Device and method for extracting nucleic acids

FIELD: molecular biology.

SUBSTANCE: the suggested innovation deals with the fact that nucleic acids should be isolated directly out of the sample without pipetting stage but with the help of interconnected reservoirs being prepared beforehand. The above-mentioned vessels should be applied either separately or being interconnected according to standard microtitrating format. The sample should be mixed with a lyzing buffer and nucleic acids are bound with matrix in closed system including, at least, two interconnected reservoirs. Forced movement of sample's mixture and buffer back and forth from one reservoir into another one for several times through narrow passage provides their thorough intermixing. The method provides quick and safe isolation of nucleic acids.

EFFECT: higher efficiency.

44 cl, 4 dwg, 1 ex

 

The scope of the invention

The present invention relates to so-called dry method work with samples and reagents, and in particular, to the field of sample preparation, quantitative and qualitative extraction, purification and amplification of nucleic acids (DNA or RNA, including various types of nucleic acids) of organic samples, such as blood, serum, urine, cell suspension, samples amplificatoare in vitro and samples of biopsies.

The level of technology

There are many different protocols for the isolation and purification of nucleic acids. Most methods aim at the selection of high-purity samples that are suitable for use in amplification by PCR (polymerase chain makes reactions).

One of the currently used protocols in which a Split Second™ DNA Preparation Kit (Boehringer Mannheim GmbH), includes the following stages. First tube to microcentrifuge poured into the first buffer solution. The sample, such as whole blood person, add in a test tube. The tubes are placed on a rocker for 10 min and then centrifuged 5 min at 2500 rpm on microcentrifuge. Then select and discard the supernatant, and the precipitate resuspended in buffer solution. Resuspending precipitate is then centrifuged 3 min at 2500 rpm After the second center is fugiranje select and discard supernatant. Sediment resuspended in the second buffer and the mixture is thoroughly shaken (on the installation of the Vortex). After incubation for 5 min on a water bath at 65°S, sample can be used directly for PCR.

Another Protocol is to purify the DNA using hottopicsnow (divisive) agent and particles of silicon dioxide, as described by Dr. J. Kleiber (preparation of DNA templates, Boehringer Mannheim GmbH, FRG). Suspension of silica added to lyse buffer in the tube microcentrifuge, and shaken. Then to lyse buffer containing particles of silicon dioxide, add blood treated with EDTA (ethylenediaminetetraacetic Crotoy), and the mixture is shaken. After 10 min of incubation, during which the tube should be regularly turn to prevent settling of the particles of silicon dioxide, the mixture is centrifuged. After removal of the supernatant, the precipitate of silicon dioxide with nucleic acids is subjected to a series of washes: a first buffer for washing (guanidinylation, Tris/HCl), then ethanol (70%) and finally acetone. Each stage leaching requires mixing by shaking, centrifugation and removal of supernatant. After the last washing the precipitate of oxide of silicon with a nucleic acid is dried by heating to 56°C for 10 minutes. Finally, the mixture is centrifuged 2 min at 10,000×g. After that the supernatant from the holding purified nucleic acid and can be used for PCR.

It is not difficult to notice that multiple stages of currently used protocols are time consuming and require constant attention. Extracted volumes required to pipette in a large number of tubes to microcentrifuge, and these tubes are subjected to various influences, such as centrifugation, mixing by shaking and incubation.

Working with samples taken from the human body, regardless of whether they are biopsy samples, blood or serum samples of other body fluids, such as urine or saliva, cell suspensions or in vitro amplificatoare samples, includes many practical difficulties and considerations. Laboratory personnel should not only be protected from pathogenic agents that may be contained in the samples, the samples must be protected from contamination, as coming from the other samples, and the staff working with them.

A large number of stages, usually used for the purification of nucleic acids, increases the risk of transferring nucleic acids from a sample in the sample. When deciding on the basis of the results extremely sensitive polymerase chain reaction (PCR) or other amplification systems, the smallest contamination can lead to false positive results is the ATA.

Work with human blood involves a specific problem. Because you cannot use heparinised blood (heparin inhibits PCR), the samples have a tendency to collapse in containers where they are in the tips of the pipettes and test tubes for microcentrifuge. Next, the blood has a tendency to stick to surfaces and dry with the formation of tiny flakes that are easily transported by air. And finally, we should not neglect the psychological discomfort or stress when working with potentially infectious samples of human blood.

The present invention aims at improving, for example, simplifying purification of nucleic acids from organic samples in General, and in particular, sample preparation of nucleic acids from whole (unprocessed) human blood.

In particular, nucleic acids presents in the form of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA, in turn, is divided into plasmid DNA and genomic DNA. RNA is usually divided into the messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA). These groups are often referred to as "nucleic acids". It would be desirable to create an easy and reliable way qualitative extraction of nucleic acids, i.e. distinguish between different types of kleinova acids.

In many chemical or pharmaceutical applications, the reagent or pharmaceutical agent is stored and delivered to the user in dried form and must be reconstituted by mixing it with a suitable amount of an appropriate solvent. The solvent is often water or an aqueous solution such as physiological saline. To use this technique mainly in order to increase the stability and the shelf life of the reagent or pharmaceutical agent. In some cases, the reaction between the components contained in the reagents become impossible or significantly slow down in dried condition and are initiated when the material is restored.

When you restore dried substance must be ensured thorough mixing. Gradients of concentration, lack of mixing and possibly the remaining solid particles can lead to errors and the risk of continued use of the recovered mixture. You must also take into account the risk of contamination of dried substances, especially when the recovery is made by the open method.

Examples of applications in which contaminating material can completely change the result include analytical and biochemical applications the Oia, in which the genetic information from the sample is subjected to amplification. Important reactions of this type include polymerase chain reaction (PCR), ligase chain reaction (LCR), LCR interval (gapped-LCR-reaction), amplification of nucleic acids based on their sequence (first NASBA), self-sustained replication (SSR), amplification, mediawindow transcription (TMA), amplification preemptive threads (SDA), and amplification of the target, the signal amplification, Hybrid CaptureRreaction, or any combination of the reactions described above. The present invention is to minimize or fully eliminate the possibility of contamination during preparation of the sample for the above and other applications where contamination is causing the risk of error.

Another objective of the present invention is a method and device for the recovery of lyophilised substances, for example, reagents for biochemical analysis or pharmaceutical agents that could provide a higher degree of security, reliability and ease of use than currently available systems.

The closest known analogs

In the patent US 5330916 described method of extraction of cell components and the vessel, which is suitable for use in this way. The so-called bilateral vessel for extractively two branches, separated from each other by a filter, and a movable proterty the piston, which is located in one of the cells. When the movement of the piston the sample is subjected to mechanical forces that cells in the sample are broken and their contents flow into the liquid phase. Specified liquid phase passes through the filter into the second compartment, while the organic components and the remains of the cells remain in the first division.

In the patent US 5786182 described double-chamber vessel disposable for amplification reactions, in which the first chamber contains a mixture of reagents for amplification, and the second chamber contains an enzyme for amplification. These two chambers are connected by a channel for passage of fluid, which can be opened or through which you can move the sample in the mechanical application of vacuum and the like, the invention consists in the fact that we share the reagents with different resistance to heat, i.e. heat sensitive enzymatic reagent and stable when heated reagent for amplification, and predusmatrivayut the ability to connect them together in a selected point in time.

The vessel according to patent US 5786182 designed for amplification reactions and does not allow alternate filling of the chambers that comprise this two-chamber vessel, as it is not suitable for the extraction of nucleic acids, i.e. there are no tools for thorough mixing of sample and reagent.

Method and vessel according to US 5330916 represents the closest existing similar in regard to the extraction of nucleic acids. However, it is not possible to make changes in the procedures for extraction, replacement buffers, sequential extraction, etc. with a sufficient degree of ease and reliability.

Summary of invention

The present invention solves the above problem by means of the devices and methods according to the attached claims.

According to the present invention, an apparatus for the extraction of nucleic acids, in which said system includes a first vessel (3) for placing the sample and at least one second vessel (4)containing at least one reagent (2), these vessels are connected through a narrow channel (7, 8) with removable, and means (5, 6) for displacement of the sample from the first vessel into the second and Vice versa.

The present invention also relates to a method for extracting nucleic acids from a sample, according to which the specified pattern is placed in a first vessel, which is connected with the possibility of disconnecting with the second vessel, with the specified second vessel contains a pre-prepared reage is t, then the sample is pumped into the second vessel and back again, repeating this until sufficient stirring, after which the vessel containing the mixture is combined with the third vessel containing a pre-prepared reagent, and mixing again.

Further examples of the invention and their advantages will become apparent or can be easily derived from the description and examples, including the accompanying drawings.

Brief description of drawings

The present invention will be disclosed in more detail in the description and examples given below, with reference to relevant drawings, in which:

figure 1 shows the cross-section of two vessels, a and b, according to one example implementation of the invention;

figure 2 shows two vessels according to figure 1, connected to each other according to the invention;

figure 3 shows a vessel with a cap for separating matrix, carrier nucleic acid; and

figure 4 shows a design with figure 3, is placed in a centrifuge tube and the tube microcentrifuge (C), respectively, for example, for emptying using centrifuges.

Description of the invention

The method of purification, extraction and enzymatic processing of nucleic acids according to the present invention makes it possible to work with a sample of “dry” and the necessary buffers, because studieperiode becomes unnecessary.

The sample, for example, blood volume, serum, urine, saliva, cell suspensions, for example, from a biopsy sample, or sample amplificatory in vitro, is placed in the first space. This is the first space may be the space contained in the test tube, VacutainerR, syringe or preferably the first space, which is contained in the device according to the invention, described below. The sample is then brought into contact with lytic buffer, which is also preferably contains a matrix, capable of binding nucleic acids. Suitable lytic buffer is a salt solution, also containing a detergent commonly used agents, such as Tris (Boehringer Mannheim GmbH) and EDTA (Merck). Suitable matrix are particles of glass or silicon oxide, diatomaceous earth, glass fiber, nylon fiber suspension pulp, paramagnetic beads, latex beads, etc. Matrix may also have a coating, for example, it may be coated with streptavidin or any other material for separating the strands of nucleic acids or threads of only one nucleic acid.

This first buffer, preferably containing solid matrix contained in the second space having a narrow channel. When injected into the second space sample forms a mixture with lytic buffer and matrix. This mixture is then forced through the above channel, and she enters the space, which may be the first space, if the sample was initially placed in the device according to the present invention. When passing through a narrow channel or opening between the two spaces or vessels, the mixture is efficiently stirred. The mixture is then forced back into the second space and the procedure is repeated a number of times, ensuring thorough mixing and lysis of the cells in the sample. After a sufficient number of movements through the channel mixture becomes almost homogeneous. During this mixing the nucleic acid bound to the matrix contained in lytic buffer. When the stirring stops, the mixture is either in the first or in the second space.

Matrix with associated nucleic acids are then separated from the bulk of solution. In the case of using the matrix, consisting of particles, such a separation may be performed if to close the space, which contains a matrix, a membrane or a separator cap, as described below, and centrifuged device. The matrix will remain on the membrane or in the separation cap.

When using a matrix made of a paramagnetic particle separation reached by the application of a magnetic field around the vessel or to the rolling element or Rosny, used for displacement of the contents of vessels from one to another.

The mixture soteriou lyse buffer and the matrix, then mixed with buffer reagents contained in the third space or receptacle attached to the second vessel or space. Passing the mixture back and forth between these two spaces through a narrow channel, once again ensure thorough mixing and flushing. When mixing is complete, the mixture is or in the second or in the third space, and everything else is discarded. Finally, the purified nucleic acid can be removed from the matrix using a buffer for elution or, in the case of paramagnetic matrix, the off paramagnetic fields.

By using different matrices can be extracted different types of nucleic acids. This is implemented by the selection of the matrix material, the matrix structure, packaging and other

structural and physical properties. This process also depends on the selection of appropriate chemical properties and/or chemical or biological pretreatment, such as processing to improve affine properties, the coating matrix antibodies, affinity groups, streptavidin, Biotin. Specific nucleic acid sequences can be extracted with the use is of a complementary nucleic acid, hybridizing on the matrix. Thus, for example, it is possible to carry out the extraction of specific viral nucleic acids, as part of the preparation of the sample to a diagnostic test.

The buffer or buffers for extraction can be chosen or adapted for the extraction of certain types of nucleic acids. According to one example implementation of the invention, the sample is passed back and forth between two vessels, one of which contains a matrix, specially adapted or conducive to the adsorption of one species of nucleic acids, while the other vessel contains a matrix in a special way adapted or conducive to the adsorption of other species of nucleic acid and buffer solution, suitable for both matrices. Thus, the extracted nucleic acids are physically separated, which improves the kinetics of the reaction and leads to better results, both qualitatively and quantitatively.

According to an alternative way of implementing this method, the matrix can be added after carrying out lysis.

The device according to the present invention contains at least two vessels, which have moving areas of the walls or elements to displace the contents of each of the vessels, and connective elements that are appropriate to the relevant part, Melsheimer, one of the other vessel and preferably other vessels.

According to a preferred method of implementation, one of the vessels contains a solid matrix, rigidly fixed in him or have physical barriers to the abandonment of the vessel. The matrix can serve as the insertion of granular or fibrous material, such as glass particles or silicon dioxide, glass fiber, nylon fiber, cellulose or diatomaceous earth, which can be subjected to sintering or compression another way, in order to give it the shape of the insert and thus to eliminate the possibility of entrainment her out of the vessel in the direction of fluid flow during emptying of the vessel. The matrix can be a specific image selected, adapted, or modified to reflect the physical/structural and/or chemical/ biological properties, as described above. Alternatively, when using paramagnetic particles, the movement of the particles of the matrix can be adjusted by the application to the vessel's magnetic field.

The matrix may also be a coating on the inner surface of the walls of one of the vessels, alternatively, the wall of one of the vessels may be made from glass, acrylic, polystyrene, or other material able to reversibly bind, for example, nucleic acids under certain conditions. At last lyrics by the tea is preferred creating a fuzz or an increase in the active surface of the walls otherwise.

According to one example implementation of this invention features the combination of the vessels are determined by the thread, the shape or size of their tips and holes. The first vessel may, for example, be provided with both external and internal thread. After mixing in the first vessel over the second throw vessel and the first vessel is connected with the third vessel by means of its external thread.

In addition, using color codes, tactile labels and the like, it is possible to determine the order of use of vessels. Imposing physical constraints (different thread, different sizes sedinami parts, etc) on the connectivity of the vessels, it is possible to minimize the possibility of error.

The device according to the present invention is preferably made of a suitable thermoplastic. Examples of such materials include, but are not limited to, polypropylene (PP), polystyrene (PS), polyethylene (PE), high density polyethylene (HDPE), polycarbonate (PC), polyacetate (PA), polymethyl methacrylate (PMMA) and poly (vinylidene fluoride) (PVF). The choice of material is determined not only chemical and temperature requirements for reagents and buffers that will bust processing, or to reactions that will have the device, but also by considerations of economy is AI, such as material costs, production technology, etc.. One of the suitable methods of production is pressing under pressure. Vacuum press molding is another possible method of production. The device according to the invention, of course, is done under conditions which exclude the possible presence of contaminants, which may affect the course of the reaction or reactions, for use in which it is intended.

The device shown in figure 1 in the form of two vessels 3 and 4. For illustrative purposes, the vessels depicted in the form of products, just like a normal syringe, having a main body, a movable piston 5 and 6, and the outlet or tip 7 and 8. Around the vents placed annular ledge 9 or 10. Since the annular protrusion on the vessel 3 has an internal thread 11 and the annular ledge on the vessel 4 has a corresponding external thread, they can be firmly connected with each other, providing a tight connection holes 7 and 8. Annular projections and the thread can also be used to attach the cap to the vessel to protect the integrity of their contents, ensuring the sterility of their inner surfaces, etc.

To illustrate, the volume of liquid contained in the vessel 3 and is indicated by broken lines, can be viewed as a sample, for example, education is the EC whole blood. The liquid in the vessel 4 in this case is a lytic buffer also preferably contains a matrix.

However, in order to control the movement of the pistons, the vessels made with the first annular constriction 18 in the middle end of the vessel and the second annular constriction 19 on the remote end of the vessel. The first protrusion 18 will have a significant resistance and thus will indicate to the user that the piston is near its lowermost position. However, the piston can push through the first protrusion 18, for example when emptying the vessel. The second shoulder 19 will prevent the piston from retracting out of the vessel, for example by mistake or as a result of too high pressure generated in the vessel.

Figure 2 two vessels 3 and 4 is shown connected through a threaded annular projections described above. The connection opens a path for fluid flow between the spaces of two vessels passing through the constriction or "neck"formed the outlets of the vessels. Because the connection is tight, both vessel limit one total. When lowering of the piston 5, the sample contained in the vessel 3, is forced through a narrow channel in the vessel 4, which contains lytic buffer and the matrix 2. Because the connection is tight, the piston 6 moves back due to the pressure exerted by the piston 5. When most of the clicks is SCA emptied into the vessel 4, push the piston 6, preceding sample plus buffer in the vessel 3. Thus, the content is pumped back and forth with the formation of the mixture. When using najprimitivniji blood that is necessary for the purposes of PCR, it was shown that such mixing prevents the clotting of blood. After mixing, the mixture is left in one of the vessels, or 3, or 4.

According to one example implementation, the vessel 4 contains a matrix, is designed to prevent the ash from the vessel, for example, the insertion of the optical fiber or sintered particles of silicon dioxide, etc. while it is preferable emptying contents 4 after thorough mixing in the vessel 3, or other equivalent vessel so that the vessel remained only the matrix with associated nucleic acids. The vessel 3 is then thrown together with all its content.

Then the vessel 4 attach the third vessel (not shown), which contains a buffer for washing, and the pumping procedure is repeated. When flushing is completed, the vessel 4 free from everything but the matrix with associated nucleic acids, and a third vessel containing the used wash buffer is discarded.

Nucleic acids can then be eluted from the matrix, for example, filling the vessel 4 buffer for elution, who prefer the Ino contained in the fourth vessel, according to this invention. Alternatively, the vessel containing the buffer for elution, attached to the vessel 4 and the elution is carried out by pumping the contents back and forth between the two vessels.

If you intend to use the extracted nucleic acid as a template for amplification process such as PCR, to the vessel 4 can be attached to the fourth vessel, which contains lyophilized reagents for amplification. The contents of the vessel 4, including elyuirovaniya DNA, then push in the fourth vessel and is pumped back and forth, in order to achieve effective dissolution and homogenization of the reactants. In this way the implementation prepare complete, the reaction mixture comprising the matrix amplification, and transfer it directly into the vessel for PCR reactions. This provides many advantages, such as easier handling and reduced risk of introducing contamination, etc.

The vessels can also be emptied by centrifugation. Figure 3 shows the method of implementation in which the vessel 3 contains a mixture of buffer and matrix. At this stage of the nucleic acid bound to the matrix, while adding a buffer for elution. Separating the cap 13 with the output of the pores 14, connected to the outlet 7 of the receptacle 3. The cap 13 may be attached to the thread is olcavage ledge 9 (not shown). When centrifugation buffer for elution leaves the vessel through the pores 14, the matrix remains in the separation cap. Erwerbende nucleic acid can then be used for amplification, such as PCR.

Figure 4 illustrates two ways, one when the vessel is equipped with a dividing cap 13 is placed in a centrifuge tube 17, and the other, when the vessel is used for emptying its contents into the tube microcentrifuge 16. During centrifugation, the matrix remains in the vessel or delayed separation of the cap, while the buffer and elution of nucleic acids pass through the weekend pores 14 and moved into the tube 16 or 17. The tubes 16 and 17 can then be used in stages, for example, required for the PCR Protocol.

The first vessel, which is the container for the sample, can also be the primary receiver of the sample. Assumes that the first vessel fit for selection of the sample. According to one method of implementing the first vessel adapted for holding hypodermal needle. In this way the blood sample can be selected directly in the first vessel. In another method of implementation of the first vessel fit for selection of the strips or gel colony with Cup agar. Then the vessel was equipped with a nozzle for puncturing strips or nozzle cleared for the I colony cups with agar. Pumping the contents of the vessels is particularly suitable for the extraction of nucleic acids from a gel or clone grown on the agar plate was assessed.

According to another method of implementing the apparatus and method according to the invention can be adapted to a parallel format. In laboratories where work with a large number of samples, method of implementation, which includes the use of a variety of vessels, such as 96, 384 or more vessels of a type appropriate to microcentrifuge or tablets for micrometrology may be in the form of so-called standard format with microtitre or other parallel formats. This set, for example, 8×12 vessels, preferably can be molded as one piece by molding under pressure. The corresponding pistons also gather in one unit with the formation of rows and columns in the same format, and will also be combined into a single unit by molding under pressure. The above-described pumping can be performed manually. The preferred use of a mechanical device. They may be mechanical or pneumatic device, comprising a set of pistons. In this way the implementation of the outlet openings 7 and 8 and annular projections 9, 10 with the thread, should be modified in a way that enables a tight and secure is connected to the e without twisting. Torque movement is difficult to implement when the vessels are collected in the form of a lattice, as in this implementation. In other words, the containers must be tightly connected by vymanivaniya vessels and pressing them to each other. This can be done using tightly incoming into each other connections (fittings, flanges, locking mechanisms, etc. by Changing the shape of annular projections 9 and 10, it is possible to get the machine type "push-close" and "pull-open".

Example

In this example, we used a set of mutually connected syringes. Tested various matrix particles of silicon oxide, glass and nylon fibers. Used lyse buffer consisting of a salt solution and detergent. Buffer for washing consisted of soy solution and ethanol.

Sample najprimitivniji whole human blood was collected in the first syringe or directly from the patient using hypodermal needle attached to the syringe, or from the intermediate container, VacutainerRcontaining sample from the patient.

Prepare a second syringe containing particles of silicon dioxide, suspended in lytic buffer. Then the syringe containing the blood sample, combined with a second syringe, and the sample of blood was released in lytic buffer. Mixed contents of both syringes were then pumped in there and education is but between the two syringes. Every move through the narrow interception between the United syringes contributed to the mixing of the sample with a lytic buffer and ensure thorough mixing of the particles of silicon oxide in solution. Such pumping continued for several minutes and ended when the sample and lyse buffer have been completely in one of the syringes. Another syringe was disconnected and threw.

When using saline with detergent for cell lysis, the sample najprimitivniji blood was found that the processing method according to the invention effectively prevents collapsing of blood.

The syringe containing the sample, a silicon oxide and lyse buffer, was added a third syringe containing buffer for washing. Then the buffer wash was pumped back and forth between the second and third syringes. After mixing the combined solution was left in the second syringe.

When using granular matrix, freely moving in the contents of the vessel, the matrix was separated from the solution by attaching the separating cap to the tip of the syringe, the premises of the latest in a centrifuge tube and centrifugation (as shown schematically in the accompanying Figure 4). The matrix remained in the separation of the cap, while the solution was removed. You spend far the Shui washing or eluted nucleic acid from the precipitate, in separating the cap.

If the matrix is the tab of glass fiber - or, as in the second example, carried out by the inventor, nylon thread some length - no need to use any separation of the cap. Instead, the matrix was washed and was suirable in situ, by attaching different syringes containing the necessary buffers to the syringe containing the matrix. Finally, the matrix was suirable nucleic acid. The result of purification was confirmed by exposing suirvey sample gel electrophoresis. A distinct single band showed that the purification of nucleic acids was successful.

Although this invention is described in relation to preferred implementations that represent the best currently known to the inventor ways, it should be understood that within the scope of the invention, which is defined in the accompanying claims, various changes and modifications are obvious to a person skilled in this field.

1. Device for the extraction of nucleic acids from organic samples, characterized in that the said device comprises a first vessel for accommodating the sample, the first piston for extruding the sample from the first vessel and at least one second vessel containing ENISA least one pre-prepared reagent and having a second piston for extruding the specified reagent from the second vessel, the first and the at least one second vessel is connected with the possibility of breaking through the narrow channel, the first and second vessels restrict a single closed volume.

2. The device according to claim 1, characterized in that the specified pre-prepared reagent specified in the second vessel is a lyophilized reagent.

3. The device according to claim 1, characterized in that the said second vessel further comprises a matrix capable of binding nucleic acids.

4. The device according to claim 1, characterized in that the specified pre-prepared reagent in the second vessel is a lytic buffer, with the specified second vessel further comprises a matrix capable of binding nucleic acids.

5. Device according to any one of claim 3 or 4, characterized in that the above matrix is a granular matrix, able to move freely in the vessel.

6. Device according to any one of claim 3 or 4, characterized in that the matrix consists of primary particles or fibers, which form a secondary shape that does not allow them to leave the vessel.

7. The device according to claim 5, characterized in that the said matrix is selected from particles of glass or silicon oxide, diatomaceous earth, glass fibers, nylon fibers, the pulp suspension, W is recov paramagnetic and latex beads.

8. The device according to claim 3, characterized in that the above matrix is processed, increasing its specific affinity for nucleic acids.

9. The device according to claim 3, characterized in that the above matrix is retained in the vessel by a magnetic field acting on the magnetic components of the matrix.

10. The device according to claim 3, characterized in that the above matrix represents the insertion of the glass fibers.

11. The device according to claim 8, characterized in that the said matrix is coated with streptavidin.

12. The device according to claim 8, characterized in that the specified matrix bears hybridisable nucleic acids.

13. The device according to claim 1, wherein the first vessel includes coupling means for coupling with corresponding means on the second vessel or other vessels.

14. The device according to item 13, characterized in that the said connecting means through its size and/or function to determine the order of joining of the second and subsequent vessels.

15. The device according to claim 1, characterized in that the vessels and subsequent vessels, which can be for them to join, have physical properties, such as tactile labels, color codes, and the like, determining the sequence of their use.

16. The device according to claim 3, characterized in that it contains the CTD is audica separating the cap, which holds the solid matrix.

17. Device according to any one of claims 1 to 16, characterized in that the vessels are connected with the formation of the lattice, corresponding to the standard format microtitre.

18. Device for separating matrix linking the nucleic acid from the liquid, when the specified device contains one end for connection to a device according to claim 1 and a closed end provided with at least one sometimes, characterized in that the at least one time or each time is above the lower inner surface of the specified device, thereby determining the amount, not being emptied by centrifugation.

19. The device according to 17, characterized in that it is firmly attached to one end of the device according to claim 1 and placed in a centrifuge tube.

20. Device according to any one of p and 19, characterized in that the vessels are connected with the formation of the lattice, corresponding to the standard format microtitre.

21. Extraction of nucleic acids from organic sample, comprising placing the sample in a vessel, the impact on the sample to mechanical forces and the movement of the sample into another container, characterized in that the method is carried out using a device according to any one of claims 1 to 16.

22. The method according to item 21, wherein the specified pattern is about Ascom blood, serum, urine, saliva, cell suspension, or a biopsy sample.

23. The method according to item 21, wherein the specified pattern is a sample of whole human blood.

24. Extraction of nucleic acids from organic sample, comprising placing the sample in a vessel, the impact on the sample to mechanical forces and the movement of the sample into another container, characterized in that it uses the device according to any one of p and 18.

25. The method according to paragraph 24, characterized in that said sample is a blood sample, serum, urine, saliva, cell suspension, or a biopsy sample.

26. The method according to paragraph 24, wherein the specified pattern is a sample of whole human blood.

27. Extraction of nucleic acids from a sample, amplified in vitro, comprising placing the sample in a vessel, the impact on the sample to mechanical forces and the movement of the sample into another container, characterized in that it uses the device according to any one of claims 1 to 16.

28. The method according to item 27, characterized in that said sample is a blood sample, serum, urine, saliva, cell suspension, or a biopsy sample.

29. The method according to item 27, wherein the specified pattern is a sample of whole human blood.

30. Extraction of nucleic acids from a sample, amplified in vitro, including the size of the giving of the sample in the vessel, the impact on the sample to mechanical forces and the movement of the sample into another container, characterized in that it uses the device according to any one of p and 19.

31. The method according to item 30, wherein the specified sample is a blood sample, serum, urine, saliva, cell suspension, or a biopsy sample.

32. The method according to item 30, wherein the specified pattern is a sample of whole human blood.

33. Method of recovery of lyophilized reagent comprising placing the sample in a vessel, the impact on the sample to mechanical forces and the movement of the sample into another container, characterized in that it uses the device according to claim 1.

34. The recovery method reagent for one of the following reactions: polymerase chain reaction (PCR), ligase chain reaction (LCR), LCR interval (gapped-LCR-reaction), nucleic acid amplification based on their sequence (first NASBA), self-sustained replication (SSR), amplification, mediawindow transcription (TMA), amplification preemptive threads (SDA), and amplification of the target, the amplification of the signal, Hybrid CaptureRreaction, or any combination of these reactions, characterized in that it uses the device according to claim 1.

35. The method of extracting nucleic acids from a sample, wherein the specified pattern is in the first vessel with the first on the Shen, the first vessel is connected with the possibility of separating from the second vessel having a second piston, with the specified second vessel contains a pre-prepared reagent, and the first and second vessels together limit one volume, after which the sample is pumped into the second vessel and back, and this operation is repeated until then, will not occur until after sufficient stirring, after which the vessel containing the mixture is combined with the third vessel containing a pre-prepared reagent and mixing again.

36. The method according to p, characterized in that the said second vessel contains a lytic buffer and matrix capable of binding nucleic acids.

37. The method according to p, characterized in that the said third vessel contains a buffer for washing.

38. The method according to any of PP-37, characterized in that the purified sample is removed from the vessel by means of centrifugation.

39. The method according to any of PP-37, characterized in that the matrix is separated from the liquid in which it is suspended, using centrifugation using devices p.

40. The method according to any of PP-37, characterized in that the matrix is separated from the liquid in which it is suspended, using a magnetic field.

41. The method according to any of PP-37, characterized in that the sample is one of the following: arr is Scam blood, serum, urine, saliva, cell suspension and a biopsy sample.

42. The method according to any of PP-37, characterized in that the sample is a sample of whole human blood.

43. The method of extracting specific types of nucleic acids, comprising placing the sample in a vessel, the impact on the sample to mechanical forces and the movement of the sample into another container, characterized in that it uses the device of claim 1, when the specified device contains a matrix that is processed in such a way as to facilitate the linking of these types of nucleic acids with the specified matrix.

44. The method of extracting specific types of nucleic acids, comprising placing the sample in a vessel, the impact on the sample to mechanical forces and the movement of the sample into another container, characterized in that it uses the device of claim 1, with said device includes a matrix containing the complementary sequence of nucleic acid, which it hybridized.



 

Same patents:

FIELD: medicine, urology.

SUBSTANCE: one should conduct subcutaneous prevocational tuberculin test and, additionally, both before the test and 48 h later it is necessary to perform the mapping of prostatic vessels and at decreased values of hemodynamics one should diagnose tuberculosis. The information obtained should be documented due to printing dopplerograms.

EFFECT: more reliable and objective information.

1 ex, 1 tbl

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

The invention relates to medicine, namely to laboratory methods of skin diseases and can be used to diagnose common progressive psoriasis
The invention relates to the field of medicine, but can also be used in veterinary medicine to determine the level of nonspecific resistance of the organism (UNRO) both human and animal

The invention relates to medicine and relates to damage mitochondrial DNA as a prognostic sign of atherosclerotic coronary heart disease

The invention relates to medicine, namely, neurology, and can be used for the diagnosis of multiple sclerosis

The invention relates to medicine, in particular for the diagnosis of infectious diseases

The invention relates to medicine, namely to predict the severity and duration of the current shock in adults and children

FIELD: biotechnology, veterinary science.

SUBSTANCE: invention relates to therapeutic vector used in therapy of infectious diseases in cats that comprises at least one foreign nucleic acid each of that (a) encodes protein taken among the group consisting of feline protein CD28 represented in SEQ ID NO:8 or its immunogenic moiety; feline protein CD80 represented in SEQ ID NO:2 or 3, or its immunogenic moiety; feline protein CD86 represented in SEQ ID NO:6 or its immunogenic moiety, or feline protein CTLA-4 represented in SEQ ID NO:10 or its immunogenic moiety; and (b) nucleic acid that is able to be expressed in insertion of vector in the corresponding host. Indicated therapeutic vector is used in effective dose as component of vaccine against infectious diseases in cats for their immunization and in methods for enhancement or inhibition of immune response in cats and reducing or eradication of tumor in cats. Invention provides stimulating the activation and proliferation of T cells and to enhance effectiveness of control of infectious diseases in cats.

EFFECT: valuable biological properties of recombinant virus.

41 cl, 13 dwg

The invention relates to genetic engineering, biotechnology, medicine, pharmacology

The invention relates to biotechnology, in particular to the immunoglobulin E (IgE), IgE antagonists, anti-IgE antibodies capable of binding to human IgE, and to a method of improving polypeptides, including anti-IgE antibodies

FIELD: biotechnology, veterinary science.

SUBSTANCE: invention relates to therapeutic vector used in therapy of infectious diseases in cats that comprises at least one foreign nucleic acid each of that (a) encodes protein taken among the group consisting of feline protein CD28 represented in SEQ ID NO:8 or its immunogenic moiety; feline protein CD80 represented in SEQ ID NO:2 or 3, or its immunogenic moiety; feline protein CD86 represented in SEQ ID NO:6 or its immunogenic moiety, or feline protein CTLA-4 represented in SEQ ID NO:10 or its immunogenic moiety; and (b) nucleic acid that is able to be expressed in insertion of vector in the corresponding host. Indicated therapeutic vector is used in effective dose as component of vaccine against infectious diseases in cats for their immunization and in methods for enhancement or inhibition of immune response in cats and reducing or eradication of tumor in cats. Invention provides stimulating the activation and proliferation of T cells and to enhance effectiveness of control of infectious diseases in cats.

EFFECT: valuable biological properties of recombinant virus.

41 cl, 13 dwg

Up!