteins, promoters and the like.

The oligonucleotide of the present invention contains at least a sequence which hybridizes with the target nucleic acid sequence and has a length of 13 to 35 bases, preferably 16 to 30 bases.
It is a base.

The label attached to the oligonucleotide is
Any substance may be used as long as the nucleic acid-specific label specifically binds to the extension product after the extension reaction and then interacts with the label. A fluorescent dye that produces a FRET reaction is preferred. FITC, 6-FAM, HE as fluorescent dye
X, TET, TAMRA, Texas Red, Cy3, C
There are y5, rhodamine and the like, but it is not limited to this as long as it interacts with the nucleic acid specific label and can be specifically detected.

The oligonucleotide extension reaction is carried out by labeling a target nucleic acid denatured into a single strand with an oligonucleotide primer, four kinds of deoxynucleoside triphosphates (dNT).
By reacting P) with DNA polymerase, a primer extension reaction occurs using the target nucleic acid as a template to synthesize a complementary strand of the nucleic acid sequence.

In the present invention, the nucleic acid-specific labeling substance may be any substance as long as it specifically binds to the nucleic acid subjected to the elongation reaction of the labeled oligonucleotide and interacts with the label of the oligonucleotide, preferably the extended double-stranded nucleic acid. Labels are preferred. The double-stranded specific label includes, but is not limited to, Cybergreen I intercalating into a double-stranded chain, ethidium bromide, acridine orange, thiazole orange, oxazole yellow, rhodamine and the like.

The interaction between the oligonucleotide label and the nucleic acid-specific label in the present invention needs to be an action that can be detected only when the action occurs and cannot be detected by the presence of each label alone. Fluorescence resonance energy transfer (FRET) is generally used as the interaction.

[0016] Another reaction mode is a hybridization method. A method in which a labeled oligonucleotide probe is hybridized and detected is generally used for detection of a nucleic acid sequence, but a labeled probe and a nucleic acid-specific label are allowed to act on a target nucleic acid to label the hybridized probe and the nucleic acid-specific By detecting the interaction of the labels, it becomes possible to detect a specific nucleic acid sequence.

For example, when a fluorescent-labeled oligonucleotide probe is hybridized and a double-stranded nucleic acid-specific fluorescent label is allowed to act on the probe, the difference between the two hybridized with the probe is 2
A specific signal is generated by the double-stranded nucleic acid-specific fluorescent dye.
At this time, FRET occurs between the fluorescent label of the probe and the hybridized probe-specific signal.

When the target nucleic acid is not contained in an amount sufficient for detection when measured by the above method, a nucleic acid fragment containing the polymorphic sequence is previously amplified by the amplification reaction shown below. It is also possible to set it.

In another mode of the present invention, it is possible to detect during an amplification reaction such as PCR. When carrying out an amplification reaction with a forward primer and a reverse primer, a specific labeled probe is put in, and when the primer anneals during the amplification cycle, the probe also simultaneously forms a double strand in a target nucleic acid sequence-specific manner. At this time, it is possible to detect during the amplification reaction by detecting the interaction between the probe label and the nucleic acid-specific label.

The amplification method in the present invention can be basically performed by a conventional method, and usually, a chromosome containing a specific nucleotide polymorphism site denatured into a single strand or a fragment thereof, Four types of deoxynucleoside triphosphates (dN
TP), DNA polymerase, and a set of primers are used to amplify the target nucleic acid as a template.

As the nucleic acid amplification method, PCR, NASB
A (Nucleic acid sequence-basedamplification metho
d; Nature, Volume 350, page 91 (1991)), L
CR (International Publication 89/12696, Japanese Patent Laid-Open No. 2-2
934), SDA (Strand Displacement Amplif)
ication: Nucleic acid research Volume 20, 169
1 page (1992)), RCR (International Publication 90/1069), TMA (Transcription mediated amplification)
method; J. Clin. Microbiol. Vol. 31, p. 3270
(1993)) and the like.

Among them, the PCR method is carried out by repeating a cycle consisting of three steps of denaturation, annealing and extension in the presence of a sample nucleic acid, four kinds of deoxynucleoside triphosphates, a pair of primers and a thermostable DNA polymerase. This is a method of exponentially amplifying the region of the sample nucleic acid sandwiched by the pair of primers. That is, the nucleic acid of the sample is denatured in the denaturing step, each primer is hybridized in the subsequent annealing step with a region on the single-stranded sample nucleic acid complementary to the primer, and in the subsequent extension step,
Using each primer as a starting point, a DNA strand complementary to each single-stranded sample nucleic acid serving as a template is extended by the action of DNA polymerase to form a double-stranded DNA. By this one cycle, one double-stranded DNA is amplified into two double-stranded DNAs. Therefore, if this cycle is repeated n times, theoretically, the region of the sample DNA sandwiched between the pair of primers is 2
Amplified ⁿ times. Since the amplified DNA region exists in a large amount, it can be easily detected by a method such as electrophoresis.
Therefore, by using the gene amplification method, it is possible to detect a very small amount (a single molecule is possible) of a sample nucleic acid, which could not be detected in the past, and it is a technique that has been very widely used recently. .

As another mode in the present invention, it is also possible to detect during an amplification reaction such as PCR. When an amplification reaction is performed with a labeled oligonucleotide and a reverse primer, it is possible to detect the interaction with the label generated by the binding of the nucleic acid-specific label when the labeled oligonucleotide primer is extended and two differences are formed. Is. Further, this detection may be performed after the amplification reaction.

Kit In the present invention, the kit includes a specific nucleic acid detection kit containing at least a labeled oligonucleotide, a DNA polymerase, four kinds of deoxynucleoside triphosphates (dNTP) and a nucleic acid-specific labeling reagent. There is a kit in which after the primer reacts with the target nucleic acid, the nucleic acid-specific label binds and the resulting interaction is measured. When detecting by amplification, a reverse primer may be further included. Further, each of the primers may be labeled in advance with a fluorescent substance or the like as described above.

As described above, in the method of the present invention, the target nucleic acid can be detected by measuring the interaction that occurs after reacting the target nucleic acid with the labeled oligonucleotide and the nucleic acid-specific label. Therefore, according to the method of the present invention, it is possible to clearly detect a target nucleic acid in a sample nucleic acid by a uniform method without requiring a complicated detection operation such as removing an unreacted labeling substance as in the conventional method. .

[0026]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples. Example 1 Detection of β3 adrenergic receptor gene (1) Detection of β3 adrenergic receptor gene
Using synthetic Perkin Elmer DNA Synthesizer 392 Gore nucleotides at the phosphoamidite method, oligonucleotides (hereinafter having the nucleotide sequence shown in SEQ ID NO: 1-2, oligo 1
Designated as ~ 3) was synthesized. The synthesis follows the manual, and various oligonucleotides are deprotected with ammonia water at 55 ° C.
It was carried out overnight. Purification of the oligonucleotide was carried out on a Perkin Elmer OPC column. Or a DNA synthesis outsourcing company (Nippon Bioservices, Sawasdee,
We requested GENSET KK, Amersham Pharmacia Biotech Co., Ltd., etc. Oligo 1 is the sense strand,
Oligo 2 is the antisense strand and is used in combination as an oligonucleotide in the amplification reaction. In addition, oligo
1 and 2 are used by labeling if necessary.

(2) β3 adrenergic recept by PCR method
or gene detection Amplification reaction by PCR method Using a DNA solution extracted from human leukocytes by the phenol / chloroform method as a sample, the following reagents were added, and human β3 adrenergic receptor under the following conditions:
The gene was detected.

[0028] was prepared 25μl solution containing the following reagents reagent. Taq DNA Polymerase Reaction Solution Oligo 1 (5 'is labeled with Texas Red) 5 pmol Oligo 2 5 pmol x 10 Buffer 2.5 μl 2 mM dNTP 2.5 μl 25 mM MgCl ₂ 1.5 μl Taq DNA Polymerase 1.3 U Extracted DNA Solution 100 ng

Amplification conditions 95 ° C. for 5 minutes 95 ° C. for 30 seconds, 62.5 ° C. for 30 seconds, 72 ° C. for 30 seconds (35 cycles) 72 ° C. for 2 minutes. 5 μl of amplification reaction solution for detection by FRET was added to Cyber Green I (Molecular probes).
100 μl of the solution of the company) and reacted at room temperature for 10 minutes. This amplified human β3 adrenergic rec
Syber Green I is incorporated into the eptor gene fragment.
Next, in a dark room, fluorescent plate reader (Dainippon Pharmaceutical Co., Ltd.)
The amount of fluorescence intensity was measured with. It took a few minutes. From the obtained fluorescence intensity, the fluorescence intensity amount of the sample was calculated using the following calculation formula. [Formula 1] FL (fluorescence intensity of sample) = FLb-FLs FLb: fluorescence intensity of Negative Control (no sample added) FLs: fluorescence intensity of each sample

[0030]

[Table 1]

As described above, a base-specific amplification reaction is carried out by using a fixed amount of oligonucleotide, and a nucleic acid-specific label is allowed to act on the amplification reaction substance, thereby easily and easily without any complicated operation. It was possible to rapidly and clearly determine the presence of the target nucleic acid gene.

[0032]

As described above, the present invention provides a method capable of clearly and simply detecting a target nucleic acid sequence in a sample nucleic acid. Since the method of the present invention does not require a complicated operation as in the conventional methods, quick and easy reproducible results were obtained.

[0033]

[Sequence list]                                SEQUENCE LISTING <110> TOYO BOSEKI KABUSHIKI KAISHA <120> Nucleic acid detection method <130> 01-0939 <141> 2001-11-07 <160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 17 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide described in example 1 <400> 1 gtcatcgtgg ccatcgc 17

[0034] <210> 2 <211> 22 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide described in example 1 <400> 2 acgaacacgt tggtcatggt ct 22

[Brief description of drawings]

[Figure 1] Fluorescence intensity of each sample in detection of β3 adrenergic receptor gene

   ─────────────────────────────────────────────────── ─── Continued front page    F term (reference) 4B024 AA11 CA01 CA09 HA13 HA14                 4B063 QA11 QQ42 QQ52 QR56 QR62                       QS25 QS34 QX02

Claims (20)

[Claims] 1. A labeled oligonucleotide specific to a specific nucleic acid sequence contained in a sample and a nucleic acid-specific label are allowed to act, and the specific nucleic acid sequence is identified by the interaction between the label of the oligonucleotide and the nucleic acid-specific label. How to detect. 2. The method according to claim 1, wherein the nucleic acid-specific label is double-stranded nucleic acid-specific. 3. The method according to claim 1, wherein the nucleic acid-specific label is a fluorescent dye. 4. The method according to claim 1, wherein the label of the oligonucleotide is a fluorescent dye. 5. The method according to claim 1, wherein the interaction is fluorescence resonance energy transfer. 6. The method according to claim 1, wherein the specific nucleic acid sequence contained in the sample is previously amplified. 7. The method according to claim 1, wherein an amplification reaction is carried out using a labeled oligonucleotide, and the measurement is carried out during and / or after the amplification reaction. 8. A labeled oligonucleotide specific to a specific nucleic acid sequence contained in a sample is caused to act as a primer to carry out an extension reaction, and then an extension product is allowed to act with a nucleic acid-specific label, thereby A method for detecting the specific nucleic acid sequence by the interaction between a label and the nucleic acid-specific label. 9. The method according to claim 8, wherein the nucleic acid-specific label is double-stranded nucleic acid-specific. 10. The method according to claim 8 or 9, wherein the nucleic acid-specific label is a fluorescent dye. 11. The method according to claim 8, wherein the label of the oligonucleotide is a fluorescent dye. 12. The method according to claim 8, wherein the interaction is fluorescence resonance energy transfer. 13. The method according to claim 8, wherein the specific nucleic acid sequence contained in the sample is previously amplified. 14. The method according to claim 8, wherein an amplification reaction is carried out using a labeled oligonucleotide primer, and the measurement is carried out during and / or after the amplification reaction. 15. A method for detecting a specific nucleic acid sequence contained in a sample, comprising the steps of: 1) causing a labeled oligonucleotide specific to the nucleic acid sequence to act as a primer to carry out an extension reaction. 2) After the extension product is denatured into a single strand, an oligonucleotide complementary to the extension product is allowed to act to perform an extension reaction to produce a double-stranded nucleic acid. 3) A double-stranded nucleic acid-specific fluorescent dye is allowed to act, and the interaction between the fluorescent dye and the label of the oligonucleotide is measured.
A method comprising: 16. The method according to claim 15, wherein the label of the oligonucleotide is a fluorescent dye. 17. The method of claims 15 and 16 wherein the interaction is fluorescence resonance energy transfer. 18. The method according to claim 15, wherein the specific nucleic acid sequence contained in the sample is previously amplified. 19. The method according to claim 15, wherein steps 1 and 2 are repeated to carry out an amplification reaction, and the measurement is carried out during and / or after the amplification reaction. 20. A kit for detecting a specific target nucleic acid molecule present in a sample containing at least a labeled oligonucleotide, a polymerase and a nucleic acid specific label.