JP6481364B2 - Nucleic acid removal method - Google Patents

Description

The present invention relates to a method for removing contaminant nucleic acids in a nucleic acid amplification method by PCR. The present invention can be used not only for research but also for clinical diagnosis and environmental examination.

PCR (polymerase chain reaction) is (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of primer to template single-stranded DNA, (3) DNA polymerase This is a method of amplifying a target nucleic acid in a sample by repeating the cycle with three steps of extension of the primer using.
The target nucleic acid can be amplified hundreds of thousands of times even from a small amount of sample such as several copies, and it is widely used not only in the research field but also in the forensic field such as genetic diagnosis and clinical diagnosis, or in microbial testing in food and the environment. It has come to be used.

However, since PCR is a very sensitive detection method, nucleic acids mixed in the reaction system cause nonspecific amplification and false positives. In particular, when a primer that amplifies a region highly conserved in microorganisms such as a 16S RNA region or an ITS region is used in microorganism identification, amplification may be observed without adding template DNA. This is derived from nucleic acid mixed in DNA polymerase or dNTPs, and if such a contaminating nucleic acid is present, there is a possibility that an erroneous test result is output.
In addition, in such a PCR cycle in which such a contaminating nucleic acid exceeds 30 cycles, non-specific amplification of unknown cause was caused.

In general, a DNA polymerase used for PCR is purified from a wild cell such as bacteria or archaea, or is purified from a host such as Escherichia coli by a gene recombination technique. However, it has been found that nucleic acids such as host genomes, RNA, and plasmids used for gene recombination may remain even with highly purified DNA polymerases.
In addition, dNTPs that are essential for PCR reactions are known to be easily contaminated by environmental bacteria due to their high energy, and because they are produced from salmon sperm genomes, they may contain biologically derived contaminant nucleic acids. . In addition, there are no products that define nucleic acid contamination with respect to PCR-promoting buffers and PCR-promoting factors such as BSA, and contamination nucleic acids may be contained due to contamination by environmental bacteria.

In order to remove these contaminant nucleic acids from the reaction system, various nucleic acid removal methods have been studied, and after DNase is acted on, the nucleic acid is decomposed, and then DNase is heated to denature and inactivate (Non-Patent Document 1). And a method for decomposing nucleic acids by applying ultraviolet rays (Non-patent Document 2). However, in the method using DNase, the reaction may be inhibited because DNase is not completely heat-denatured after nucleic acid removal, or DNase does not act well on DNA when DNA is bound to DNA polymerase. There is a problem that does not lead to complete nucleic acid removal. Similarly, even in the method using ultraviolet rays, there still remain problems such as ultraviolet rays damaging the DNA polymerase or failing to completely decompose the nucleic acid.

For these reasons, there has been a demand for a technique for efficiently removing contaminant nucleic acids without affecting the reactivity in a PCR reaction system.

WO2012-102208 EP2495338

Electrophoresis. 2001 Dec; 22 (20): 4316-9. Methods in Enzymology Vol. 218, 1993, 381-388 Appl. Environ. Microbiol. 2008, 74 (8): 2537. PLOS ONE. February 2014, Volume 9, Issue 2, e82624

Therefore, the present invention provides an efficient method for removing nucleic acid in a PCR reaction solution. More specifically, the present invention provides a method for removing contamination nucleic acid without affecting the reaction system and preventing false positive and non-specific amplification by contamination nucleic acid. Furthermore, the other object of this invention is to provide the reagent kit suitable for said objective. In summary, an object of the present invention is to provide a nucleic acid removal method and a removal reagent in a PCR reaction solution that prevent false positive and non-specific PCR.

In order to achieve the above object, the nucleic acid removal method of the present invention is a nucleic acid removal method characterized in that a photosensitive crosslinking agent is allowed to act on a solution containing at least one component constituting a PCR reaction solution.

That is, the present inventor makes a photosensitive crosslinking agent act on a solution containing at least one component constituting a PCR reaction solution in a PCR reaction solution containing (or possibly containing) a contaminating nucleic acid. The inventors have found that it is possible to remove contaminant nucleic acid and prevent amplification by the contaminant nucleic acid, and the present invention has been achieved.

In the present invention, the photo-sensitive cross-linking agent refers to a reagent known to be strongly bound to DNA when exposed to light, such as EMA and PMA.
EMA, PMA, and the like are used to distinguish between dead and live bacteria, but such a reagent that binds strongly to DNA is considered to inhibit PCR and is usually removed before PCR (Non-patent Documents) 3) or it is used after sufficiently diluted (Patent Document 1). In addition, there is an example (Non-patent Document 4) in which nucleic acid removal of a polymerase with a photo-sensitive cross-linking agent was performed (Non-patent Document 4), but also here, a purification process was added and PCR was performed after removing the photo-sensitive cross-linking agent. Yes.
Therefore, no example has been reported in which a photo-sensitive cross-linking agent is added to a solution containing at least one component constituting the PCR reaction solution and light is irradiated in the solution to act on the photo-sensitive cross-linking agent.

The representative invention of the present application is as follows.
[Claim 1]
A method for removing contaminant nucleic acids contained in a PCR reaction solution, the method comprising the step of adding a light-sensitive cross-linking agent in a solution containing at least one component constituting the PCR reaction solution to act on light energy. .
[Section 2]
Item 2. The method according to Item 1, wherein the means for applying light energy is irradiation with visible light.
[Section 3]
Item 3. The method according to Item 1 or 2, wherein the contaminant nucleic acid is DNA and / or RNA.
[Claim 4]
Item 4. The method according to any one of Items 1 to 3, wherein the photosensitive crosslinking agent is one or more selected from the group consisting of erydium monoazide (EMA), propidium monoazide (PMA), EMA derivatives, and PMA derivatives.
[Section 5]
Item 5. The method according to Item 4, wherein the total concentration of EMA and EMA derivative contained in the solution containing at least one or more components constituting the PCR reaction solution is 2 μg / ml or more.
[Claim 6]
Item 5. The method according to Item 4, wherein the total concentration of PMA and PMA derivative contained in the solution containing at least one component constituting the PCR reaction solution is 1 μM or more.
[Claim 7]
A reagent for performing nucleic acid removal by the method according to any one of Items 1 to 6.
[Section 8]
A PCR reaction solution in which nucleic acid is removed by the method according to any one of Items 1 to 6.
[Claim 9]
A method for performing gene amplification by adding a primer to the PCR reaction solution obtained by the method according to any one of Items 1 to 6.

According to the present invention, contaminant nucleic acid present in a PCR reaction solution can be removed. Contamination of nucleic acids into the PCR reaction solution has been a big problem especially in microorganism identification. The present invention enables accurate microorganism identification and can be said to be a very useful technique particularly in the clinical field. In addition, it can be widely used in quality control fields such as domestic water, food, and cosmetics.

Inhibitory effect on PCR reaction in EMA and PMA (Taq PCR) Inhibitory effect on PCR reaction in EMA and PMA (PCR of KOD) Removal of contaminating nucleic acid from PCR reaction solution in EMA and PMA (Taq PCR) Inhibitory effect on PCR reaction in EMA and PMA (PCR of KOD) Inhibitory effect on PCR reaction in EMA and PMA (PCR of TTH) Evaluation of contamination nucleic acid removal ability in EMA and PMA Confirmation of contaminant nucleic acid to PCR factors

Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention.
One embodiment of the present invention is a method for removing contaminant nucleic acids contained in a PCR reaction solution, wherein a photosensitive crosslinking agent is added in a solution containing at least one component constituting the PCR reaction solution. The method includes a step of applying light energy.

The photosensitive crosslinking agent in the present invention is not particularly limited. For example, ethidium monoazide (EMA), propidium monoazide (PMA), psoralen compound (psoralen), 4′-AMDMIP (4′-aminomethyl-4,5) '-Dimethylisosporalen), AMIP (5-aminothysopsoalen), 5-MIP (5-methylsoposalene), 8-methoxypsoralen, etc.), propidium iodide, EMA derivatives (for example, those described in Patent Document 2) and PMA derivatives ( For example, it is one or more selected from the group consisting of those described in Patent Document 2. Preferred is EMA, PMA or a derivative thereof.

In the nucleic acid removal method of the present invention, the means for applying light energy to the photosensitive crosslinking agent is not particularly limited. Examples of the light energy include visible light, ultraviolet light, and infrared light, and light energy can be applied by irradiating the light-sensitive crosslinking agent with these.
In the case of irradiating visible light, the wavelength of light used for the light irradiation treatment is not particularly limited. For example, single wavelength light having a wavelength of 350 to 700 nm or multiwavelength light including a wavelength of 350 to 700 nm can be used. . The light intensity and the irradiation time of the light irradiation treatment can be appropriately set according to the photosensitive crosslinking agent and the light source to be used. For example, when using EMA, a lamp of 1 to 1000 W is used, The light irradiation treatment can be performed under the condition of setting the distance of 1 to 50 cm and irradiating for 1 to 20 minutes. Furthermore, an LED (Light Emitting Diode) lamp with low power consumption and high brightness has been developed and can be used in the present invention. In addition, the light irradiation treatment of the present invention is preferably performed at a low temperature such as on ice.

As for the concentration of the photosensitive crosslinking agent in the present invention, for example, in the case of EMA or an EMA derivative, the total concentration is desirably 2 μg / ul or more. Moreover, if it is PMA or a PMA derivative, it is desirable that the total concentration of them is 1 μM or more.
On the other hand, the upper limit of the total concentration of EMA and EMA derivatives is desirably 50 μg / ul or less. The upper limit of the total concentration of PMA and PMA derivative is preferably 10 μM or less. Preferably, the upper limit of the total concentration of EMA and EMA derivatives is 20 μg / ul or less. The upper limit of the total concentration of PMA and PMA derivative is preferably 4 μM or less.

An RT-PCR reaction solution is also included in the PCR reaction solution to which the nucleic acid removal method of the present invention is applied.
In the nucleic acid removal method of the present invention, the composition of the PCR reaction solution or the RT-PCR reaction solution is not particularly limited. For example, the composition containing the following (a) to (e) is mentioned.
(A) Buffer (b) Metal salt (c) dNTPs
(D) DNA polymerase (e) Photosensitive crosslinking material

In the composition, (a) to (d) are not particularly limited.
(A) Examples of the buffer include Tris and Bicine.
(B) Examples of the metal salt include magnesium, manganese, potassium and the like.
(C) As dNTPs, for example, dATP, dGTP, dCTP, and dTTP need only be included in a suitable balance, but other dNTPs may be included. For example, dUTP can be included instead of dTTP.
(D) The DNA polymerase for PCR may be either α-type or PolI-type. Examples of α-type include KOD and Pfu, and examples of PolI-type include Taq and TTH. The amino acid sequence of the DNA polymerase may be further modified.
As the DNA polymerase for RT-PCR, a polymerase having RT (reverse transcription) activity such as the above-mentioned DNA polymerase and RT enzyme derived from MMLV or RT enzyme derived from AMV may be used in combination. Alternatively, a DNA polymerase having both DNA synthesis activity such as TTH and RT activity may be used.

In the nucleic acid removal method of the present invention, the timing for adding the photo-sensitive cross-linking agent in the PCR reaction solution or the RT-PCR reaction solution and the timing for applying the light energy include all of the above (a) to (d). It is not limited to adding a light-sensitive crosslinking agent to the composition and performing light irradiation.
For example, a missing part may be added after adding a light-sensitive crosslinking agent to the composition containing any one or more of the above (a) to (d) and performing light irradiation. In this case, it is preferable to use a component that has been confirmed in advance to be free of contamination as a component to be added after adding a light-sensitive crosslinking agent and performing light irradiation.

Another embodiment of the present invention is a reagent for performing nucleic acid removal by the method described above. Reagents for carrying out the nucleic acid removal method of the present invention include a light-sensitive cross-linking material and a PCR reagent, and the others are not particularly limited. Examples of the photosensitive crosslinking agent include one or more selected from the group consisting of EMA, PMA, psoralen compounds, 4′-AMDMIP, AMIP, 5-MIP, propidium iodide, EMA derivatives and PMA derivatives, preferably EMA. , PMA or their derivatives. The concentration is not particularly limited, but is preferably 2 μg / ul to 50 μg / ul for EMA and EMA derivatives. Moreover, if it is PMA or a PMA derivative, 1 micromol-10 micromol are preferable.
Components other than the photocrosslinking agent are not particularly limited. Factors that promote PCR, such as single-stranded DNA-binding protein, dUTPase, and PCNA, and factors that are related to improved specificity, such as DMSO and glycerol, may be included.
In addition, the form of the reagent of this invention is not specifically limited, A so-called kit form is also included.

Another embodiment of the present invention is a PCR reaction solution from which nucleic acid has been removed by the above-described method, and further a method for performing gene amplification by adding a primer to the PCR reaction solution. The PCR reaction solution that has been subjected to nucleic acid removal in the present invention is a PCR reaction solution that has been subjected to nucleic acid removal by the action of a light-sensitive crosslinking material, and is not particularly limited. The form of the PCR reaction solution of the present invention is not particularly limited, and includes a so-called kit form.

Hereinafter, the present invention will be specifically described by way of examples. The description of the following examples does not limit the invention in any way.
Example 1
Inhibitory effect on PCR reaction in EMA and PMA Taq DNA Polymerase (Taq) or KOD DNA Polymerase (KOD) is used to amplify Hbg gene 746 bp to PCR reaction system, respectively, EMA and PMA are added to inhibit The resulting concentration was confirmed.

For the Taq PCR reaction system, a mixture of rTaq DNA Polymerase (TOYOBO) and Anti-Taq High (TOYOBO) was used.
A light irradiation sample in which a light-sensitive crosslinking agent was added to the PCR reaction solution and allowed to act on the light energy was prepared by the following procedure.
(1) A PCR reaction solution containing 2.5 U of enzyme mixed with Buffer, 2 mM dNTPs, and antibody attached to 1 × rTaq DNA Polymerase was prepared.
(2) In the reaction solution prepared in (1) above, EMA (ANASPEC) dissolved in sterilized water is adjusted to a final concentration of 100, 50, 20, 10, 5, 2, 0 μg / ml, or PMA (Biotium ) Was added to 20, 10, 4, 2, 1, 0.4, 0 μM.
(3) Thereafter, the solution prepared in (2) above was irradiated with light for 5 minutes at an irradiation distance of 20 cm using a halogen lamp (Iwasaki Electric).
(4) A 50 μl PCR reaction solution was prepared by adding 10 pmol primer (SEQ ID NOs: 1 and 2) and 10 ng human genomic DNA (Roche) to 50 μl of the light-irradiated sample obtained above.
The reaction was performed at 94 ° C. for 2 minutes, followed by PCR using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 35 cycles of 94 ° C., 10 seconds → 62 ° C., 30 seconds → 68 ° C., 1 minute. It was.

For the KOD PCR reaction system, KOD-PLUS-Ver. 2 (TOYOBO) was used.
The light irradiation sample was obtained in the same manner as in the case of Taq except that the PCR reaction solution was prepared by the following procedure (1).
(1) A PCR reaction solution containing 1 × attached Buffer, 2 mM dNTPs, 1.5 mM MgSO ₄ , and 1 U enzyme was prepared.
The reaction was performed at 94 ° C. for 2 minutes, followed by PCR on PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 98 ° C., 10 seconds → 65 ° C., 10 seconds → 68 ° C., 1 minute repeated 35 cycles. It was.

After completion of the reaction using the Taq or KOD PCR reaction system prepared above, electrophoresis was performed on a DNA-1000 gel using MaltiNA (Shimadzu Corporation), and a DNA fragment near 746 bp was confirmed.
As a result, amplification by Taq PCR was not observed due to inhibition at 20 μg / ml in EMA and 4 μM or more in PMA (FIG. 1). On the other hand, PCR of KOD was more resistant to inhibition than Taq, and amplification was confirmed up to 20 μg / ml in EMA and up to 4 μM in PMA. At concentrations higher than that, KOD also resulted in inhibition (FIG. 2). It was shown that PCR can be performed without inhibiting the respective reaction systems of Taq and KOD when the concentration is below these concentrations.

(Example 2)
Removal of contaminating nucleic acid from PCR reaction solution in EMA and PMA (Taq PCR)
In Taq PCR, it was examined whether bacterial-derived nucleic acids contaminating the PCR reaction system could be removed at EMA final concentrations of 2 μg / ml and 5 μg / ml, PMA final concentrations of 1 μM, and 0.4 μM, which were not inhibited.
Usually, when PCR is carried out using primers that amplify the 16S rRNA common region of bacteria, amplification is observed even in a control (NTC) without a template. If the contaminant nucleic acid has been removed, amplification should not be confirmed by NTC.
As in Example 1, a mixture of rTaq DNA Polymerase (TOYOBO) and Anti-Taq High (TOYOBO) was used. The light-irradiated sample in which a light-sensitive cross-linking agent was added to the PCR reaction solution and allowed to act on the light energy was added to the PCR reaction solution containing 2.5 U of enzyme mixed with Buffer, 2 mM dNTPs attached to 1 × rTaq DNA Polymerase, and antibody. Then, EMA (ANASPEC) dissolved in sterile water was added to a final concentration of 5, 2 μg / ml, or PMA (Biotium) was added to 1, 0.4 μM to prepare a light irradiation sample. Thereafter, using a halogen lamp (Iwasaki Electric Co., Ltd.), light irradiation was performed for 5 minutes at an irradiation distance of 20 cm, and 10 μmol of a primer (SEQ ID NOs: 3 and 4) for amplifying the 16S rRNA common region was added, thereby 50 μl of a PCR reaction solution. Was prepared. In order to confirm the control without the light-sensitive cross-linking material and the inhibition by the light-sensitive cross-linking material, 5 ng of E.I. A control supplemented with the E. coli genome was also performed at the same time. The reaction was performed at 94 ° C. for 2 minutes, followed by PCR at 94 ° C., 10 seconds → 50 ° C., 10 seconds → 68 ° C., 1 minute for 40 cycles on a PCR system GeneAmp 9700 (Applied Biosystem). It was.
After completion of each reaction, electrophoresis was performed on a DNA-1000 gel using MaltiNA (Shimadzu Corporation), and a DNA fragment around 800 bp was confirmed (FIG. 3).

As a result, amplification was confirmed even with NTC in the control to which no photosensitive crosslinking material was added. This is thought to be due to contaminant nucleic acids contained in the PCR reaction solution. E. In the control to which the E. coli genome was added, amplification was confirmed even when EMA or PMA was added. This indicates that there is no inhibition by EMA or PMA. On the other hand, when EMA was added at 5, 2 μg / ml and PMA was added at 1 μM, no amplification was observed, indicating that the contaminated nucleic acid could be removed. When PMA is added at 0.4 μM, amplification is thinly observed. This suggests that the contamination nucleic acid could not be completely removed due to insufficient action of the photosensitive crosslinking material.

(Example 3)
Contaminating nucleic acid removal from PCR reaction solution in EMA and PMA (KOD PCR)
Similar to Taq, in KOD PCR, it was examined whether bacterial-derived nucleic acids contaminating the PCR reaction system could be removed at an EMA final concentration of 20 μg / ml and PMA final concentration of 4 μM, where inhibition was not caused.
The study was conducted using KOD-Plus-Ver. 2 (TOYOBO) was used. EMA (ANASPEC) dissolved in sterilized water in a PCR reaction solution containing 1 × Buffer, 2 mM dNTPs, 1.5 mM MgSO ₄ , 1 U enzyme to a final concentration of 20 μg / ml, or PMA (Biotium) A light irradiation sample was prepared by adding to 4 μM. Thereafter, using a halogen lamp (Iwasaki Electric Co., Ltd.), light irradiation was performed for 5 minutes at an irradiation distance of 20 cm, and 10 μmol of a primer (SEQ ID NOs: 3 and 4) for amplifying the 16S rRNA common region was added, thereby 50 μl of a PCR reaction solution. Was prepared. In order to confirm the control without the light-sensitive cross-linking material and the inhibition by the light-sensitive cross-linking material, 5 ng of E.I. A control supplemented with the E. coli genome was also performed at the same time. The reaction was performed at 94 ° C. for 2 minutes, followed by PCR with PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 98 ° C., 10 seconds → 50 ° C., 10 seconds → 68 ° C., 1 minute repeated 40 cycles. It was.
After completion of each reaction, electrophoresis was performed on a DNA-1000 gel using MaltiNA (Shimadzu Corporation), and a DNA fragment around 800 bp was confirmed (FIG. 4).

As a result, as in Example 2, amplification was confirmed even in NTC in the control to which no photo-sensitive crosslinking material was added. This is thought to be due to contaminant nucleic acids contained in the PCR reaction solution. E. In the control to which the E. coli genome was added, amplification was confirmed even when EMA or PMA was added. This indicates that there is no inhibition by EMA or PMA. On the other hand, when EMA was added at 20 μg / ml and PMA was added at 4 μM, no amplification was observed, indicating that the contaminated nucleic acid could be removed.

Example 4
Removal of contaminating nucleic acid from PCR reaction solution in EMA and PMA (TTH PCR)
TTH DNA Polymerase (TTH) retains reverse transcription activity in the presence of manganese and can be amplified from RNA. It was examined using TTH whether contaminant nucleic acid could be removed by RNA.
For the examination, RNA-direct Realtime PCR Master Mix (TOYOBO) was used. A light-irradiated sample was prepared by adding EMA (ANASPEC) dissolved in 2 × MasterMix with sterilized water to a final concentration of 10, 5 μg / ml, or PMA (Biotium) to 2, 1 μM. Thereafter, using a halogen lamp (Iwasaki Electric Co., Ltd.), light irradiation was performed for 5 minutes at an irradiation distance of 20 cm, and 10 μmol of a primer (SEQ ID NOs: 3 and 4) for amplifying the 16S rRNA common region was added, thereby 50 μl of a PCR reaction solution. Was prepared. In order to confirm the control without the light-sensitive cross-linking material and the inhibition by the light-sensitive cross-linking material, 5 ng of E.I. A control supplemented with the E. coli genome was also performed at the same time. The reaction is 90 ° C, 15 seconds, 50 ° C, 20 minutes reverse transcription reaction, 95 ° C, 30 seconds pre-reaction, 95 ° C, 10 seconds → 50 ° C, 10 seconds → 76 ° C, 1 minute, 40 cycles. RT-PCR was performed on a PCR system GeneAmp 9700 (Applied Biosystem) according to the schedule.
After completion of each reaction, electrophoresis was performed on a DNA-1000 gel using MaltiNA (Shimadzu Corporation) to confirm a DNA fragment of about 800 bp (FIG. 5).

As a result, amplification was confirmed even with NTC in the control to which no photosensitive crosslinking material was added. This is considered to be due to contamination nucleic acid (DNA or RNA) contained in the PCR reaction solution. E. In the control to which the E. coli genome was added, amplification was confirmed even when EMA or PMA was added. This indicates that there is no inhibition by EMA or PMA. On the other hand, when EMA was added at 10 and 5 μg / ml and PMA was added at 2 and 1 μM, no amplification was observed, indicating that the contaminated nucleic acid could be removed. This result shows that the photosensitive cross-linking material also acts on RNA contained in the reaction system.

(Example 5)
Evaluation of contamination nucleic acid removal ability in EMA and PMA It was confirmed how much nucleic acid can be removed by EMA and PMA.
For the examination, KOD FX (TOYOBO) was used, and EMA was prepared by adding 10, 1, and 0.1 ng of human genome (Roche) to PCR reactions containing 1 × Buffer, 4 mM dNTPs, and 1 U of enzyme, respectively, and dissolving in sterile water. A light irradiated sample was prepared by adding (ANASPEC) to a final concentration of 20 μg / ml or PMA (Biotium) to 4 μM. Thereafter, using a halogen lamp (Iwasaki Electric Co., Ltd.), irradiating with light at an irradiation distance of 20 cm for 5 minutes, and adding 10 pmol of a primer (SEQ ID NO: 5 and 6) for amplifying the Hbg region 481 bp, a 50 μl PCR reaction solution was prepared. Prepared. A control without a light-sensitive crosslinking material was also performed at the same time, and a control for PCR without light irradiation was also performed. The reaction was performed at 94 ° C. for 2 minutes, followed by PCR using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 98 ° C., 10 seconds → 65 ° C., 10 seconds → 68 ° C., 1 minute repeated 40 cycles. It was.
After completion of each reaction, electrophoresis was performed on a DNA-1000 gel using MaltiNA (Shimadzu Corporation), and a DNA fragment near 481 bp was confirmed (FIG. 6).

As a result, in the control obtained by PCR without light irradiation, amplification of 481 bp of Hbg was confirmed from 10, 1, and 0.1 ng of the human genome even when EMA and PMA were included. This shows that there is no inhibition of PCR by EMA or PMA at this concentration. On the other hand, in the light-irradiated one, the control containing no photo-sensitive cross-linking material can confirm the amplification of 481 bp of Hbg from the human genome of 10, 1, 0.1 ng, respectively, whereas the sample containing EMA, PMA Then, amplification could not be confirmed. This indicates that the nucleic acid was removed by EMA and PMA. Normally, it is unlikely that a genome exceeding 10 ng will be contaminated, but it has been shown that if the present invention is used, contamination of about 10 ng can be completely removed.

(Example 6)
Confirmation of contaminant nucleic acid to PCR factor KOD-Plus-Ver. It was confirmed from which factor the contamination in 2 (TOYOBO) was brought from.
[1] PCR reaction solution containing 2 mM dNTPs, 1.5 mM MgSO ₄ , 1U enzyme [2] 1 × attached buffer, 1.5 mM MgSO ₄ , PCR reaction solution containing 1 U enzyme [3] 1 × attached Buffer reaction solution containing Buffer, 2 mM dNTPs, and 1 U enzyme [4] 1 × Attached PCR reaction solution containing Buffer, 2 mM dNTPs, 1.5 mM MgSO ₄ was dissolved in EMA (ANASPEC) dissolved in sterile water. A light-irradiated sample was prepared at a concentration of 20 μg / ml. Then, using a halogen lamp (Iwasaki Electric Co., Ltd.), the light was irradiated for 5 minutes at an irradiation distance of 20 cm. In [1], the attached Buffer was 1 ×, in [2], 2 mM dNTP, [3 ] Is added 1.5 mM MgSO ₄ , 1 U of enzyme is added to [4], and 10 pmol primer (SEQ ID NOs: 3 and 4) is added to amplify the 16S rRNA common region. Prepared. A control containing no light sensitive crosslinker was also performed. The reaction was performed at 94 ° C. for 2 minutes, followed by PCR with PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 98 ° C., 10 seconds → 50 ° C., 10 seconds → 68 ° C., 1 minute repeated 40 cycles. It was.
After completion of each reaction, electrophoresis was performed on a DNA-1000 gel using MaltiNA (Shimadzu Corporation), and a DNA fragment around 800 bp was confirmed (FIG. 7).

As a result, amplification was confirmed in the control not containing EMA as in Example 3. On the other hand, in the case where EMA was added and the nucleic acid was removed, amplification was confirmed only in the case where dNTP was added later [2] and the enzyme was added later [4]. This indicates that contaminating nucleic acids were present in dNTPs and enzymes.
If there is contamination in PCR factors such as dNTPs as well as polymerase as in this case, amplification will occur in NTC no matter how much the polymerase is purified. The present invention can be said to be very effective in removing nucleic acids present in the PCR reaction system.

The present invention is useful in the biotechnology-related industry, and particularly useful in the technology related to DNA synthesis.

Claims (11)

A method for removing contaminant RNA nucleic acid contained in a PCR reaction solution, wherein light is contained in a solution containing at least one component constituting the PCR reaction solution (except when the solution contains EA1 proteinase) . A method comprising the step of adding a sensitive cross-linking agent and applying light energy.   The method according to claim 1, wherein the means for applying light energy is irradiation with visible light. Light-sensitive crosslinking agent, ethidium monoazide (EMA), propidium monoazide (PMA), is one or more selected from the group consisting of EMA derivatives and PMA derivatives A method according to claim 1 or 2. The method according to claim 3 , wherein the total concentration of EMA and EMA derivatives contained in the solution containing at least one or more components constituting the PCR reaction solution is 2 µg / ml or more. The method according to claim 3 or 4, wherein the total concentration of EMA and EMA derivatives contained in a solution containing at least one or more components constituting the PCR reaction solution is 20 µg / ml or less. The method according to claim 3 , wherein the total concentration of PMA and PMA derivative contained in the solution containing at least one component constituting the PCR reaction solution is 1 μM or more. The method according to claim 3 or 6, wherein the total concentration of PMA and PMA derivative contained in the solution containing at least one component constituting the PCR reaction solution is 4 µM or less. The method according to any one of claims 1 to 7, wherein the PCR reaction solution further comprises at least one DNA polymerase selected from the group consisting of α-type DNA polymerase and PolI-type DNA polymerase. The method according to any one of claims 1 to 8, excluding the case where the PCR reaction solution contains a thermophilic proteinase. Reagent comprising a light sensitive cross-linking agents for carrying out the method in contaminant RNA nucleic acid removal according to any of claims 1 to 9. A method for carrying out gene amplification by adding a primer to the PCR reaction solution obtained by the method according to any one of claims 1 to 9 .