KR100318222B1 - Target Nucleic Acid Detection Method - Google Patents

Abstract

The present invention relates to (i) a polymerase, a first primer with or without a non-adjacent segment for the first priming sequence, in the presence of an oligonucleotide that cannot function as a primer for the polymerase, and a second priming Amplifying the target nucleic acid to obtain an amplification product using a second primer with or without non-adjacent segments for the sequence, wherein the oligonucleotide is attached to at least five consecutive nucleotides of the first primer. Having at least 5 contiguous nucleotides completely complementary to; And (ii) detecting the presence of the target nucleic acid by monitoring amplification of the target nucleic acid.

Description

Target nucleic acid detection method

Background of the Invention

Recent developments in polymerase chain reaction ("PCR") have provided an important means for the detection of nucleic acid sequences present at low concentrations. (Mullis, KB et al., US Pat. Nos. 4,683, l95 and 4,683,2O2) In PCR, target sequences having boundaries defined by two oligonucleotide extension primers are amplified through repeated enzyme cycles to further amplify. Provide additional templates for the reaction. Thus, a small number of target sequences can be exponentially amplified and easily detected.

The main vulnerability of PCR is the large number of by-products resulting from non-specific prime events, such as random priming of nucleic acid templates and / or self priming of extension primers. Thus, where multiple amplification cycles are required for amplification of target sequences present at relatively low concentrations, the occurrence of non-specific priming events significantly interferes with PCR sensitivity.

Another related vulnerability of PCR is that a separation step is required before detection of the amplified target. According to standard PCR conditions, the separation of amplified target sequences from products of non-specific priming events is a prerequisite for the detection of amplified target sequences. The lack of a unique amplification reaction, ie, the reaction where amplification and detection occur in the same reaction vessel, has been an obstacle to the automation of PCR methods. In addition, the need for a separation step also allows the PCR mixture to be contaminated by the separation process. The possibility of contamination significantly limits the applicability of PCR to routine clinical diagnosis.

Attempts have been made to develop a homegenous assay for amplification and detection. One such attempt was described by Holland et al. (1991, Proc. Natl. Acad. Sci. USA 88: 7276), wherein 5 '→ 3' of Taq polymerase was used to generate detection signals simultaneously with PCR amplification. Assays using exonuclease activity. However, subsequent separation steps are required to detect the exonuclease production signal. Higuchi et al. (Higuchi, l992, Bio / Technology 10: 413) describe a unique detection process based on the increased fluorescence monitor of ethidium bromides when the ethidium bromides are inserted into the double stranded amplification product. Since these detection methods are not target sequence specific, they are susceptible to the presence of any double stranded DNA. Another approach is to use fluorescence polarization to detect hybridization of fluorescent nucleic acid probes or fluorescent primer extension products. (Garmen, A.J. et al., European Patent Publication No. 382,433). This method of detection can distinguish hybridized or extended probes with high molecular weight (and thus reduced fluorescence polarization) from unreacted probes. However, the presence of unhybridized probes with high polarization greatly affects the observed polarization, providing a high background.

Non-radioactive energy transfer by proximity of two fluorescent moieties can be used as an effective signal detection scheme. Pure immunoassays based on fluorescence energy transfer have been described. (Patel et al., European Patent Application No. 137,515).

Fluorescence energy transfer has also been designed to detect nucleic acid hybridization (Heller et al., European Patent Application No. 070,685, US Patent No. 4,996,143). In this application, a fluorescence energy transfer signal is generated when probes carrying different fluorescent moieties are brought in close physical proximity by hybridization to a strand of the target nucleic acid.

Summary of the Invention

One aspect of the invention relates to a method for detecting a target nucleic acid comprising the following steps: (i) a polymerase, a first priming sequence, in the presence of an oligonucleotide that cannot function as a primer for the polymerase Amplifying the target nucleic acid to obtain an amplification product using a first primer with or without a non-adjacent segment for and a second primer with or without a non-adjacent segment for a second priming sequence. Wherein said oligonucleotide has at least 5 (preferably, at least 8) contiguous nucleotides that are completely complementary to 5 (preferably, at least 8) contiguous nucleotides at the enemy of the first primer. ; And (ii) detecting the presence of the target nucleic acid by monitoring amplification of the target sequence.

The first and second primers, corresponding to the primer pairs used in the PCR method, hybridize to the first and second priming sequences, respectively, in the target nucleic acid before being extended by the polymerase. As used herein, the term "polymerase" refers to a catalyst that has DNA polymerase activity and is capable of both substitution of "downstream" single-stranded oligonucleotides.

The above oligonucleotides are "blocking oligonucleotides" of the present invention. For convenience, any nucleotide having (a) at least five consecutive nucleotides that are not completely complementary to at least five consecutive nucleotides of the primer (with or without adjacent segments) and cannot be used as a PCR primer. Referred to herein as "blocking oligonucleotides". Blocking oligonucleotides block primers in the sense that they hybridize with the primers to reduce the likelihood that the primers will bind to their priming sequences in the target nucleic acid. In a preferred embodiment of the method of the invention, the amplification step is carried out in the presence of additional blocking oligonucleotides for the second primer.

5-base complete complementarity is formed between the blocking oligonucleotide and one of the contiguous or non-contiguous segments of the primer. The contiquous segmend of the primer is a sequence complementary to the priming sequence within the target nucleic acid. In contrast, the noncontiguous segment of the primer is a non-complementary sequence for the priming sequence in the target nucleic acid.

Preferred lengths of each of the primers and blocking oligonucleotides of the invention are in the range of 10-50 nucleotides (more preferably 15-40 nucleotides). The operative concentration of the blocking oligonucleotides depends greatly on several factors, such as the degree of complementarity between the blocking oligonucleotides and their corresponding primers, the assay temperature, and the like, in any case, without any unnecessary experimentation. Can be easily determined. In view of the molar ratio of blocking nucleotides to its corresponding primers, it is generally acceptable that the range is 0.3-5.0 (or 0.5-2.5).

The detecting step can be performed by monitoring the amplification product of the target nucleic acid on the gel after electrophoresis. Otherwise, the two fluorophores are covalently bound to the second primer (or second primer) and the corresponding blocking oligonucleotide, respectively, so that one of the two fluorophores is the donor fluorophore and the other is the receptor fluorophore. When the first primer and blocking oligonucleotide hybridize, the donor fluorophore and receptor fluorophore can be brought into close proximity to allow resonance energy transfer between them. If the blocking oligonucleotides and their corresponding primers are thus fluorophore labeled, the fluorescence emission change of the receptor fluorophore can be monitored upon irradiation by the excitation light of the donor fluorophore to quantify the amplification of the target nucleic acid. This is because this change is a function of the degree to which the blocked primer is dissociated from the blocking oligonucleotide and subsequently inserted into the amplification product of the target nucleic acid.

Another aspect of the invention relates to a kit for detecting a target nucleic acid. For example, the detection kit of the present invention may comprise (i) a pair of primers (10-50) to be used with a polymerase for amplification of a target nucleic acid, each with or without a non-adjacent segment for its priming sequence. Bases, or 15-40 bases); And (ii) a blocking oligonucleotide capable of blocking one of the two primers (10-50 bases, or 15-40 bases). Preferably, the blocking oligonucleotide (s) and corresponding primer (s) are fluorophore labeled in the manner described above. The blocking oligonucleotides and their corresponding primers are duplexed (i.e., when fluorophore labeled, "specific detection duplex", FIG. 1, middle portion and 2, top and description below) It may be provided as. It is also preferred that the kit further comprises one or both of the following reagents: Additional blocking oligonucleotides for blocking polymerase and other primers.

In another embodiment, the kits of the present invention comprise (i) a first fluorophore labeled oligonucleotide (10-50 bases, or 15-40 bases) that cannot be used as a primer in amplification by the polymerase; And (ii) a second fluorophore labeled oligonucleotide (5-30 bases, or 7-15 bases) with free 3'OH. Both oligonucleotides hybridize to each other by at least five (preferably, at least 8) consecutive, completely complementary nucleotides, and the first oligonucleotide is 1-12 bases at the 3 'end of the second oligonucleotide ( Preferably, the first and second fluorophores, protruding by 4-8 bases), moreover, one is a donor fluorophore and the other is a receptor fluorophore, so close to allow resonance energy transfer therebetween. . The first and second oligonucleotides may be provided in a duplex, ie, a "universal detection duplex" (see description below). Preferably, the kits of the present invention further comprise other reagents such as, for example, ligase, kinases, and polymerases with or without 5 '→ 3' exonuclease activity. Ligase, kinase (or polymerase with or without 5 '→ 3' exonuclease activity) can be used to link any selected primer to the universal detection duplex, converting the latter to the specificity detection duplex. .

Specificity and universal detection duplexes as described above, as well as "immobilized" oligonucleotides that form one strand of two duplexes, are included within the scope of the present invention.

Other features and advantages of the invention will be apparent from the following drawings and description of the preferred embodiments and from the appended claims.

First, the drawings will be described.

1 is one embodiment of the method of the invention.

2 is another embodiment of the method of the invention.

3 is a photograph of a gel demonstrating the effect of blocking oligonucleotides on the reduction of nonspecific priming events.

4 is a graph showing the use of fluorophore labeled blocking oligonucleotides for monitoring specific priming events.

5 is a graph showing the resolution and sensitivity in the detection of two different target sequences using two probes embodied by the present invention.

Description of the Preferred Embodiments

As used herein, “amplification” broadly employs a polymerase and a pair of primers in an amount greater than the amount initially present for any particular nucleic acid sequence, ie, “target nucleic acid sequence” or “target nucleic acid”. Means how to produce.

The invention also relates to a method of detecting the presence of a particular nucleic acid sequence by monitoring the insertion of the primer (s) into the amplification product of a particular nucleic acid. In the present invention, blocking oligonucleotides are used to prevent nonspecific priming events during the amplification process. In a preferred embodiment, the amplified target sequence is detected by monitoring by fluorescence the dissociation of the blocking oligonucleotides from its complementary primers.

Amplification relies on hybridization of each pair of primers to a specific priming sequence, which is the sequence within the original nucleic acid template that most preferably hybridizes with the highest degree of complementarity to the primer. In addition to specific priming sequences, the original nucleic acid sequences may include nonspecific priming sequences to which primers may hybridize. Non-specific priming events, i.e. events that result in amplification of non-specific nucleic acid template sequences, are due to intramolecular interactions between the primers and reactions such as hybridization of the primer to template sequences other than the priming sequence of the primer. Self priming, such as "dimerization" (chou et al., In Nucleic Acid research (1992) 20: 1717).

Primers are oligonucleotides that, whether purified from restriction enzyme digests or prepared by organic methods, can serve as a starting point for the synthesis of primer extension products complementary to the target sequence when placed under appropriate conditions. In addition to the sequences complementary to the priming sequences specific to the original nucleic acid template, the primers of the present invention may also include their 5 'region sequences that are not adjacent to the priming sequence of the original template. That is, the adjacent segment is the stretch at the 3 'region of the primer, which sequence is complementary to the priming sequence specific at the original nucleic acid target, while the non-adjacent segment is the stretched portion at the 5' region of the primer. As such, this sequence is non-complementary to the priming sequence of the original nucleic acid template.

As used herein, original nucleic acid template refers to a purified or unpurified nucleic acid in a sample that contains or is suspected of containing a target sequence. Thus, the method may comprise DNA, RNA or DNA: RNA hybrids. The original nucleic acid template is not limited to DNA or RNA that ultimately is translated into protein products, and may include non-coding nucleic acid sequences located between non-coding nucleic acids or coding sequences. To minimize the likelihood of occurrence of non-specific priming events, the present invention provides blocking oligonucleotides that compete with the non-specific priming sequence for hybridization with primers. The blocking oligonucleotides of the present invention "can't function as primers for the polymerase". That is, it cannot serve as a starting point for the synthesis of extended products. Blocking oligonucleotides may be used to remove or modify 3 'terminal hydroxy groups, such as addition of terminal 3'-dideoxynucleotides, 3'-phosphorylation, 3' amino, to steric hindrance to polymerases from catalyzing the extension reaction. Termination and conjugation of large volume molecular moieties such as rhodamine near the 3 'end can be modified such that they cannot function as primers for extension reactions. Representative conditions for extending product synthesis are described in the Examples.

In most cases where the 3 'terminal portion of a primer protrudes or extends beyond its blocking oligonucleotide, in the 5' → 3 'direction of the primer when the primer hybridizes to its blocking oligonucleotide as a "primer: blocking oligonucleotide duplex" It is desirable to exclude unintended extension of. In this case, it should be pointed out that the generation of primer: blocking oligonucleotide duplex is a reversible process. Thus, duplexes can be dissociated and recombined repeatedly under suitable conditions (eg, temperature increase and subsequent temperature decrease).

The term "blocking oligonucleotide" refers to an oligonucleotide capable of hybridizing with a primer to broadly prevent non-specific priming events. Since blocking oligonucleotides have sufficient complementarity to their corresponding primers, it is possible to stabilize primers that do not participate in specific priming events at appropriate concentrations to prevent non-specific priming events. "Sufficient complementarity" means that the two sequences must have sufficient nucleotide complementarity to allow hybridization to occur. Preferably, blocking oligonucleotides are present for each primer at the start of the amplification reaction.

It will be understood by those skilled in the art that the degree and concentration of complementarity for hybridization between the two strands to occur is an interdependent variable. Thus, the blocking oligonucleotides may be completely complementary to their corresponding primers, or otherwise, if the blocking oligonucleotides are present at a concentration sufficient to prevent binding of the primers to nonspecific sequences It may be incompletely complementary. Generally, nucleic acid samples are double-stranded. Therefore, it is necessary to separate these two strands prior to extension product synthesis. In the examples provided below, heat denaturation was used to separate the strands of the nucleic acid sample. However, other agents for separating nucleic acid strands are also known, including enzyme preparations such as RecBCD helicase. See Muskavitch et al. (1981, Enzyme 14: 233). Primers and their blocking oligonucleotides can be added in duplex to nucleic acid samples or in two separate strands. If primers and blocking oligonucleotides are added to the duplex, the steps that cause denaturation of the nucleic acid sample will likewise separate the primer: blocking oligonucleotide duplex into its respective strands.

After strand separation, the reaction mixture is subjected to appropriate hybridization conditions that increase the hybridization of each primer with its priming sequence in the template to form a primer: template complex or with the blocking oligonucleotide to form a primer: blocking oligonucleotide duplex. Placed under Suitable hybridization conditions are well known in the art and some of these are set forth in the examples below.

Once the primers hybridize to the priming sequence of the template, the reaction mixture is placed in suitable extension reaction conditions that allow the primers to act as an initiation of the extension reaction, ie the polymerization reaction. Hybridization to the template of the primer and extension of the primer need not proceed in two different reactions. Thus, hybridization and extension can proceed simultaneously by combining the conditions required for hybridization with the conditions required for extension. Suitable extension reaction conditions, which are well known in the art, are conditions that lead to the synthesis of extension products at the 3 'end of the primer hybridized to the nucleic acid template upon addition of nucleotides in the presence of a catalyst such as DNA polymerase. (Eg, temperature, pH and ionic strength). Representative extension reaction conditions are also presented in the Examples below.

Catalysts for the extension reaction are generally DNA polymerases, such as thermostable polymerases such as Thermos aquaticus or Thermos thermophilus (Kong et al., J. Biol). Chem. (1993) 268: 1965). In order to practice the present invention, it is important that the polymerase can perform substitution of oligonucleotides hybridized to the template "downstream" of the sequence to which the primers hybridized. Under appropriate extension reaction conditions, the polymerase continually adds nucleotides at the 3 'end of the extension primer and substitutes, if any, a blocking oligonucleotide hybridized "downstream" of the primer to complement the target sequence of the template. And an extension product comprising the sequence of blocking oligonucleotides.

In the case of a double stranded nucleic acid template, two extension products are first synthesized, each extension product corresponding to one strand of the target sequence. The newly synthesized extension product is not only a copy of the target sequence, but also includes a primer sequence with or without the extension of the non-contiguous sequence. The newly synthesized extension products are then separated into the original template strand and used as the next template to contain additional extension products. Next, the segments of nonadjacent primers to the priming sequence of the original template, if any, are replicated and integrated into the extension product. It should be noted that each extension product that can be used as a template in subsequent extension reactions consists of two covalently bonded moieties: (i) the entire primer, including the non-adjacent segment, if present, and (ii) the target An amplified nucleic acid sequence complementary to one strand of the sequence. Completion, hybridization, and extension / substitution of strand separation constitutes one cycle of layerwidth reaction. Thus, the concentration of the extension product increases at an exponential rate with the completion of the increasing amplification cycle.

In a preferred embodiment, both the primer and its blocking oligonucleotides are each labeled with a donor fluorophore and a receptor fluorophore, which are brought in close proximity via hybridization, for example, to allow resonance energy transfer. As the extension proceeds, the availability of the labeled primer decreases because the labeled primer is inserted into the extension product. This reduces the amount of labeled primers available for hybridization to labeled blocking oligonucleotides. As the extension proceeds in a repeat cycle, an unlabeled extension product is prepared that can bind to the primers, which compete with the labeled blocking oligonucleotides for hybridization with the primers. Thus, in this preferred embodiment, the blocking oligonucleotide performs two dynamics: (1) blocking molecules for elimination or reduction of nonspecific priming events, and (2) signal probes involved in the detection process.

When fluorophores are used in the practice of the present invention, two fluorophores, a donor and a receptor are bound to a blocking oligonucleotide and its corresponding primers so that when the blocking oligonucleotides and primers hybridize to each other in duplex form, they The resonance energy transition is within the distance. The fluorophores are preferably at least one nucleotide apart from each other, but at a distance of 100 μs or less (Clegg, Methods in Enzymology 211: 353). It is also preferred that the fluorophores are bound to internal nucleotides. If desired, more than two fluorophores may be used. The fluorophores may also be the same or different. The overlap of the emission spectrum of the donor fluorophore and the excitation state spectrum of the receptor fluorophore allows for energy transfer between them. Thus, changes in signal due to disruption of energy transfer are produced when the primer: blocking oligonucleotide duplexes denature or dissociate, such as during an extension reaction.

If desired, fluorophore labeled blocking oligonucleotides may be made preferentially that are not complementary (ie, at least 5 base pairs of complementarity) to both the primer and target sequences of the original template. A "universal detection duplex" consists of a blocking oligonucleotide and a second fluorophore labeled oligonucleotide, wherein the blocking oligonucleotide is complementary to and protrudes from the 3 'end of the second oligonucleotide. Before mixing the primers specific for the extension reaction with the original template, the blocking oligonucleotides are brought closer to the complementarity of the primers through a ligation process, where the 3 'end of the second oligonucleotide is 5 'Covalently attached to the end. That is, the term "universal detection duplex" is used to describe oligonucleotides formed between blocking oligonucleotides and their complementary oligonucleotides that do not function as primers. For obvious reasons, the two fluorophores (or more) of the universal detection duplex should be in close proximity. In a particularly preferred embodiment, the donor fluorophore and the receptor fluorophore are bound to each oligonucleotide of the universal detection duplex with weak stability, and subsequent linkages to the duplexes of the primers provide even more stable duplex-primers. As a result, the energy transfer efficiency between the two fluorophores is improved.

Unlike the universal detection duplex, the "specificity detection duplex" is convenient for use in amplification / detection of target nucleic acids. See also Figure 1, the filled circle (●) and the rectangle (■) represent two fluorophores and the hollow circle (○) represents the 3 'end of the neutralized blocking oligonucleotide. In the embodiment shown in FIG. 1, it should be noted that the labeled primers comprise nonadjacent segments for their priming sequences. Moreover, pre-asymmetric amplification, which preferentially amplifies the strand to which the labeled primer binds, is performed to increase sensitivity. The asymmetric reaction can be induced by providing unequal amounts of primers, ie, excess one primer and a limited amount of another primer, or by providing primers that are not equal in length to the complementary portion for the specific priming sequence. Can be. Other factors, such as differences in the synthesis rate of each template strand, can also contribute to the asymmetric nature of amplification.

The optimal primer ratio for asymmetric amplification is an experimentally determined factor (Shyamala et al., 1989. J. Bacteriol. 171: 1602).

Another type of “specificity detection duplex” is used in the method shown in FIG. 2. In this embodiment, two unlabeled specificity detection duplexes (ie, two blocking oligonucleotide: primer pairs) are used. Neither primer has a nonadjacent segment for its priming sequence. Each hollow circle represents the neutralized 3 'end of the blocking oligonucleotide. An important downstream blocking oligonucleotide substitution process, not shown in FIG. 1, is shown in FIG.

The method of the present invention can be carried out in an apparatus that simultaneously performs the process of detecting the presence of an amplified target without undergoing amplification and separation of the nucleic acid sequence in the nucleic acid template. The apparatus comprises (1) an oligonucleotide container for containing oligonucleotides; (2) a reaction vessel containing a nucleic acid sample, the vessel being connected to the oligonucleotide vessel so that the contents in the oligonucleotide vessel can be automatically delivered to the reaction vessel: (3) a control system for mixing the oligonucleotide and the sample; (4) a temperature controller for adjusting the temperature of the reaction vessel; (5) an irradiation source; And (6) a detector for detecting radiation emitted from the reaction vessel. Preferably, the reaction vessel is a well of a multi-well microtiter plate. In general, a radiation source is a device capable of generating photons of a desired wavelength. Such devices are well known to those skilled in the art to which the present invention pertains and are commercially available. (Eg, tungsten lamps or ionizing laser lamps). Detectors are devices that can convert photon energy into electrical signals. In other words, such devices are well known and can be obtained from a number of sources. Embodiments include photomultipliers and semiconductor-based photon detectors.

It is particularly preferred that the device further comprises a data processor for processing the signal data received by the detector. The apparatus may include a control mechanism to terminate the amplification reaction when a predetermined level of radiation corresponding to the pre-selected level of amplification is detected by the detector. Thus, the device provides an “on-line” monitor of the amplification reaction in a manner similar to the fermentation product on-line monitor used for mass recombinant protein synthesis. Thus, using a unique amplification and detection method in conjunction with the devices disclosed herein, there is no need to sample the mixture to determine the extent to which target sequence amplification has occurred, thereby preventing the reaction vessel from being contaminated.

Without further effort, it is believed that one of ordinary skill in the art to which this invention pertains can utilize the invention to the fullest extent based on the description herein. Accordingly, the specific embodiments below are for illustrative purposes only and should not be construed as limiting the remainder of the disclosure herein in any way.

Example I. Effect of blocking oligonucleotides on reduction of nonspecific priming events

To illustrate the effect of blocking oligonucleotides on priming efficiency, 127 bp segments of the human SRY gene (Sinclair et al., 1990, Nature 346: 240) were amplified in the presence and absence of blocking oligonucleotides. The sequence of the target defining primer is as follows; Primer (A) 5'-AACTC AGAGA TCAGC AAGCA GCTGG GATAC-3 'and Primer (B) 5'-ACTTA TAATT _TR CGGGT ATTTC T _TR CTCT GTGCA-3'. Each T _TR represents a Texas red conjugated amino-C6-dT residue (Glenn Reserarch, Sterling, VA) and was conjugated to primer (B) according to the method proposed by the supplier (Molecular Probes, Inc. Eugene, OR. USA). The blocking oligonucleotides used are complementary to the 24 bases of the primer (A) and are 5'-CAGCT GCTTG CTGAT CTCTG AGTTL-3 ', where L indicates the 3'-amine terminus [3'-amine-ON CPG , Clontech, Palo Alto, CA].

All nucleotides are Operon Technologies Inc. (Alameda, Calif.) And then purified by electrophoresis on 8 M urea / l5% acrylamide gel. Specific bands of oligonucleotides were eluted by electrophoresis and quantified by UV absorbance at 260 nm.

Male genomic DNA used as target DNA was extracted from whole blood by DTAB / CTAB method (Gustinicich et al., 1991, Biotechniques 11: 298), quantified by UV 260 nm absorbance, and 50 ng / μl yeast tRNA The carrier was washed successively with TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0).

Amplification was performed in 96-well polycarbonate microtiter plates (Costar, Cambridge, Mass.) Using AG-9600 Thermal Station (MJ Research, Watertown, Mass.). 50 mM Tris-HCI, pH 8.7, containing 0.3 units of Tth DNA polymerase (Carballeira et al., 1990, Biotechniques 9: 276-281) and varying amounts of target DNA (10 ² , 10 ³ and 10 ⁴ ) Primers with or without the same amount of blocking oligonucleotide in 10 μl of the reaction mixture of 40 mM KCl, 5 mM NH ₄ Cl, 2 mM DTT, 0.1% Triton X-100, 100 μM dNTP ng was added.

Yeast tRNA and salmon sperm DNA were used as negative controls. 9% acrylamide electrophoresed at 10 V / cm in TBE buffer after a total of 35 thermal cycles (25 seconds at 94 ° C., 20 seconds at 60 ° C., and 40 seconds at 72 ° C.) was performed. Analysis on the gel. The molecular marker used for gel electrophoresis was the HpaII cleavage product of pBR322 DNA. The gel was simply stained with ethidium bromide and photographed under UV light. The photograph is shown in FIG.

Referring to Figure 3, the contents of each lane of the gel are indicated, where tRNA refers to yeast transfer RNA, SS stands for salmon sperm DNA, M stands for molecular size marker, and numbers represent targets. Represents the amount of molecules. The symbol "-" or "+" indicates the absence or presence of blocking oligonucleotides. The intensity of the 127 bp SRY specific sequence was more pronounced for the reaction in the presence of blocking oligonucleotides, even at lower target doses. In contrast, the intensity of the primer dimer was more pronounced for the reaction in the absence of blocking oligonucleotides. Thus, the amplification efficacy of specific target sequences was directly influenced by the extent of interference by non-specific priming events, such as the formation of primer dimers, which are much richer in reactions without blocking oligonucleotides.

Example II. Use of fluorophore labeled blocking oligonucleotides to monitor amplification of target nucleic acids

This example demonstrates that blocking oligonucleotides can reduce nonspecific priming as well as be used to monitor the amplification process when labeled by fluorophores. The target DNA, blocking oligonucleotides and primers used in this example are complementary to the 20 bases of primer (B) and the indicator probe having the sequence 5'-AGAGA GAAAT ACCCG AATTAL-3 'has a fluorescence energy with primer (B). They are basically the same as those used in Example I, except that they are included to participate in metastasis. "L" represents 3'-amine terminal [3'-amine-ON CPG, Clontech, Palo Alto, CA). After purification, the indicator probe was added to a provider [Molecular Probes, Inc. Eugene, OR. USA) was further conjugated with fluorescein according to the method proposed by.

Amplification was performed in 96 well polycarbonate microtiter plates (Techne Inc., Cambridge, England) using MW-1 Thermocycler (Tehne Inc., Cambridge, England). 50 mM Tris-HCl, pH 8.7, 40 mM KCl, 5 mM NH ₄ Cl, 2 mM DTT, 0.1% Triton X-100, 100 μM, containing 0.6 units of Tth DNA polymerase and 10 ² copies of target DNA To 10 μl of reaction mixture of dNTP, 20 ng of each of the primers with or without the same amount of blocking oligonucleotide was added. Salmon sperm DNA was used as a negative control. An indicator probe was added to the reaction after a total of 24 heat cycles (42 seconds at 96 ° C., 42 seconds at 53 ° C., and 60 seconds at 72 ° C.) was performed. Each reaction mixture taken in cycle 25 was subjected to the first 630 nm fluorescence reading using a microplate fluorometer (Model 7600, Canbridge Technologies, Inc., Watertown, Mass., USA). Subsequent readings were made in cycles 30 and 35. Fluorescence reading data is plotted and shown in FIG.

The bar graph in FIG. 4 illustrates the extent of fluorescence signal change in different cycles of amplification with and without blocking oligonucleotides initially. The decrease in fluorescence signal indicates dissociation of the primer: blocking oligonucleotide duplex, in the absence of non-specific priming events, which is proportional to the extent of insertion of the primer into the amplified target sequence. The reaction without Tth DNA polymerase was used as a control for the fluorescence signal (denoted No Amp "). Simultaneously analyzing 10 ² copies of SRY sequence and salmon sperm DNA to observe target specificity and non-specific signals. As shown in the figure, in the late amplification step (cycle 35), the presence of blocking oligonucleotides is dependent on the difference in fluorescence changes between the two salmon sperm DNA controls, each with and without blocking oligonucleotides. As demonstrated by, non-specific priming events were reduced.

Example III. Increased energy transfer through linkage of universal detection duplex and primer

In this example, two sets of universal oligonucleotides were used to illustrate the energy transfer enhancement induced by the linkage process in which the complementary oligonucleotides are linked to primers having their blocking oligonucleotides as a template.

All oligonucleotides were synthesized according to the method described in the previous examples, purified and conjugated to fluorophores. For ligation reactions, the same molar amount of complementary oligonucleotides and primers as 200 ng of blocking oligonucleotides was added to 0.15 units of T4 DNA ligase (New England Biolab, Beverly, Mass.), 0.5 units of T4 polynucleotide kinase (New England Biolab). , Beverly, MA), 20 μl of reaction mixture containing 1 mM ATP and buffer (70 mM Tris-HCI, pH 7.6, 10 mM MgCl ₂ , and 5 mM DTT). The linking reaction was carried out by incubating for 1 hour and 30 minutes at 40 ° C., and the reaction was terminated by heating at 95 ° C. for 10 minutes. Before and after each reaction, fluorescence intensities were measured at 630 nm emission and at 495 nm excited state for aliquots of the reaction mixture.

Set A: The blocking oligonucleotide has the sequence 5'-T _TR TTTT GAACA GGCCT TGT _TR G-3 ', and its complementary oligonucleotide has the sequence of SEQ ID NO: 5'-CAAGG CCT _F G-3', with the primer underlined. Has the sequence 5'-TTCAA ACCTG TGCTT AGGGC ACTG-3 'with bases complementary to the priming sequence. T _TR means Texas red conjugated amino-C6-dT residue and T _F means fluorescein conjugated amino-C6-dT residue. See Examples I and II above for their binding to oligonucleotides.

The fluorescein readout of the reaction mixture before ligation was 9OO ± 23 and the readout after ligation was 1336 ± 211. Therefore, the set induced energy transfer in set A was 1.48 times.

Set B: blocking oligonucleotide has the sequence 5'-T _TR TGGT CTCGA CCTCC TTGT _TR G-3 ', and its complementary oligonucleotide has the sequence of SEQ ID NO: 5'-CAAGG AGGT _F C G-3'; The primer has the sequence 5'-AGACC ACCTG TGCTT AGGGC ACTG-3 'with bases complementary to the underlined priming sequence.

The fluorescein readout of the reaction mixture before ligation was l 489 ± 58 and the readout after ligation was 3019 ± 94. Therefore, the set induced energy transfer in set B was 2.03 times.

Example IV. Application of Detection Duplexes to Monitor Asymmetric Amplification of Target Nucleic Acids

To demonstrate the application of the detection duplex, we used either (A) primer: blocking oligonucleotide duplex or (B) universal detection duplex bound to the primer to monitor the amplification process. In the case of duplex (A), the blocking oligonucleotide is complementary to both the primer and the original target sequence. In the case of duplex (B), the primers were linked to the complementary oligonucleotides of the blocking oligonucleotides to establish complementarity between the primers and blocking oligonucleotides. Both duplexes (A) and (B) were subjected to the same amplification reaction. Segments of human acute myeloma leukemia breakpoint associated sequence (AML-1) and human X-chromosome specific amelozinene (AMG-X) were used as amplification targets to illustrate this application.

Primer: Preparation of Blocking Oligonucleotide Duplex

Duplex (A): The sequences of the primers and blocking oligonucleotides used for AML-1 are 5'-ACGGG CATAC GCATC ACAAC AA-3 'and 5'-TTGAT GCGTA TCCCC GTTT-3', respectively, for AMG-X The ones used are 5'-AGACT GAGTC AGAGT GGCCA GGC-3 'and 5'-TCCAC TCTGA CTCAG TCTTA-3', respectively. Primers and blocking oligonucleotides were conjugated with fluorescein and Texas red, respectively, in the underlined positions. The 3 'end of the blocking oligonucleotide was neutralized for extension by terminating the 3'-dideoxy.

Duplex (B): The sequence of the primer for AML-1 is 5'-GGGAC GCACG GGGAA ACGCA TCACA ACAA-3 'and the primer for AMG-X is 5'-GGGAC GCAGA CTGAG TCAGA GTGGC CAGGC-3'. Each primer was linked to a detection duplex similar to that shown in Example III. The increase in energy transfer for AML-1 and AMG-X was 2.62 and 2.42 times, respectively, at linkage.

Asymmetric Amplification and Detection

For the AML-1 target, the excess primer is 5'-TGTTT GCAGG GTCCT AACTC AATCG-3 'and the restriction primer is 5'-GCGGC GTGAA GCGGC GGCTC G-3'; For AMG-X targets, the excess primer is 5'-TGATG GTTGG CCTCA AGCCT GTG-3 'and the restriction primer is 5'-TGGGA TAGAA CCAAG CTGGT CAG-3'.

The male genomic DNA used as target was extracted from whole blood according to the method of Example I.

Target sequences were asymmetrically amplified by 20 cycles prior to addition of the primer duplex. Asymmetric amplification was performed with 1 unit of Tth DNA polymerase, 50 mM Tris-HCl, 40 mM KCl, 5 mM NH ₄ Cl, 100 μM dNTP with an excess primer to restriction primer ratio of 7.5 (1.2 μM vs 0.16 μM). , 5 μl MgCl ₂ and 0.1% Triton X-100 in 10 μl of reaction mixture. Cycling conditions were the same as used in Example I. Reactions were carried out in 96 well polycarbonate microtiter plates (Costar, Cambridge, Mass.) On AG-9600 Silver Block Thermal Station (MJ Research, Watertown, Mass.). 4 ng of primer duplex each was added to the reaction wells at room temperature to initiate simultaneous amplification and signal detection. As baseline for calibration, the initial 63O nm fluorescence emission was measured using 486 nm excitation in cycle 21. Subsequent signal measurements were made in cycle 28 and all subsequent cycles.

The results collected from detection in cycle 3O are shown in FIG. 6, demonstrating the quantitative fractionation and detection sensitivity by duples (A) and (B). As shown in the figure, the decrease in fluorescence intensity reflects the original target dose.

Other embodiments

From the foregoing description, those skilled in the art will be able to readily identify the essential features of the present invention, and various changes and modifications of the present invention in order to adapt the present invention to various uses and conditions without departing from the spirit and scope of the present invention. You can make them. Accordingly, other embodiments are also within the scope of the claims herein.

Claims (34)

A method for detecting a target nucleic acid comprising the following steps: In the presence of oligonucleotides that cannot function as primers for the polymerase, for the polymerase, the first primer with or without the 5 ′ segment non-complementary to the first primer sequence, and for the second priming sequence. Amplifying the target nucleic acid to obtain an amplification product using a second primer with or without non-complementary 5 ′ segment, wherein the oligonucleotide is attached to at least five consecutive nucleotides of the first primer. Having at least 5 contiguous nucleotides completely complementary to: Detecting the presence of the target nucleic acid by monitoring amplification of the target nucleic acid. The method of claim 1, Wherein said detecting step is performed by monitoring said amplification product of the target nucleic acid on a gel after electrophoresis. The method of claim 1, Two fluorophores are covalently bonded to the first primer and the oligonucleotide, respectively, one of the two fluorophores being a donor fluorophore and the other a receptor fluorophore, the first primer and the oligonucleotide When is hybridized, the donor fluorophore and the acceptor fluorophore are bound so close as to allow resonance energy transfer between them; Furthermore, the detecting step is performed by monitoring a change in fluorescence emission of the receptor fluorophore upon irradiation with the excitation light of the donor fluorophore, wherein the change is such that the first primer is dissociated from the oligonucleotide to the amplification product of the target nucleic acid. Method that is a function of the degree inserted into it. The method of claim 1, Wherein said first primer comprises a 5 'segment that is non-complementary to said first priming sequence. The method of claim 4, wherein And said oligonucleotide has at least five consecutive nucleotides that are completely complementary to at least five consecutive nucleotides in said non-complementary 5 'segment of said first primer. The method of claim 1, And wherein the first primer does not comprise a 5 'segment that is non-complementary to the first priming sequence. The method of claim 1, The amplification step is performed in the presence of an additional oligonucleotide that is unable to function as a primer for the polymerase, wherein the additional oligonucleotide is completely complementary to at least five consecutive nucleotides of the second primer. A method having at least 5 contiguous nucleotides. The method of claim 1, Wherein said first primer, said second primer and said oligonucleotide each comprise 10-50 nucleotides. The method of claim 8, Wherein said first primer, said second primer and said oligonucleotide each comprise 15-40 nucleotides. The method of claim 1, And said oligonucleotide has at least 8 contiguous nucleotides that are completely complementary to at least 8 contiguous nucleotides of said first primer. The method of claim 1, The concentration of said oligonucleotide is 0.3-5.0 times the concentration of said first primer. The method of claim 11, The concentration of said oligonucleotide is 0.3-2.5 times the concentration of said first primer. The method of claim 12, Wherein said first primer, said second primer and said oligonucleotide each comprise 10-5O nucleotides. The method of claim 12, And said oligonucleotide has at least 8 contiguous nucleotides that are completely complementary to at least 8 contiguous nucleotides of said first primer. The method of claim 13, Wherein said oligonucleotide has at least eight contiguous nucleotides that are at least eight contiguous nucleotides of said first primer completely complementary. The method of claim 15, Two fluorophores are covalently bonded to the first primer and the oligonucleotide, respectively, one of the two fluorophores being a donor fluorophore and the other a receptor fluorophore, the first primer and the oligonucleotide When is hybridized, the donor fluorophore and the acceptor fluorophore are bound so close as to allow resonance energy transfer between them; Furthermore, the detecting step is performed by monitoring a change in fluorescence emission of the receptor fluorophore upon irradiation with the excitation light of the donor fluorophore, wherein the change is such that the first primer is dissociated from the oligonucleotide to the amplification product of the target nucleic acid. Method that is a function of the degree inserted into it. A 5 'segment that is either non-complementary with respect to the first priming sequence to be used with the polymerase for amplification of the target nucleic acid, and a 5' segment that is non-complementary with respect to the second priming sequence; A second primer with or without: and An oligonucleotide comprising at least five (at least five) consecutive nucleotides that cannot function as a primer to the polymerase and are completely complementary to at least six consecutive nucleotides of the first primer; The target nucleic acid detection kit of claim 1, wherein the first primer, the second primer and the oligonucleotide each comprise 10-50 nucleotides. The method of claim 17, And wherein said first primer and said oligonucleotide are provided in a duplex. The method of claim 17, And the oligonucleotide has at least 8 contiguous nucleotides that are completely complementary to at least 8 contiguous nucleotides of the first primer. The method of claim 19, Wherein said first primer, said second primer and said oligonucleotide each comprise 15-40 nucleotides. The method of claim 19, And said kit further comprises a polymerase. The method of claim 19, The kit may not function as a primer for the polymerase and contains 10-50 nucleotides, further having at least 8 contiguous nucleotides completely complementary to at least 8 contiguous nucleotides of the second primer And a kit further comprising an oligonucleotide. The method of claim 19, Two fluorophores are covalently bonded to the first primer and the oligonucleotide, respectively, one of the two fluorophores being a donor fluorophore and the other a receptor fluorophore, the first primer and the oligonucleotide And the donor fluorophore and the receptor fluorophore are bound so close as to allow resonance energy transfer therebetween. A first oligonucleotide having a covalently bound first fluorophore and comprising 10-5O nucleotides, the first oligonucleotide being unable to function as a primer for the polymerase in amplification of the target nucleic acid: and A second oligonucleotide having a second fluorophore covalently bound and comprising 5-30 nucleotides, wherein the second oligonucleotide has free 3 ′ OH and can hybridize to the first oligonucleotide through at least five consecutive fully complementary nucleotide pairs. A second oligonucleotide present; Wherein the first oligonucleotide protrudes 1-l2 nucleotides from the 3 'end of the second oligonucleotide, further one is a donor fluorophore and the other is a receptor fluorophore, and the first oligonucleotide is the second The kit for detecting a target nucleic acid when hybridizing with an oligonucleotide, wherein the first and second fluorophores are in close proximity to allow resonance energy transfer between them. The method of claim 24, The first oligonucleotide comprises l5-40 nucleotides and protrudes 4-8 nucleotides above the 3 'end of the second oligonucleotide, the second oligonucleotide comprises 7-15 nucleotides and the first oligonucleotide And hybridize through at least eight consecutive fully complementary nucleotide pairs. The method of claim 25, And wherein said first and second oligonucleotides are provided in a duplex. The method of claim 25, And said kit further comprises a ligase. The method of claim 25, Wherein said kit further comprises a polymerase having a kinase or 5 ′ → 3 ′ exonuclease activity. The method of claim 27, Wherein said kit further comprises a polymerase having a kinase or 5 ′ → 3 ′ exonuclease activity. The method of claim 25, And wherein said kit further comprises a polymerase with or without 5 ′ → 3 ′ exonuclease activity. A first oligonucleotide for use with a polymerase in amplification of a target nucleic acid, which may function as a primer and that may or may not comprise a 5 ′ segment that is non-complementary to its priming sequence; A second oligonucleotide that hybridizes with at least five consecutive fully complementary nucleotide pairs with the first oligonucleotides and is unable to function as a primer for the polymerase; And A donor fluorophore and a receptor fluorophore, each covalently bonded to said two oligonucleotides, wherein resonant energy transfer occurs between them when they are in proximity to each other: Wherein the first and second oligonucleotides comprise 10-50 nucleotides. The method of claim 31, wherein Wherein said first and second oligonucleotides each comprise 16-40 nucleotides, and wherein said second oligonucleotide hybridizes through at least eight consecutive fully complementary nucleotide pairs with said first oligonucleotide. A first single-stranded oligonucleotide comprising 10-5O nucleotides and unable to function as a primer for use with the polymerase in amplification of the target nucleic acid; And A first fluorophore covalently bound to the oligonucleotide; A second single-stranded oligonucleotide comprising 5-30 nucleotides and having a free 3 ′ OH, wherein the second oligonucleotide hybridizes with the first oligonucleotide through at least five consecutive fully complementary nucleotide pairs; The first oligonucleotide comprises a second oligonucleotide that protrudes 1-12 nucleotides from the 3 'end of the second oligonucleotide; And And further comprising a second fluorophore covalently bound to the second oligonucleotide, wherein the first and second fluorophores, one donor fluorophore and the other acceptor fluorophore, enable resonance energy transfer therebetween. A composition comprising fluorophores in close proximity. The method of claim 33, wherein The first oligonucleotide comprises the 15-40 nucleotides and protrudes 4-8 nucleotides above the 3 'end of the second oligonucleotide, the second oligonucleotide comprises 7-15 nucleotides and the first oligonucleotide And a composition hybridized through at least eight consecutive fully complementary nucleotide pairs.