Method detection identification and/or quantitative evaluation of material, test kit, an optical waveguide biosensor

 

(57) Abstract:

The invention relates to genetic engineering, in particular to a method of analysis using gene probes and using a biosensor, comprising a detector of oscillation damping. The invention allows to improve the sensitivity analysis and to accelerate its implementation. Get oligonucleotide, complementary to all or part of the oligonucleotide sequence, characteristic of RNA or DNA of the studied material, and immobilized on the surface of the waveguide detector damped oscillation. The content of typical sequences is enriched by amplification of specific sequences, which is carried out in the presence of immobilized oligonucleotide. The obtained immobilized oligonucleotide is treated with a solution of the material sample or DNA or RNA derived from this material under conditions that allow DNA or RNA to gibridizatsiya to the immobilized oligonucleotide. Spend the immobilized binding of the oligonucleotide with fluorescently detektivami agent. Conditions are selected so that fluorescently detected, the agent contacted the product of hybridization. Measure the amount fluorescently number and presence, identity and/or quantity of the material under investigation. The test kit includes oligonucleotide, complementary to part of the oligonucleotide sequence, characteristic of RNA or DNA of the evaluated material, and fluorescently detected agent. Oligonucleotide immobilized on the surface of the detector, the attenuation of the vibrations on the optical waveguide. 4 C. and 22 C.p. f-crystals, 1 tab., 8 Il.

The invention relates to a method for detecting the identification and/or quantitative evaluation of the tissues of plants or animals, microorganisms, or isolated from cells RNA or DNA; as well as reagents and the detection device adapted to implement the above method. This method, in particular, based on the fluorescence associated with the phenomenon of total internal reflection (AA-f), and used to assess hybridization of RNA or DNA analyzed sample with RNA or DNA associated with a waveguide detector attenuation of vibrations.

Analysis using gene probes and hybridization of nucleic acids is an alternative to the immunoassay, and is aimed at the detection and identification of biological materials. In this analysis the specificity of gene probes to their targets ravenii of these probes with the materials of the targets, in advance amplificatoare using polymerase chain reaction, can be obtained the maximum sensitivity. Existing techniques based on the use of gene probes, resulting in only a few hours, or even days. Biosensors proposed as an alternative method, which allow for rapid analysis using gene probes, but the present description such biosensor analyses relate to the use of surface plasma resonance (Evans & Charles, 1990); Bulletin of the 1st International Congress on DNA probes and immunoassay; Pollard-Knigh et al., 1990, Ann. Biol. Clin. 48, 642-646).

Previously, beyond biosensors, where the detection is based on the phenomenon AA-f (Sutherland & Dahne, (1987), J. Immunol. Meth., 74, 253-265), as biological recognition elements used proteins. For example, to study the binding of acetylcholine and cholinesterase inhibitors were used antibodies that facilitate the detection of binding of the labeled fluorescent antigen (Eldegrawi et al. (1991) Biosensors &Bioelectronics, 6, 507-516) c receptors of acetylcholine. It was also evaluated the binding of the antibody with Fc-epitopes (Poglitsch & Thompson (1990) Biochemistry, 29,248-254).

The present invention is etector of oscillation damping, and POF-waveguide adapted for the implementation of this method through the introduction of this waveguide bioculturally sensor.

The detectors damped oscillation, based on the phenomenon of total internal reflection (AA), allow to obtain a highly sensitive method for the detection of reactions on the surface of the waveguides. Such a waveguide may be of any shape, but usually it is made in the form of prisms, plates or fibers. The reaction used for the analysis of target molecules can be controlled, for example, by estimating the change in fluorescence upon binding or desorption of the fluorescent material, or by generating a fluorescent material using enzymatic or chemical reagents. In some published patents disclosed the use of detectors attenuation of waves by immunoassay systems as a biological sensing element (see, for example, U.S. patent 4582809); however, the limitations inherent in these immunoreagents not allow you to use the full capabilities of these sensors.

The present invention relates to a method for the detection, identification and/or quantitative evaluation of material vibranta, that:

(I) receive an oligonucleotide probe complementary to all or part of the oligonucleotide sequence, characteristic of RNA or DNA of the investigated material immobilized on the surface of the waveguide detector damped oscillation;

(II) immobilized oligonucleotide from stage (I) is subjected to interaction with the sample or DNA or RNA material obtained from this sample, under conditions in which DNA or RNA having a characteristic oligonucleotide sequence hybridize with the specified immobilized oligonucleotide;

(III) immobilized oligonucleotide stage (I) or nukleinovokisly material obtained from a sample, is subjected to the binding of a fluorescently detektivami agent before, during or after hybridization with DNA or RNA, as indicated in stage (II), so that the indicated fluorescently detected, the agent contacted hybridizing product, and was not associated with dehybridization immobilized oligonucleotide;

(IV) using the detector of wave decay estimate the number of fluorescently detected agent associated, as described in stage (III), and in accordance with this assessment determine the presence, identity and/or quantity is orescence detektivami agent is a fluorescently detected intercalating dye, able to integrate into the duplex hybridization product. In this embodiment of the method of the present invention, the dye is not associated with dehybridization immobilized oligonucleotides. This method of determining the presence of the hybridization product is ideal when using immobilized sequences with length sufficient to allow specific hybridization used in hybridization conditions. Analyzed thus the sample may have a constant amount of DNA/RNA or that content may be redundant in the specific amplification of a sequence, for example, using polymerase chain reaction (PCR) or ligase chain reaction (LCR).

In the second preferred embodiment of the present invention indicated detectable agent is a fluorescently detected oligonucleotide that is capable of hybridisierung with hybridizing characteristic sequence or which can act as a specific primer for amplification reaction sequence, for example, polymerase chain reaction, providing the production of fluorescently-program of amplificatoare on damping.

In her second preferred embodiment, which is described above and uses the detected oligonucleotide, the present invention relates to a method for the detection, identification and/or quantitative evaluation of material selected from the tissues of plants or animals, microorganisms, or free non-cellular RNA or DNA, namely, that:

(I) receive an oligonucleotide probe complementary to all or part of the oligonucleotide sequence, characteristic of RNA or DNA analyzed material, and immobilized on the surface of the waveguide detector damped oscillation;

(II) receive a fluorescently-detected oligonucleotide, which is complementary to all or part of the rest of the characteristic sequence;

(III) immobilized oligonucleotide from stage (I) is treated with a solution containing the material, or derived from DNA or RNA material, under conditions which are characterized by oligonucleotide sequence will hybridisierung with the specified immobilized oligonucleotide (IV), the solution of the material (III) is replaced by a solution containing fluorescently detected complementary oligonucleotide opredelennyuyu linked oligonucleotide in accordance with this assessment determine the presence, identity and/or amount of target material in the sample.

In another preferred variant of its implementation of the present invention relates to a method for the detection, identification and/or quantitative evaluation of the tissues of plants or animals, microorganisms, or free cellular RNA or DNA, which is that:

(I) receive an oligonucleotide probe complementary to part of the oligonucleotide sequence, characteristic of RNA or DNA analyzed material, and immobilized on the surface of the waveguide detector damped oscillation;

(II) receive a fluorescently detected oligonucleotide which is identical to all or part of the rest of the characteristic sequence;

(III) using fluorescently detected oligonucleotide stage (II) as one of the primers carry out a specific reaction amplification sequence on the sample material, which amplificates present any characteristic sequence, and this sequence is embedded fluorescently detected agent;

(IV) immobilized on the waveguide complementary oligonucleotide from stadiumvision product with the specified immobilized oligonucleotide;

(V) using the detector damped oscillation, estimate the number of fluorescently detected of the oligonucleotide, hybridizing with immobilized oligonucleotide, and in accordance with this assessment determine the amount of the amplified material originally present in the sample.

Suitable and preferred specific reaction amplification used in stage (III) is a polymerase chain reaction, however, can also be used and other reactions well known in the art, such as ligase chain reaction.

In all variants of implementation of the present invention, the waveguide is preferably placed in terms of temperature control, for example in the camera control or thermostat so that you can adjust the stiffness of the hybridization of complementary sequences of RNA or DNA targets or they can be cycles of denaturation, hybridization and extension chain polymerase chain reaction.

Fluorescently detected complementary oligonucleotides can be obtained in various forms, for example, in the form of the oligonucleotide, labeled or fluorescent label; or molecules, what can generate a fluorescent material. Therefore, prior to the stage of detecting fluorescence may require additional stages, such as stage of processing fluorescent material that is able to communicate with the specified material, or stage of processing a substrate for the enzyme or catalytic reaction.

Complementary sequence stage (I) may have a length sufficient for specific binding to RNA or DNA target, but it must remain a sufficient portion of the unbound RNA or DNA sequence that is associated with detektivami the oligonucleotide stage (III). Usually immobilized sequence is approximately half the length of the target sequence, but with increasing the length of the target sequence, this ratio may be smaller.

In a preferred embodiment of the present invention using a program oligonucleotide compared to the sequence of the oligonucleotide can be complementary to the entire or almost the entire rest of the target sequence, but with the increase in the characteristic length of the target posledovatel is consistent oligonucleotide is complementary sequences, toward the end or at the end of the target sequence, and fluorescently detected oligonucleotide is complementary to the sequence located closer to the other end or the other end of the target sequence. Therefore, in order to these ends characteristic of a target sequence to be more accessible, preferably at the stage of hybridization to carry out the hydrolysis of RNA or DNA using restrictively endonucleases. Similarly, in the second preferred embodiment of the present invention it is necessary that the detected sequence had only such a length that would be sufficient for use as PCR primers for a desired specificity, but no more. It should be noted that both PCR primers can be fluorescently detectivesyme that will enhance the level of fluorescence on the surface of the waveguide, and thereby increase the efficiency corresponding to the damping. In all cases, the preferred length of the reagent is 5-30, preferably 10-25, and in particular, 15-20 bases.

It should be noted that these complementary sequences neobyazatelno can easily determine the stiffness terms for a statistically acceptable use defective coupling of oligomers (for example, sequences complementary to 90%) and can get quite good results by adjusting the temperature hybridization, which uses such "defective paired sequence.

Requirements for using exactly complementary sequences vary depending on the uniqueness of the sequence designed for the study. For example, a sequence that has a high percentage of compatibility reasons with other potentially present sequences, requires a greater degree of complementarity than its relative differences. Thus, if the sequence under consideration is not fully identified, or it is changed, the technician can easily determine statistically acceptable oligomers for stages (I) and (III).

In the second version of my implementation of the present invention relates to a test kit for use in the method of this invention, containing: (1) an oligonucleotide probe complementary to all or part of the oligonucleotide sequence, characteristic of RNA or DNA of the studied material; and (2) fluorescently detection is Telenesti RNA or DNA of the studied material.

When selecting components (1) and (2) should be guided by the considerations set out above or in the claims; and the collection of these components may be, but not necessarily, supplemented with reagents necessary for immobilization of oligomer (1) on the waveguide and is described in the following discussion of the methods of the present invention.

In the third version of my implementation of the present invention relates to a waveguide suitable for use in biological sensors and characterized in that it contains on its surface immobilized oligonucleotide, complementary to all or part of the oligonucleotide sequence, characteristic of RNA - or DNA-sequence of the investigated material. In addition, kits containing these waveguides are designed for use in the method of the present invention described in the claims.

In the fourth version of my implementation of the present invention relate to the biosensor, including the detector, the attenuation of the oscillations, characterized in that it contains a waveguide, which has immobilized on the surface of the oligonucleotide, complementary to all or part of the oligonucleotide posledovaniem, be in the form of prisms, plates or fibers.

It should be noted that in all embodiments of the present invention can be conjugated to several typical sequences using several types of immobilized nucleotides and fluorescently detectable binding agents in this particular method and the device.

Advantages of the method of the present invention in comparison with similar systems immunoassay are that:

(a) a more dense packing of immobilized component on the surface of the waveguide allows to increase the sensitivity and dynamic range of the method and gives the possibility to construct a simpler surface multispecificity;

(b) binding between the analyzed sample and immobilized and derivationally the oligonucleotides can be adjusted by adjusting the hybridization conditions and the length of the oligonucleotides, which, ultimately, allows to obtain the desired specificity and sensitivity of the test, meet the relevant requirements;

(C) conventional immunoassays are limited by the availability and stability of reagents, a variety of antigenic properties, the research Institute analysis using gene probes has significant advantages;

(d) fluorescent immunoassays have several disadvantages associated with exposure to environmental contamination and nonspecific binding, whereas in the analysis of the present invention using a gene probe the necessity of using higher temperatures (>60oC, instead of the 37oC) allows you to reduce unwanted adsorption of impurities on the surface of the detector;

(e) due to the low dissociation constants obtained for nukleinovokisly complexes, compared with immune complexes, can be fully implemented highly sensitive technology detector attenuation of vibrations.

As the samples can be used natural materials (e.g., available RNA - or DNA-oligonucleotides), or materials, pre-processed, for example, by cell lysis, DNA/RNA concentration or amplification (e.g., via polymerase chain reaction using Taq polymerase), or by degradation using restriction enzymes (e.g., to obtain oligonucleotides of the desired size).

Possible applications of the method, kits and devices of the present invention support who may find many other applications of the present invention.

In Fig. 1 shows the main elements used in the examples of the detector, the attenuation of the oscillations of Fig. 2 is a graph of voltage as a function of time, obtained after hybridization of labeled with fluorescein 20-measure with a complementary sequence, covalently associated with the optical fiber of Fig. 3 - curves "concentration-response", illustrating specific hybridization of complementary and control oligonucleotides. These curves are constructed in the form of graphs based signal (mV/min) concentration (nm) of the oligomer of Fig. 4 - influence of the flow rate of the sample solution passing through the temperature-controlled chamber containing the fiber, the signal of hybridization. This effect is illustrated in the form of a curve of signal (mV/min) flow rate (ml/min); Fig. 5 - effect of temperature on the rate of hybridization; this effect is illustrated in the form of a curve of signal (mV/min) temperature (oC) to 50 ng/ml sample 20-measure, labeled with fluorescein, and Fig. 6 - pH-profile hybridization of nucleotides on the optical fiber waveguide is represented as curve signal (mV/min) from pH; Fig. 7 - binding (8 nm-solutions) oligomers complementary to the IG. 8, a-h diagram illustrating at the molecular level two variants of the method of the present invention.

Methods.

Because the oligonucleotides are long-chain molecules, the length of which is comparable with the depth of the zone of attenuation, to assess the effect of probe length on detection used a short, labeled with fluorescein oligonucleotides which have hybridisable with any end of the oligomer from 204 bases (fragment, amplified from the protective antigenocide gene (Bacillus anthracis) associated with its other end surface of the waveguide.

Materials.

The glutaraldehyde, 3-aminopropyltriethoxysilane (APT); tween-20; Ficoll; formamide; polyvinylpyrrolidone; fraction V of bovine serum albumin (BSA), streptavidin, horseradish peroxidase (HRP-SA); 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), sodium chloride and trinatriytsitrat were obtained from Sigma Chemical Company, Poole, UK. Mononatriumfosfaatti, dinatriumfosfaatti, dodecylsulfonate sodium (LTOs), hydrogen peroxide, hydrochloric acid and acetone were pure chemical reagents (AR-standard) and were supplied from BDH Polle, UK. The ammonium hydroxide was obtained from Aldrich, Gillingham Dorset, UK, and ethanol from Hayman Ltd. the E. DNA oligonucleotides with C6-related, fluorescein-, amino-, or Biotin-modified ends, obtained from British Bio-Technology Ltd, Abingdon, UK, presented in table. 1.

The optical fiber detector of oscillation damping (prototype No. 19) and quartz optical fiber (1 x 65 mm) was obtained from ORD, Nrth Salem, New Hampshire, USA (see Fig. 1). The light from source 1 in the form of a tungsten filament 6 W passes through three collimator lens 2, between which is a blue filter 3 (485 mm) and the mask with the point of puncture 4 (0.8 mm), and gets on a device for the separation of the beam (50:50) 5, which periodically directs the beam through a focusing lens 6 in oligonucleotides the waveguide 7. Fluorescent radiation from any linked (e.g., hybridized or intercalation) of the fluorescent material excited by hitting the light goes in the opposite direction from the waveguide 7, through the lens 6, and passing through a device for the separation of the beam falls on the green filter (530 nm) 8, from which the radiation passes through the device for interrupting the flow 9 and the focusing lens 10, gets, finally, the detector device 11 is connected to the measuring device, such as a voltmeter (not shown).

The output voltage from the voltmeter PE, fitted shirt, and is made of a glass capillary tube, the ends of which are provided with covers made of stainless steel and silicone gaskets. The temperature was regulated by passing water from the water bath through a glass shirt. Temperature monitoring was performed using thermocouples. The optical fiber was purified, and then spent covalent linking with glutaraldehyde according to the method described in Tijssen (1985), "Practice and Theory of enzyme immunoassays": pp 322-323, vol 15 Laboratory Techniques in Biochemistry and Molecular Biology. Ed: Burdon and van Knippenburg: Pub. Elsevier.

Fibers were silanediol a 2% solution of APTS in acetone for 24 h at room temperature, washed with acetone, and dried at 50oC, immersed for 1 h in 1% glutaraldehyde in water, washed in PBS, and finally was left overnight at 4oC 10 µg/ml solution of the oligonucleotide with aminocom.com. For the analysis of optical fiber with immobilized oligonucleotide was washed in deionized water, dried by blotting, were made in the flow cell. To optimize the used conditions, adapted from the method of Anderson & Young (1985), nukleinovokisly hybridization was carried out according to the method Hames and Higgins, Ed. IRL Press, Oxford, pp. 73-111. The temperature of the water in the jacket was brought to 65oand 1 ml of Tween-20 was added to 600 ml of sterile deionized water, the final pH of 6.8) was pumped using a peristaltic pump fibers in the flow cell (25 μl) at 0.5 ml/min until then, until a stable base line. Then with the same flow rate injected target marked on the end with fluorescein oligonucleotide in prehybridization solution and followed by hybridization, which was recorded as an increase of the output signal to a recording device. Five minutes prehybridization solution was again sent to the flow cell and increased the temperature to 80oC. After 10-15 minutes the whole linked oligonucleotide was decarbonators, after which the temperature was lowered to 65oC for holding another cycle.

First, the fibers were connected by a short (16-20 measures) oligonucleotides. To investigate the possibility of using longer probes synthesized amino-terminal 204-Mer by PCR carried out on the target sequence using a single primer (20-Mer), marked the end of the amino (oligo. 5453), and another primer (20-Mer), marked the end of Biotin (oligo. 1222). Unbound aminomethyl primer was removed using streptavidin, immobilized nimera. Aminomethane chain from 204 foundations were released from the complementary chain and pellets by heating for 10 min at 95oC followed by rapid cooling in a mixture of water with ice, after which the pellets were removed by centrifugation. The amount of biogas produced thereby 204-measure was enough to get 0,85 mg/ml solution used to bind to the fibers.

Analysis of oligomers.

In the initial experiment with 20-Merom were used high levels of target and control oligonucleotides. The solution (200 nm, 1 μg/ml) labeled with fluorescein control oligonucleotide (oligo. 4950) was injected with a flow rate of 1 ml/min and at a temperature of 60oIn flow cell containing fiber with immobilized oligonucleotide (oligo. 4946). Immediately after injection was observed shift in the baseline of 80 mV, then the output signal remained constant up until again was not introduced prehybridization solution, resulting in the output signal returned to its previous level. In contrast, with the introduction of oligo. 4947, which is complementary oligo. 4946 immobilized on the fiber, at a concentration of 160 nm (also with a speed of 1 MGK plateau when the signal 1,140 mV above the baseline level. By passing prehybridization solution at 90oC through the flow cell was again observed decrease in signal. In order to confirm the specificity of binding was shown that the control oligo. 4950 binds to an optical fiber with its complement (oligo. 4949), covalently bound to the surface of this fiber.

The process of hybridization of 8 nm labeled with fluorescein oligo. 4947 with immobilized oligo 4946 depending on the time shown in Fig. 2, and Fig. C shows curves "concentration-response" illustrates the hybridization of the labeled fluorescein oligo. 4950 with immobilized oligo. 4949, where as the control oligonucleotide used oligo. 4947. The slopes of the curves were measured after 1 min after injection of the sample in the flow cell.

The results of optimization of the conditions illustrated in Fig. 4-6. The influence of the flow velocity was assessed using solutions (50 ng/ml) oligo. 4047. The data from this study is shown in Fig. 4. Evaluated the dependence of the rate of hybridization temperature and pH and the results of this assessment are presented in Fig. 5 and 6, respectively. The maximum rate of binding was observed at 65oC and at a pH of 6.8.

Detection was carried out in the nanomolar range with a linear dependence between response and concentration; similar results were obtained for immunoassay using a specific biosensor.

For analyses requiring high sensitivity, can be introduced some improvements in the methods of analysis and equipment.

For detection ¾ multiple fluorophores, or fluorescent granules or fluorescent intercalating dyes.

Biosensor analysis can be combined with the technique of DNA amplification, such as PCR, which is carried out before the stage of hybridization on the waveguide, preferably using labeled with fluorophore primers (as in one of the preferred variants of the method of the present invention, described above), which improves the detection of the product. It also enables the direct monitoring of PCR reactions using the biosensor. To conduct biosensor analysis only requires 60 seconds or so, which is several orders of magnitude faster than is necessary for routine analysis using gene probes, and much faster than the time of routine tests, for which it takes about 1 h (Engleberg, 1991, ASM News 57 (4) 183-186).

Fast regeneration of the detector surface by removing related nucleic acids by heating allows to solve one of the problems faced by professionals in the performance of immunoassays for such systems, and allows to obtain results within 60 that may be the which the optical fibers, as has already been installed, can be used for several days without being seen, however, any loss in sensitivity.

Were used both 5' and 3' B-labeled oligonucleotide; moreover, this factor did not exert any noticeable influence on the results of the analysis. There were also the binding oligonucleotides with the proximal and distal ends of the oligonucleotide from 204 grounds. Total length ranged from 70 nm (assuming that one step double-stranded DNA helix, which has 10 bases in the helix is 3.4 nm) to 200 nm (considering the distances of the intermediate links to replays-O-P-O-C-C-C along the frame of the molecule). The depth of the zone of attenuation, defined as the distance at which the intensity falls to 1/e of its original value, was approximately 100 nm, which is comparable with the length 204-measure. Because hybridization labeled with fluorescein distal and proximal oligonucleotides were obtained similar signals (for distal of the oligonucleotide were obtained slightly larger signals), it can be assumed that the orientation of the long oligonucleotide chain was not perpendicular to the surface of the waveguide.

Only a limited colitis with optical fibers, than, obviously, was due to its relatively low concentration on the surface of the waveguide. This factor can explain the rapid achievement of the plot of the plateau in the curves of the dependence of the signal on time in Fig. 7 in comparison with the equivalent curves in Fig. 2 (where the immobilized gene probes being covalently bound of > 100-x-concentrated solution had a higher concentration). If the binding criteria not take the rate of change of the signal, and the height of the plateau, limiting the number of immobilized probes, giving the saturation can be achieved faster.

The implementation of the present invention at the molecular level is illustrated in Fig. 8, where a-c and d-f are for the first and second preferred variants of the present invention, respectively. In and oligonucleotide sequence (A), which is the complementary part of the oligonucleotide sequence specific to the target DNA sequence (B), was immobilized on the exorbitant waveguide detector device using the methods described above. In b immobilized oligonucleotide was treated under conditions of hybridization solution sample or target DNA placenta is asany the sample solution was replaced (in hybridizers conditions) solution containing labeled with fluorescein oligonucleotide (C), which is complementary to the remaining dehybridization part of the target oligonucleotide, and who had likewise hybridisable. After hybridization fluorescein gives the radiation in the zone of attenuation of the waveguide.

In d complementary to sequence (A) was immobilized on the exorbitant waveguide detector, as described above; in e, the sample solution was treated in the conditions of the PCR, two primers (D) and (E), each of which corresponds to a characteristic sequence of one of the two ends of the target characteristic sequence (B). Primer to the end of the sequence opposite to the end that's hybrid with immobilized oligonucleotide (A), was labelled with fluorescein, resulting in a received labeled with fluorescein target sequence (F). In f immobilized sequence (A) was treated with a solution of the sample in hybridizers conditions, resulting labeled with fluorescein target sequence (F) hybridisierung sequence (A), and could be detected due to fluorescence.

In a preferred variant of the natives methods specific amplification, immobilized oligonucleotide (A) is chosen so that it was unable to connect with the primers (D) and (E), resulting in excessive amounts of these primers will not result in the suspension of immobilized area. For example, in this embodiment of the method, the oligonucleotide (A') will hybridisierung with section (F), which occupies a more Central position in the sequence (B) than primer (D), resulting in end (F), not carrying the fluorophore will be as shown in Fig. 8,g.

Variant of the method of the present invention using the intercalating dye, although not reflected in the specific examples given describe, but its implementation can best be illustrated by Fig. 8, a and b, where these stages can be carried out in the presence of the dye or the subsequent addition. Suitable for this purpose dyes, as well as the conditions for their application are described in the literature, see, for example, Latt and Wohileb; Chromosoma (Berl) 52, 297-316 (1975), Jorgenson et al., Chromosoma (Beri. ) 68, 287-3-2 (1978) and Jennings and Ridler; Biophysics of Structure and Mechanism, 10, 71 - 79 (1983).

1. Method detection identification and/or quantitative evaluation of material selected from the tissues of plants or animals, mikroorganismen all or part of the oligonucleotide sequence, characteristic of the RNA or DNA of this material, and immobilized on the surface of the waveguide detector damped oscillation;

(II) processing of immobilized oligonucleotide obtained in stage (I), a solution of the sample material, or DNA or RNA derived from this material, under conditions in which DNA or RNA having a characteristic oligonucleotide sequence will be hybridisierung with immobilized oligonucleotide;

(III) binding to immobilized oligonucleotide obtained in stage (I), or nukleinovokisly material obtained from the sample with a fluorescently detektivami agent before, during or after hybridization to DNA or RNA under the conditions specified in the stage (II), so that this fluorescently detected, the agent contacted the product of hybridization and was not associated with dehybridization immobilized oligonucleotide;

(IV) measuring the amount of a fluorescently detected agent linked as described in stage (III), using the detector, the attenuation of the oscillations and the establishment of the relationship between the measured quantity and presence, identity and/or quantity of the investigated material, characterized in that the content of the features is given in the presence of immobilized oligonucleotide stage (I).

2. The method according to p. 1, characterized in that the amplification of specific sequences is carried out in a solution in contact with the waveguide detector attenuation of vibrations.

3. The method according to p. 1 or 2, characterized in that the fluorescently detektivami agent is a fluorescently detected intercalating dye that is able to integrate into the duplex product of hybridization, and the specified media is not associated with dehybridization immobilized oligonucleotides.

4. The method according to p. 3, characterized in that the amplification of a specific sequence represents a reaction amplification using polymerase chain reaction (PCR) or ligase chain reaction (LCR).

5. The method according to PP.1, 3, or 4, wherein the fluorescently detektivami agent is a fluorescently detected oligonucleotide that is capable of hybridisierung hybridized with a characteristic sequence of stage (II).

6. The method according to p. 1, or 3 or 4 or 5, characterized in that reaction amplification of specific sequences as primers using fluorescently detected agent.

7. The method according to p. 6, characterized in that Prime time"ptx2">

8. The method according to p. 1 or 5, characterized in that exercise

(I) the receipt of the oligonucleotide, complementary to part of the oligonucleotide sequence, characteristic of RNA or DNA of the investigated material immobilized on the surface of the waveguide detector damped oscillation;

(II) the production of fluorescently detected of the oligonucleotide, which is complementary to all or part of the rest of the characteristic sequence;

(III) treatment of immobilized oligonucleotide obtained in stage (I), the solution containing the material, or DNA or RNA derived from this material, under conditions which are characterized by oligonucleotide sequence will hybridisierung with immobilized oligonucleotide;

(IV) replacing the solution of the material from stage (III) a solution containing fluorescently detected complementary oligonucleotide from stage (II); and

(V) measuring the amount of bound fluorescently detected of the oligonucleotide, using the detector, the attenuation of the oscillations and the establishment of the balance between this measured quantity and presence, identity and/or amount of target material in the sample.

9. The method according to ucleotide sequence, characteristic of the RNA or DNA of the studied material, and immobilized on the surface of the waveguide detector damped oscillation;

(II) the production of fluorescently detected of the oligonucleotide, which is identical to all or part of the rest of the characteristic sequence;

(III) providing a reaction amplification of a specific sequence at a sample of the material, where as one of the primers used fluorescently detected oligonucleotide stage (II), and the result of this reaction amplificates present any characteristic sequence into which a fluorescently detected agent;

(IV) processing immobilized on the waveguide complementary oligonucleotide stage (I) of the reaction mixture from stage (III) under conditions that may lead to hybridization of the amplified product with the specified immobilized oligonucleotide;

(V) assessment of fluorescently detected of the oligonucleotide that is hybridized to the immobilized oligonucleotide, using the detector, the attenuation of the oscillations and the establishment of the balance between this assessment and the amount of the amplified material, original PR is up in the form of a prism, plate or fiber.

11. The method according to any of paragraphs.1 to 10, characterized in that the waveguide is thermocontroller conditions.

12. The method according to PP.11, characterized in that thermocontroller conditions are achieved in the camera control.

13. The method according to any of paragraphs.1 - 12, characterized in that the fluorescently detected complementary oligonucleotides obtained by tagging the respective oligonucleotides fluorescent label or a group that can communicate fluorescent material or an enzyme or a catalyst capable of generating fluorescent material.

14. The method according to p. 13, wherein the complementary oligonucleotide been labelled by fluorescein.

15. The method according to p. 13, characterized in that the oligonucleotide target enzyme or a catalyst capable of generating fluorescent radiation, and before measuring fluorescence using the detector of the damped oscillation type substrate for this enzyme or catalyst.

16. The method according to PP.1 - 15, characterized in that the material is treated restrictively endonucleases to split its RNA or DNA.

17. The method according to the Lina from 5 to 30 bases.

18. The method according to p. 17, characterized in that the oligonucleotides have a length from 10 to 25 bases.

19. The method according to p. 18, characterized in that the oligonucleotides have a length of from 15 to 20 basis.

20. A test kit comprising an oligonucleotide probe complementary to part of the oligonucleotide sequence, characteristic of RNA or DNA detected, identified and/or quantified estimate of the material, characterized in that it further comprises a fluorescently detected agent, and the oligonucleotide immobilized on the surface of the detector, the attenuation of the vibrations on the optical waveguide.

21. Set on p. 20, characterized in that as fluorescently detected agent using intercalating dye.

22. Set on p. 20, characterized in that as fluorescently detected agent using an oligonucleotide probe complementary to all or part of the rest of the characteristic sequence.

23. Set according to any one of paragraphs. 20 to 22, characterized in that the oligonucleotide is not immobilized on the waveguide surface and further comprises reagents suitable for immobilization on the waveguide.

24. Optical waveguide, summability oligonucleotide, which is complementary to all or part of the oligonucleotide sequence, characterizing RNA or DNA sequence, which is the object of the study.

25. Biosensor containing the optical detector, the attenuation of the waves and the light guide, characterized in that the fiber is placed in a controlled environment, is able to adjust the stiffness of the hybridization and/or cycles of polymerase chain reaction.

26. Biosensor according to p. 22, characterized in that on the whole surface of the optical fiber immobilized oligonucleotide, which is complementary to all or part of the oligonucleotide sequence, characterizing RNA or DNA sequence, which is the object of the study.

 

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The invention relates to biotechnology, genetic engineering

The invention relates to molecular biology and may find application in medicine and molecular biology for the analysis of gene expression, diagnosis and detection mechanisms of pathology at the genetic level

Molecular probe // 2107730

The invention relates to molecular biology and recombinant DNA technology

The invention relates to a method of determining the presence or absence of one or more sequences of variant nucleotides by extracapillary or lack thereof, and kits for the determination

The invention relates to the provision of animal cells with biologically active substances, more particularly to a method of transferring nucleic acid into the cells of animals

The invention relates to Microbiology and molecular biology and concerns a method for the differentiation of different vaccine strains of Brucella, the causative agent of brucellosis in cattle and can be used in research and veterinary laboratories

FIELD: medicine, psychiatry.

SUBSTANCE: one should isolate DNA out of lymphocytes of peripheral venous blood, then due to the method of polymerase chain reaction of DNA synthesis one should amplify the fragments of hSERT locus of serotonin carrier gene and at detecting genotype 12/10 one should predict the risk for the development of hallucino-delirious forms of psychoses of cerebro-atherosclerotic genesis.

EFFECT: more objective prediction of disease development.

3 ex

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