JP2004057055A - Method for preventing contamination of nucleic acid, embedded primer used in the method and kit with the primer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for preventing the occurrence of a contamination with an impure nucleic acid in a nucleic acid-elongating reaction. <P>SOLUTION: This method for preventing the contamination of the nucleic acid is characterized by having the steps of (1) making a nuclease and an embedded primer comprising an embedding agent and a primer embedded in the embedding agent to exist in a reaction system for carrying out an elongation reaction, (2) maintaining the reaction system described in (1) under conditions for suitably degrading the nucleic acid with the nuclease, (3) enhancing the temperature of the reaction system to inactivate the nuclease, after the maintenance of (2), and maintaining the system under conditions for allowing the primer to flow from the embedded primer, and (4) maintaining the reaction system described in (3) under conditions for giving the suitable elongation reaction, after the maintenance of (3), in the method for using a template nucleic acid and the primer in the presence of a polymerase to elongate the nucleic acid. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a contamination prevention method, an embedding reagent used in the method, and a kit including the same.
[0002]
[Prior art]
In a genetic test method serving as a basis for a definitive diagnosis, in particular, in a nucleic acid amplification test (eg, polymerase chain reaction; hereinafter, referred to as a PCR method), it is important to control measurement accuracy and to guarantee reliability. When using the nucleic acid amplification method, the most important point is contamination. Even if only one molecule of the genome is mixed, it is amplified several hundred thousand times after the reaction, which leads to false diagnosis such as false positive. As a conventional pollution prevention measure, a method using uracil-N-glycosidase (UNG) is known. In this method, an amplification reaction is performed using dUTP instead of dTTP, and after the reaction, decomposition is performed using UNG. However, the UNG method can control the contamination of the amplification product, but cannot deal with the contamination of a small amount of the non-amplification product.
[0003]
Therefore, at present, there is a demand for a pollution control method capable of dealing with all types of pollution.
[0004]
[Problems to be solved by the invention]
In view of the above situation, an object of the present invention is to provide a means for preventing the occurrence of contamination by nucleic acid contaminants in a nucleic acid extension reaction, particularly an amplification reaction.
[0005]
[Means for Solving the Problems]
The present invention for achieving the above object,
In a method of extending a target nucleic acid by a polymerase using a template nucleic acid and a primer,
(1) Presence of a nuclease and an embedding agent and an embedding primer having a primer contained therein in a reaction system for performing the extension reaction;
(2) maintaining the reaction system according to (1) above under conditions where the nuclease appropriately degrades nucleic acids;
(3) maintaining the conditions under which the nuclease is inactivated by raising the temperature of the reaction system after the maintenance of the above (2) and the primer is allowed to flow out of the embedding primer;
(4) maintaining the reaction system according to (3) under the condition that an appropriate elongation reaction is obtained after maintaining the above (3);
A method for preventing nucleic acid contamination, comprising:
An embedding primer comprising: an embedding agent which is fluidized by heat; and a primer held inside the embedding agent; and a container; and the above-described embedding accommodated in the container. A nucleic acid extension reaction kit comprising at least a primer.
[0006]
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention relates to a reagent for preventing contamination in a nucleic acid test and a method for controlling the accuracy of a nucleic acid test.
[0007]
According to the present invention, there is provided a method for preventing the occurrence of contamination by nucleic acid contaminants in a nucleic acid extension reaction by using an embedding primer and a nuclease.
[0008]
1. Explanation of terms The term "extend a nucleic acid" as used herein means that a nucleic acid of interest is formed by allowing a polymerase to act using a template nucleic acid and a primer complementary to a part of its base sequence. Extension refers to both such extension and amplification in which such extension is performed repeatedly.
[0009]
As used herein, the term "embedding primer" refers to a primer provided (ie, encapsulated) within an embedding agent. As used herein, "embedding" refers to the fact that the primer is wrapped in an embedding agent, thereby being isolated from the outside and stably existing.
[0010]
As used herein, "conditions under which an appropriate extension reaction can be obtained" refers to various conditions for the polymerase to synthesize a nucleic acid chain, for example, conditions such as temperature, pH, salt concentration, and dNTP, which make the activity of the polymerase appropriate. Indicate a condition that is sufficient to obtain
[0011]
As used herein, the term "conditions under which a nuclease appropriately degrades a nucleic acid" refers to various conditions for a nuclease to degrade a nucleic acid, such as conditions such as temperature, pH, salt concentration, and coenzyme. Indicates a condition that is sufficient for proper acquisition.
[0012]
The term “reaction system” as used herein indicates a space for performing a reaction. For example, it may be the inside of a container and the surface of a substrate for performing a reaction therein.
[0013]
1. One embodiment of the present invention will be described below with reference to FIG. First, a nuclease is added to a reaction system in which an extension reaction is to be performed. At this time, there are substances necessary for the intended extension reaction and substances other than the template and nucleic acids used as the template, for example, substances necessary for the extension reaction such as polymerase, dNTP, buffer and embedding primer. You may let it. However, preferably, the template nucleic acid is added after inactivating the enzyme after the nuclease reaction.
[0014]
Next, the reaction system is maintained under conditions under which the nuclease can perform a nucleic acid degradation reaction. For example, in the case of 5 ′ → 3 ′ exonuclease, it may be maintained at 37 ° C. for several tens of seconds to one hour. Appropriate conditions may be selected depending on the nuclease to be selected.
[0015]
Thereafter, the reaction system is maintained under conditions where the exonuclease can be inactivated. For example, in the case of 5 ′ → 3′λ-exonuclease, it may be maintained at 75 ° C. or higher for 10 minutes. Appropriate conditions may be selected depending on the nuclease to be selected.
[0016]
Subsequently, a desired nucleic acid serving as a template is added to the reaction system to perform a desired extension reaction.
[0017]
In this embodiment, before, simultaneously with or after the addition of the nuclease to the reaction system, a substance necessary for the intended extension reaction, other than the nucleic acid used as the template, for example, polymerase, dNTP, buffer Substances required for the extension reaction, such as an agent and an embedding primer, are present. In this state, the temperature of the reaction system is changed to activate the nuclease, and thereafter, the temperature is further changed to inactivate the nuclease. The embedding primer is fluidized by the temperature at which the nuclease is inactivated. The fluidization releases the primer present inside the embedding medium into the reaction system. Performing fluidization of the embedding agent simultaneously with inactivation of the nuclease is advantageous for simplifying the method and reducing the number of steps.
[0018]
However, the addition of those reagents is not limited to the above-mentioned period. That is, the substances required for the intended extension reaction other than the nucleic acid used as the template, the reagents such as polymerase, dNTP, buffer and embedding primer are added before, simultaneously with or after the addition of the nuclease as described above. There is no limitation, and it is sufficient that the compound is present in the reaction system at the time when the desired extension reaction is started. However, undesired nucleic acid contaminants may be present in the reagent used for the extension reaction. Therefore, it is preferable to treat these reagents with nuclease.
[0019]
It is not necessary to simultaneously inactivate the nuclease and fluidize the embedding agent, and inactivate the nuclease by adjusting the temperature or changing other conditions. The temperature of the reaction system may be raised to the temperature achieved to release the primer. However, since the elongation reaction of the nucleic acid is performed after the inactivation of the nuclease and the fluidization of the embedding medium, the activation and inactivation of the nuclease and the fluidization of the embedding medium are performed so as not to inhibit the desired elongation reaction. It needs to be done.
[0020]
According to such an embodiment of the present invention, the target elongation reaction such as amplification is achieved in a state where the generation of contamination by the impure nucleic acid in the elongation reaction of the nucleic acid is prevented. According to such an aspect of the present invention, nucleic acid contamination is prevented by a simple operation, and a more reliable elongation method is provided.
[0021]
2. Embedding primer The embedding primer that can be used according to the embodiment of the present invention includes a primer used in an extension reaction known per se, and an embedding agent for embedding the primer therein. It is a reagent. By embedding with the embedding agent, the primer is isolated from the external environment and kept stable until needed.
[0022]
An example of such an embedding primer is shown in FIG. The embedding primer 1 according to the embodiment of the present invention is a reagent comprising a primer 2 used in an extension reaction known per se and an embedding agent 3 for embedding the primer therein.
[0023]
The primer that can be used in the embodiment of the present invention may be any primer that can be used in extension of a nucleic acid known per se, and may be, for example, a DNA primer or an RNA primer. In addition, primers with various modifications or labels, if desired, may be used. Further, it is desirable that the end of the primer be phosphorylated.
[0024]
The embedding agent that can be used according to the embodiment of the present invention may be any substance that exhibits the following properties depending on the temperature. That is, at a relatively low temperature, for example, preferably about 50 ° C. to about 75 ° C. or less, the solidifies and loses fluidity, and takes a solid form such as a jelly-like form. In that case, the primer embedded therein is isolated from its external environment by the presence of the embedding agent. On the other hand, when the embedding agent is exposed to a temperature at or above its fluidization temperature (or melting temperature), the solid form disintegrates into a fluid form, and as a result, its content is Release the primer into the external environment. The preferred fluidization temperature (or melting temperature) here is from about 50 to about 75 ° C or higher.
[0025]
Examples of embedding agents consist of synthetic polymers such as, but not limited to, polyvinyl alcohol, polyethylene glycol, polyacrylamide and photocrosslinkable resins, and natural polymers such as carrageenan, alginic acid, agar and agarose. Gelling agents and the like can be mentioned.
[0026]
Embedding of the primer with the embedding agent may be performed by any means known per se. For example, when polyvinyl alcohol is used as an embedding agent, gel beads may be prepared by suspending a desired primer in a polyvinyl alcohol solution and repeating freeze-thawing. The obtained gel beads may be used as an embedding primer. When agarose is used, the embedding primer can be prepared by adding the primer to an agarose solution dissolved at 70 ° C. or higher and returning the temperature to room temperature. Further, at the time of preparation, if an agarose solution is dropped into a low-temperature buffer solution, a bead-like gel can be easily prepared, so that operability is improved.
[0027]
Further, in the method for preventing the occurrence of contamination by impure nucleic acid in the nucleic acid extension reaction according to the above-described embodiment of the present invention, an example in which only the primer is embedded in the embedding agent has been described, but according to the gist of the present invention, It is sufficient that at least the primer is embedded, and therefore, reagents other than nucleases, for example, an enzyme such as a polymerase, a substrate such as dNTP, or a part thereof are embedded in an embedding agent, and as an embedding substance, May be used. In that case, the desired substance may be embedded together with the primer, or the desired substance may be embedded alone or in combination. In addition, the sense strand and the antisense strand of the primer used may be embedded together or separately.
[0028]
Such an embedding primer and an embedding substance used in combination therewith are provided as a further aspect of the present invention as an embedding reagent for elongating a nucleic acid while achieving contamination prevention.
[0029]
3. Extension Reactions The extension reactions that can be utilized in accordance with aspects of the present invention can be any means commonly known per se, used to extend nucleic acids, and any means used to amplify nucleic acids. And any such means may be used. Although not limited thereto, for example, a method utilizing a polymerase chain reaction (generally called PCR, hereinafter referred to as PCR), reverse transcription PCR, or the like can be used. Furthermore, Nucleic acid strand amplification (NASBA), Transcription mediated amplification (TMA), Ligase chain reaction (LCR), Strand displacement amplification (SDA), Isothermaland Chimeric primer-initiated Amplification of Nucleic acids (ICANN) and Rolling circle amplification (RCA Means) can also be used in accordance with aspects of the present invention.
[0030]
The nuclease that can be used according to the embodiments of the present invention can use any nuclease known per se. Examples of such nucleases include, but are not limited to, exonuclease, 3 ′ → 5 ′ exonuclease, 5 ′ → 3 ′ exonuclease, λ-exonuclease, mung bean nuclease, S1 nuclease, Bal31 nuclease, T7 gene 6 exonuclease, DNase I, RNase, etc., and λ-exonuclease is preferred.
[0031]
4. Kit comprising an embedding primer A kit comprising the above-described embedding primer is also an aspect of the present invention. FIG. 3 shows an example of such a kit. The kit 4 according to the embodiment of the present invention includes at least the embedding primer 1 and the container 5. The embedding primer 1 and the container 5 provided in the kit may be independently provided in the kit, or the embedding primer 1 may be contained in the container 5 and provided.
[0032]
The container 5 may be provided with a lid 6 to prevent drying of the embedding primer, to prevent contamination and to favor the reactions carried out therein. FIG. 3 shows an example in which the container 5 and the lid 6 are integrated, but these may be provided separately and independently. Further, the kit 4 may include a solution 7 for stably maintaining the embedding primer. However, the presence of solution 7 is not essential. Furthermore, the kit 4 may be provided with a nuclease in such a state that its activity is maintained until use, for example, the kit 4 may be provided in the container 5 or provided without being stored in the container 5. May be done.
[0033]
According to the embodiment of the present invention, the container 5 provided in the kit may be a container in which a nucleic acid elongation reaction can be performed, or may be a container for simply maintaining a reagent. As such a container, a commonly used container known per se can be used. For example, the size of the container may be about 10 μL to about 2 mL. In addition, the container may be a container made of a resin such as polypropylene, polyethylene, polystyrene, polycarbonate, and fluorocarbon, or made of glass, and may be, for example, a microtube or a test tube. The container may be a container such as a multiwell plate or a microtiter plate having a plurality of wells known per se.
[0034]
In addition to the embedding primer, other embedding reagents and non-embedding reagents described above may be provided in the kit in or out of the solution.
[0035]
5. Further Embodiment of Pollution Prevention Method A further embodiment of the above-described contamination prevention method will be described with reference to FIG. First, a nuclease and an embedding primer are added to a reaction system in which an extension reaction is to be performed.
[0036]
Next, the reaction system is maintained under conditions under which the nuclease can perform a nucleic acid degradation reaction. For example, in the case of λ-exonuclease, it may be maintained at 37 ° C. for 1 minute.
[0037]
Thereafter, the reaction system is maintained under conditions where the exonuclease can be inactivated. For example, in the case of λ-exonuclease, the temperature may be maintained at 75 ° C. for 10 minutes.
[0038]
Subsequently, a desired nucleic acid to be used as a template in the reaction system and a substance necessary for an intended extension reaction, for example, a substance necessary for an extension reaction such as a polymerase, dNTP, a buffer and an embedding primer are added, Perform the desired extension reaction.
[0039]
According to such an embodiment of the present invention, the target elongation reaction such as amplification is achieved in a state where the generation of contamination by the impure nucleic acid in the elongation reaction of the nucleic acid is prevented. According to such an aspect of the present invention, nucleic acid contamination is prevented by a simple operation, and a more reliable elongation method is provided.
[0040]
Example 1 Example 1 An embedding reagent according to an embodiment of the present invention was prepared as shown in FIG. The primer phosphorylated at the 5 ′ end was suspended in a polyvinyl alcohol solution, and freeze-thawing was repeated to prepare gel beads. The obtained gel beads are embedding primers as embedding reagents.
[0041]
Second Example A kit comprising an embedding primer according to an embodiment of the present invention as shown in FIG. 3 was prepared. In a 0.2 mL microtube, 5 ′ phosphorylated primer and 5 ′ biotinylated primer embedded in polyvinyl alcohol, and dNTP, Taq polymerase, 10 × PCR buffer, λ-nuclease were added, and the tube lid was closed. Closed. This was used as a kit including the embedding primer.
[0042]
When the kit is used to amplify a nucleic acid, a solution containing a template nucleic acid such as a template DNA may be added to the microtube at the time of use, and the reaction may be performed under desired conditions.
[0043]
Third Embodiment FIG. 5 illustrates a method for preventing contamination according to an embodiment of the present invention. Using the kit prepared as described in the example of FIG. 2, a PCR amplification reaction was performed as a nucleic acid extension reaction. In addition, templates (contaminants) of different lengths, which were amplified with the same primer in advance, were added. First, a nuclease reaction was performed at 37 ° C. for 1 minute. Next, the reaction was carried out at 75 ° C. for 10 minutes to inactivate the nuclease. Thereafter, the template nucleic acid is added into the tube, and the cycle is performed at 95 ° C. for 30 seconds, 60 ° C. for 30 seconds, 72 ° C. for 30 seconds for 40 cycles, before 95 ° C. for 10 minutes, and afterwards at 72 ° C. for 10 minutes. Processing was performed. As a result, a nucleic acid chain was synthesized. For comparison, a reaction tube from which only nuclease was removed was prepared, and the same experiment was performed. Thereafter, biotin in the reaction product was bound to the avidin-labeled magnetic fine particles using the magnetic particle to collect the reaction product. Subsequently, the resulting reaction product was converted into single-stranded nucleic acid depending on the temperature. Next, the single strand obtained by electrophoresis was identified. As a result, in the system to which nuclease was added, no band of contaminants thought to be derived from contaminating nucleic acids other than the target reaction product was observed, but in the system to which nuclease was not added, a band of contaminants was observed. Was.
[0044]
According to the method according to the embodiment of the present invention, even when the amplification product or the genome is contaminated in the tube, it is possible to remove the contamination by decomposing the nuclease. Thereby, it is possible to prevent erroneous diagnosis due to contamination beforehand.
[0045]
【The invention's effect】
According to the present invention as described above, a means for preventing the occurrence of contamination by an impure nucleic acid in a nucleic acid extension reaction is provided.
[Brief description of the drawings]
FIG. 1 is a scheme showing a nucleic acid extension reaction according to an embodiment of the present invention.
FIG. 2 schematically shows an example of an embedding primer according to an embodiment of the present invention.
FIG. 3 schematically shows an example of a kit including an embedding primer according to an embodiment of the present invention.
FIG. 4 is a scheme diagram showing a nucleic acid extension reaction according to an embodiment of the present invention.
FIG. 5 is a scheme diagram showing a procedure of a method performed in a third embodiment.
[Explanation of symbols]
1. 1. Embedding primer Primer 3. Embedding agent 4. Kit 5. Container 6. Lid 7. solution

Claims (10)

In a method of extending a target nucleic acid by a polymerase using a template nucleic acid and a primer,
(1) Presence of a nuclease and an embedding agent and an embedding primer having a primer contained therein in a reaction system for performing the extension reaction;
(2) maintaining the reaction system according to (1) above under conditions where the nuclease appropriately degrades nucleic acids;
(3) maintaining the conditions under which the nuclease is inactivated by raising the temperature of the reaction system after the maintenance of the above (2) and the primer is allowed to flow out of the embedding primer;
(4) maintaining the reaction system according to (3) under the condition that an appropriate elongation reaction is obtained after maintaining the above (3);
A method for preventing nucleic acid contamination, comprising: In a method of extending a target nucleic acid by a polymerase using a template nucleic acid and a primer,
(1) The presence of a nuclease, an embedding agent and an embedding primer comprising an embedding agent and a primer contained therein, a polymerase, and a substrate in a reaction system for performing the extension reaction therein;
(2) maintaining the reaction system according to (1) above under conditions where the nuclease appropriately degrades nucleic acids;
(3) maintaining the conditions under which the nuclease is inactivated by raising the temperature of the reaction system after the maintenance of the above (2) and the primer is allowed to flow out of the embedding primer;
(4) adding a template nucleic acid to the reaction system after maintaining the above (2);
(5) maintaining the reaction system according to the above (4) under conditions where an appropriate extension reaction can be obtained;
A method for preventing nucleic acid contamination, comprising: The method for preventing nucleic acid contamination according to claim 1, wherein the embedding primer includes an embedding agent that is fluidized by heat, and a primer that includes the embedding agent. The method according to claim 3, wherein the embedding agent is a gelling agent. The method according to claim 3, wherein the embedding agent is selected from the group consisting of polyvinyl alcohol, polyethylene glycol, polyacrylamide, a photocrosslinkable resin, carrageenan, alginic acid, agar and agarose. The method for preventing nucleic acid contamination according to any one of claims 1 to 5, wherein the nuclease is a 5 'to 3' exonuclease. An embedding primer comprising: an embedding agent that is fluidized by heat; and a primer held inside the embedding agent. The embedding primer according to claim 7, wherein the embedding agent is a gelling agent. The embedding primer according to claim 7, wherein the embedding agent is selected from the group consisting of polyvinyl alcohol, polyethylene glycol, polyacrylamide, photocrosslinkable resin, carrageenan, alginic acid, agar and agarose. A nucleic acid extension reaction kit comprising at least a container and the embedding primer according to any one of claims 7 to 9 housed in the container.

Images