JP2008017851A - Additive for promoting dna synthesis reaction - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel composition and method useful for polymerase chain reaction (PCR) upon generation of a DNA from a template nucleic acid and further DNA amplification. <P>SOLUTION: A liquid composition for reaction includes anionic substances effective for the promotion of DNA synthesis are included in an enzymatic reaction for DNA synthesis. Concretely, a liquid composition for reaction comprises divalent carboxyl ions consisting of dicarboxylates especially comprising at least one of oxalic, malonic and maleic ion. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention belongs to the field of molecular biology. The present invention relates to novel compositions and methods useful in the production of DNA from template nucleic acids and further DNA amplification. The present invention relates specifically to novel compositions and methods useful for polymerase chain reaction (PCR).

  The detection, analysis, transcription and amplification of nucleic acids are the most important manipulation methods in modern molecular biology. The application of such procedures for DNA analysis is particularly important in gene expression studies, diagnosis of infectious agents or genetic diseases, cDNA generation and retrovirus analysis and the like.

PCR used for DNA amplification is a well-known technique in the art. Specifically, the gene amplification method based on the PCR method comprises a cycle comprising three steps of denaturation, annealing, and extension in the presence of a target nucleic acid, four types of deoxynucleoside triphosphates, a pair of primers and a DNA synthase. This is a method of exponentially amplifying a target nucleic acid region sandwiched between the pair of primers by repeating (Nature, 324 (6093), 13-19 (1986)) (Non-patent Document 1). That is, the nucleic acid of the sample is denatured in the denaturation step, each primer is hybridized with a region on the single-stranded target nucleic acid complementary to each in the subsequent annealing step, and DNA is started from each primer in the subsequent extension step. A DNA strand complementary to each single-stranded target nucleic acid serving as a template is extended by the action of the polymerase to form double-stranded DNA. By this one cycle, one double-stranded DNA is amplified into two double-stranded DNAs. Therefore, if this cycle is repeated n times, the region of the sample DNA sandwiched between the pair of primers is theoretically amplified 2n times.
Preferred reaction conditions for amplification are thermocycling, i.e. changing the temperature of the reaction mixture in order to achieve each step of the PCR cycle. Thermal cycling is performed in a temperature range between about 23 ° C and about 100 ° C, preferably between about 37 ° C and about 95 ° C. Nucleic acid denaturation is usually performed between about 90 ° C and about 100 ° C, preferably at about 94 ° C. Annealing is usually performed at between about 37 ° C and about 75 ° C, preferably at about 60 ° C. DNA elongation is usually performed at between about 55 ° C and about 80 ° C, preferably at about 68-72 ° C. The number of cycling varies greatly depending on the amount of DNA product desired. The number of PCR cycles is preferably in the range of about 5 to about 99, more preferably more than about 20 cycles, and most preferably about 25 to 40 cycles.
Nature, 324 (6093), 13-19 (1986).

  The PCR technique is first performed using a DNA synthase derived from a thermophilic bacterium such as Escherichia coli, and the success rate (the probability that the target DNA is actually amplified) is low. Eventually, thermostable DNA synthases derived from thermophilic bacteria began to be used, and devices that automatically perform temperature cycling were developed to improve the PCR success rate. Furthermore, in order to improve the success rate of PCR, various improvements such as improvement of DNA synthase to be used, improvement of reaction solution composition, improvement of cycle temperature conditions, improvement of performance of temperature cycling equipment, improvement of reagent management status, etc. Has been made. As a result of the above, the PCR success rate has improved remarkably, but even now, the target DNA cannot be obtained 100%, and improving the PCR success rate has been an eternal theme for those who use PCR technology. .

For example, as an improvement of the DNA synthase for improving the PCR success rate, a technique of mixing and using two kinds of thermostable DNA synthases proposed by Barnes (Barns, WM (1994) Proc Natl. Acad. Sci. USA 91, 2216-2220). The thermostable DNA synthase used for PCR is roughly divided into two types. One is a Pol I type enzyme typified by Taq DNA polymerase and Tth DNA polymerase, and the other is an α type enzyme typified by Pfu DNA polymerase.
In general, a Pol I-type enzyme has a high DNA synthesis rate and is excellent in processivity (Processivity: the number of nucleotides synthesized after the DNA polymerase binds to a substrate DNA and then leaves). 'Because of the lack of exonuclease activity, when the wrong base was polymerized, it could not be removed, and the reaction was stopped in situ, and the target DNA could not be sufficiently amplified. On the other hand, the α-type enzyme has 3′-5 ′ exonuclease activity, so even if an incorrect base is polymerized, it can be removed and DNA synthesis can be continued. The target DNA could not be sufficiently amplified due to low secrecy.
Therefore, an enzyme (mixed-type enzyme) was born in which two kinds of enzymes were mixed for the purpose of combining the advantages of both. This enzyme has a Pol I type enzyme as a main component and DNA synthesis is mainly carried out by the Pol I type enzyme, and the α type enzyme only carries out calibration of incorrect bases. The PCR success rate could be improved by using this technique.
There have also been many studies on thermostable DNA polymerases useful for polymerase chain reaction (PCR). For example, the KOD DNA polymerase we discovered is an enzyme that combines the advantages of the above two while being a single enzyme (α-type enzyme), has a high DNA synthesis rate, is excellent in processability, and is 3′-5 ′ exo. It possessed nuclease activity. These advantages of the enzyme were also demonstrated in PCR and showed excellent PCR performance.
Furthermore, as a method for improving the enzyme, there is a method (Hot start PCR method) in which an antibody of DNA synthase is added to completely suppress the enzyme activity at room temperature. The PCR success rate is increased by suppressing the production of by-products such as primer dimers due to enzyme reaction at room temperature and protecting the degradation of primer.

Various studies have been made to improve the PCR reaction solution composition. Buffers include, for example, Tris (TRIS), Tricine (TRICINE), Bistolycin (BIS-TRICINE), Hepes (HEPES), Mops (MOPS), Tes (TES), Tapes (TAPS), Pipes (PIPES), Caps (CAPS) and others were tried. In the salt solution, for example, potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate, magnesium chloride, magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, manganese sulfate / sodium chloride, sodium acetate, lithium chloride, And lithium acetate was tried. Examples of additives include DMSO, glycerol, formamide, betaine, tetramethylammonium chloride, PEG, Tween 20, NP40, ectoine, polyols, E. coli SSB protein, phage T4 gene 32 A protein, BSA, was attempted.
The above reagents are not effective for all DNA synthetases, and are unsuitable for each enzyme. The unique reaction solution composition is studied for each enzyme, and the optimal reaction buffer is attached to each enzyme. Therefore, it was considered impossible to achieve higher performance than the attached buffer.

In the above studies, conventionally, salt solutions have focused on cationic substances (K +, Na +, NH42 +, etc.) from the viewpoint of acting on nucleic acids. However, as a result of diligent research, we have found that anionic substances are also important in determining the success or failure of PCR. Specifically, it has been found that the success rate of PCR is improved by adding a divalent carboxyl ion composed of a dicarboxylate to a PCR reaction system. In addition, oxalate, malonate and maleate ions were found to be particularly effective among divalent carboxyl ions.
In the present invention, these ions are added in the form of an inorganic salt. Specifically, zinc oxalate, ammonium oxalate, potassium oxalate, calcium oxalate, diethyl oxalate, N, N'-disuccinimidyl oxalate, dimethyl oxalate, tin oxalate, cerium oxalate, oxalate Iron oxalate, copper oxalate, sodium oxalate, nickel oxalate, bis oxalate, oxalate (2,4-dinitrophenyl), oxalate (2,4,6-trichlorophenyl), manganese oxalate, methyl oxalate Lanthanum oxalate, lithium oxalate, isopropylidene malonate, ethyl malonate, diethyl malonate, dibenzyl malonate, dimethyl malonate, thallium malonate, disodium malonate, monosodium maleate, maleic acid Diethyl, chlorpheniramine maleate, di-n-butyl maleate It can be prepared and added from reagents such as til, maleic acid mono-n-butyl ester.

That is, the present invention has the following configuration.
(1) A reaction solution composition comprising an anionic substance effective in promoting DNA synthesis in an enzymatic reaction for synthesizing DNA.
(2) The reaction liquid composition according to (1), wherein the anionic substance is a carboxyl group.
(3) The reaction solution composition as described in (1), wherein the anionic substance comprises a divalent carboxyl group.
(4) A reaction solution composition, wherein a divalent carboxyl group in (3) is added in the form of an inorganic salt.
(5) The reaction liquid composition as described in (1) to (4), wherein a divalent carboxyl group is added in the form of a salt with an alkali metal, an alkaline earth metal or ammonium in (4) .
(6) The reaction liquid composition as described in any one of (1) to (5), which contains at least one kind of oxalate ion, malonate ion, and maleate ion in an enzyme reaction for synthesizing DNA.
(7) A reaction liquid composition, wherein the oxalate ion, malonate ion, and maleate ion are added in the form of an inorganic salt in (6).
(8) In (7), the oxalate ion, malonate ion, and maleate ion are added in the form of a salt with an alkali metal, an alkaline earth metal, or ammonium. The reaction liquid composition as described.
(9) Oxalate ion, malonate ion, and maleate ion are zinc oxalate, ammonium oxalate, potassium oxalate, calcium oxalate, diethyl oxalate, N, N′-disuccinimidyl oxalate, and oxalic acid Dimethyl, tin oxalate, cerium oxalate, iron oxalate, copper oxalate, sodium oxalate, nickel oxalate, bis oxalate, oxalic acid (2,4-dinitrophenyl), oxalic acid (2,4,6- Trichlorophenyl), manganese oxalate, methyl oxalate, lanthanum oxalate, lithium oxalate, isopropylidene malonate, ethyl malonate, diethyl malonate, dibenzyl malonate, dimethyl malonate, thallium malonate, dimalonate Sodium or monosodium maleate, diethyl maleate, male (1) to (8), which are prepared and added from at least one of the group consisting of chlorpheniramine inacid, di-n-butyl maleate and mono-n-butyl maleate The reaction liquid composition.
(10) In (8), the oxalate ion, malonate ion, and maleate ion are added in a form containing at least one of potassium salt or sodium salt. The reaction liquid composition as described in 9).
(11) As a reaction solution component for DNA synthesis, a template nucleic acid, a DNA synthesizing enzyme, one or more oligonucleotide primers, one or more nucleotides or their derivatives, buffers, salts, useful for DNA synthesis The composition according to any one of (1) to (10), which contains at least one member of the group consisting of additives.
(12) The composition according to (11), wherein the template nucleic acid is RNA.
(13) The composition according to (11), wherein the template nucleic acid is DNA.
(14) The composition according to (9) or (10), wherein the DNA synthase is a reverse transcriptase.
(15) The reverse transcriptase is AMV-RT, M-MLV-RT, HIV-RT, EIAV-RT, RAV2-RT, C.I. (11), (12) selected from the group consisting of C. hydroformans DNA polymerase, SuperScript I, SuperScript II, and mutants, variants and derivatives thereof. ).
(16) The composition according to (11) or (13), wherein the DNA synthase is a DNA polymerase.
(17) The composition according to (11) or (13), wherein the DNA synthase is a heat-resistant DNA polymerase.
(18) The composition according to (11) or (13), wherein the DNA synthase is a heat-resistant α-type DNA polymerase.
(19) The composition according to (11) or (13), wherein the DNA synthase is a heat-resistant Pol I type DNA polymerase.
(20) The composition according to (11) or (13), wherein the DNA synthase is a mixed enzyme obtained by mixing a heat-resistant α-type DNA polymerase and a heat-resistant Pol I-type DNA polymerase.
(21) The DNA polymerase is Taq, EX-Taq, LA-Taq, Expand series, Platinum series, Tbr, Tfl, Tru, Tth, T1i, Tac, Tne, Tma, Tih, Tfi, Pfu, Pfubo, Pyrobest, (11), (13) description selected from the group consisting of Pwo, KOD, Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENT, DEEPVENT, and mutants, variants and derivatives thereof. Composition.
(22) The buffer is Tris (TRIS), Tolysin (TRICINE), Bistolycin (BIS-TRICINE), Hepes (HEPES), Mops (MOPS), Tes (TES), Tapes (TAPS), Pipes (PIPES) And the composition according to (1) to (13), which is selected from the group consisting of Caps (CAPS).
(23) The salt is potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate, magnesium chloride, magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, manganese sulfate, sodium chloride, sodium acetate, lithium chloride. And the composition according to (11), selected from the group consisting of lithium acetate.
(24) Further, as additives useful for DNA synthesis, DMSO, glycerol, formamide, betaine, tetramethinolemmonium chloride, PEG, Tween 20, NP40, ectoine, polyols, E. coli SSB protein, phage T4 gene 32. The composition according to (11), comprising at least one selected from the group consisting of 32 proteins and BSA.
(25) The composition according to (11), wherein the nucleotide is deoxyphosphonucleotide or a derivative thereof.
(26) The deoxyphosphonucleotide is selected from the group consisting of dATP, dCTP, dGTP, dTTP, d1TP, dUTP, α-thio-dNTPs, biotin-dUTP, fluorescein-dUTP, and sigoxygenin-dUTP (25) Composition.
(27) A method for synthesizing DNA using the composition according to any one of (1) to (26).
(28) The method according to (27), wherein the DNA synthesis method comprises a polymerase chain reaction (PCR).
(29) The method according to (28), wherein the PCR amplification comprises repeating at least 25 DNA denaturation, annealing, and DNA polymerization processes.

  As described above, according to the present invention, PCR can be performed even on a target DNA, which has been impossible until now, and the success rate of PCR can be increased. The effect of adding a divalent carboxylate could be confirmed by all kinds of DNA synthetases, and was effective not only in a simple DNA synthesis reaction but also in a PCR method.

  Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited to these examples.

Example 1.
Confirmation of addition effect of oxalate ion in mixed enzyme (EX-Taq) (Fig. 1)
The effect of addition of oxalate ions was confirmed by PCR using EX-Taq DNA polymerase. A PCR buffer attached to EX-Taq (Takara Shuzo) was used, and 0.2 mM dNTPs and a primer pair described in SEQ ID NOs: 1 and 0.3 μM were used. Using EX-Taq DNA polymerase 1.0 U and 20 ng of Genomic DNA derived from human cultured cell K562 as a template, the influence on the PCR result due to the difference in the addition concentration of oxalate ions was examined. After the previous reaction at 94 ° C. for 2 minutes, PCR was performed with GeneAmp2400 (PE Applied Biosystems) on a schedule of repeating 94 cycles of 15 ° C., 15 seconds to 60 ° C., 30 seconds to 68 ° C. and 8.5 minutes. .
When no oxalate ion was added, amplification of the target DNA was not observed at all. However, amplification of the target DNA was observed when 1 mM oxalate ion was added, and the amount of amplification was maximum when 2 to 3 mM was added. It was. Amplification of the target DNA was confirmed in the range of 1 to 4 mM oxalate ions.
From the above results, the effect of adding oxalate ions in the mixed enzyme was confirmed. The concentration range in which the effect of addition was observed was 4 mM or less, but the optimum concentration was expected to vary depending on the type and amplification size of the target DNA, the PCR buffer composition, and the like.

Example 2
Confirmation of effective salt type by PCR using mixed enzyme (EX-Taq) (Figure 2)
Next, under the same conditions as in Example 1, the types of salts (potassium chloride, potassium acetate, potassium oxalate, sodium oxalate, potassium sulfate) effective for PCR using EX-Taq DNA polymerase were investigated. In addition, the usage-amount of each salt was made into the ionic value of 4 and 6 mM which was the most effective in Example 1. Amplification was confirmed only when potassium oxalate and sodium oxalate were added as salts. No amplification was observed when other commonly used potassium salts were added. From the above results, it was suggested that the anionic oxalate ion is effective for PCR.

Example 3
Confirmation of the types of carboxylates that are effective in PCR using a mixed enzyme (EX-Taq) (Fig. 3)
Next, the types of carboxylates (potassium oxalate, sodium oxalate, potassium succinate, potassium formate) effective for PCR using EX-Taq DNA polymerase under the same conditions as in Example 2 were investigated. As in Example 2, amplification of the target DNA was confirmed only when potassium oxalate and sodium oxalate were added as salts.
Formic acid having a monovalent carboxyl group and succinic acid having a divalent carboxyl group could not confirm the same effect as oxalate. However, since potassium formate and potassium succinate, unlike oxalate, may have different concentration ranges that affect PCR amplification, a complete denial cannot be made. In certain concentration ranges, other carboxylates are likely to have an effect on PCR.

Example 4
Confirmation of addition effect of oxalate ion in PolI type enzyme (Taq) The effect of addition of oxalate ion was confirmed by PCR using Taq DNA polymerase. The PCR buffer attached to Taq (manufactured by Toyobo) (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2) was used, and 0.2 mM dNTPs and each primer pair were used in an amount of 0.3 μM. In addition, the primer pair described in SEQ ID NOs: 3 and 4 is used for PCR (FIG. 4) for amplifying 3.6 kb in the β-globin cluster, and SEQ ID NO: for PCR (FIG. 5) for amplifying myelin oligodendrocyte glycoprotein 4.5 kb. The primer pairs described in 5 and 6 were used. Taq DNA polymerase 2.5U was used, and 20 ng of genomic DNA derived from human cultured cell K562 was used as a template, and the influence on the PCR results due to the difference in the addition concentration of oxalate ions was examined. After a pre-reaction at 94 ° C. for 2 minutes, PCR was performed with GeneAmp2400 (PE Applied Biosystems) on a schedule in which 94 ° C., 15 seconds to 60 ° C., 30 seconds to 68 ° C. and 8 minutes were repeated for 30 cycles.
In any target, only smear was observed when no oxalate ion was added. However, in β-globin 3.6 kb PCR, target amplification was observed in the smear when 2 mM oxalate ion was added, and no smear was observed when 4 mM was added. Clean amplification was confirmed in the range of oxalate ions of 4 and 5 mM. On the other hand, in the myelin oligodendrocyte glycoprotein 4.5 kb PCR, amplification of the target was observed in the smear when 1 mM oxalate ion was added, and no smear was observed when 2 mM was added. Clean amplification was confirmed in the range of 2 to 5 mM oxalate ion.
From the above results, the effect of addition of oxalate ions was also confirmed in the PolI type enzyme. The concentration range in which the effect of addition is observed is about 5 mM or less, but the optimum concentration is considered to vary depending on the type and amplification size of the target DNA, the PCR buffer composition, and the like.

Example 5 FIG.
Confirmation of addition effect of oxalate ion in α-type enzyme (native Pfu) (Fig. 6)
The effect of adding oxalate ions was confirmed by PCR using nPfu polymerase. A PCR buffer attached to nPfu (manufactured by Stratagene) (20 mM Tris-HCl (pH 8.75), 10 mM KCl, 10 mM (NH 4) 2 SO 4, 2 mM MgSO 4, 0.1% Triton X-100, 100 ug / ml BSA) is used. 0.2 mM dNTPs, a primer pair described in SEQ ID NOS: 5 and 6 was used at 0.3 μM. Using nPfu DNA polymerase 2.5 U and 20 ng of Genomic DNA derived from human cultured cell K562 as a template, the influence on the PCR result due to the difference in the addition concentration of oxalate ions was examined. After the previous reaction at 94 ° C. for 2 minutes, PCR was performed with GeneAmp 2400 (PE Applied Biosystems) on a schedule of repeating 94 ° C., 15 seconds to 60 ° C., 30 seconds to 68 ° C. and 8 minutes for 35 cycles.
In the case of no oxalate ion addition, only smear was observed, but amplification of the target DNA could be confirmed in the range of 2 to 5 mM oxalate ion.
From the above results, the addition effect of oxalate ions was confirmed in the α-type enzyme. The concentration range in which the effect of addition is observed is about 5 mM or less, but the optimum concentration is considered to vary depending on the type and amplification size of the target DNA, the PCR buffer composition, and the like.

Example 6
Confirmation of effective salt type by PCR using α-type enzyme (native Pfu) (FIG. 7)
Next, under the same conditions as in Example 5, the types of salts (potassium chloride, potassium acetate, potassium oxalate, potassium sulfate) effective for PCR using nPfu DNA polymerase were investigated. In addition, the usage-amount of each salt was set to the ionic value of 4 and 6 mM which was most effective in Example 5. When the commonly used potassium salts (potassium chloride, potassium acetate, potassium oxalate, potassium sulfate) were added, amplification of the target DNA was not observed, and amplification was confirmed only when potassium oxalate was added. . From the above results, it became clear that the anionic oxalate ion is effective in PCR.

Example 7
Confirmation of effect of addition of oxalate ion on Hot start enzyme (KOD-plus) (FIG. 8)
In order to confirm the effect of addition of oxalate ions in the Hot start enzyme, examination was performed using a Hot start enzyme and KOD-plus DNA polymerase in which two kinds of KOD antibodies were added to KOD (α-type enzyme). A PCR buffer attached to KOD-plus (manufactured by Toyobo Co., Ltd.) was used, and 0.2 mM dNTPs and a primer pair described in SEQ ID NOs: 1 and 0.3 μM were used. KOD-plus DNA polymerase 2.5U was used, and 20 ng of genomic DNA derived from human cultured cell K562 was used as a template, and the influence on the PCR result due to the difference in the addition concentration of oxalate ions was examined. After the previous reaction at 94 ° C. for 2 minutes, PCR was performed with GeneAmp2400 (PE Applied Biosystems) on a schedule of repeating 94 cycles of 15 ° C., 15 seconds to 60 ° C., 30 seconds to 68 ° C. and 8.5 minutes. .
In the case of no oxalate ion addition, a number of extra bands were observed, but with the addition of potassium oxalate, the extra bands disappeared and only the target DNA could be amplified. The optimum concentration of potassium oxalate was about 3 mM, and when 5 mM was added, the amount of amplification of the target DNA was reduced.
From the above results, the effect of addition of oxalate ion was confirmed also in the Hot start enzyme. The concentration range in which the effect of addition is observed is about 5 mM or less, but the optimum concentration is considered to vary depending on the type and amplification size of the target DNA, the PCR buffer composition, and the like.

Example 8 FIG.
Confirmation of the type of salt effective in PCR using the Hot start enzyme (KOD-plus) (FIG. 9)
Next, under the same conditions as in Example 7, kinds of salts effective for PCR using KOD-plus DNA polymerase (potassium chloride, potassium acetate, potassium formate, potassium oxalate, sodium oxalate, potassium succinate, Potassium sulfate) was investigated. In addition, the usage-amount of each salt was made into the ion value 6 mM most effective in Example 7. FIG. When the commonly used potassium salts (potassium chloride, potassium acetate, potassium oxalate, potassium sulfate) are added, amplification of the target DNA is not observed, and only when potassium oxalate and sodium oxalate are added. The amplification effect of DNA was confirmed. In addition, potassium formate and succinate, which were considered to be relatively related from the chemical formula, were also examined, but the same effect as oxalate could not be confirmed. Potassium formate and potassium succinate, unlike oxalate, may have different concentration ranges that have an effect on PCR amplification, so the effect of addition to PCR cannot be completely denied. In certain concentrations, other carboxylates may also have an effect on PCR.
From the above results, it was clarified that the anionic oxalate ion also has an effect of adding to the Hot start enzyme.

Example 9
Confirmation of addition effect of malonate ion and maleate ion in Hot start enzyme (KOD-plus) (FIG. 10)
Next, under the same conditions as in Example 7, the types of salts (sodium malonate and sodium maleate) having an effect on PCR other than oxalate were investigated. When sodium malonate was added, an extra band decreased when 2 mM was added. When 6 mM was added, the extra band almost disappeared, and amplification of only the target DNA could be confirmed. In addition, in sodium maleate, a tendency to decrease the extra band was observed when 4 mM was added, and the extra band almost disappeared when 10 mM was added, confirming the amplification of only the target DNA.
From the above results, the addition effect could be confirmed also in malonate and maleate belonging to the same divalent carboxylate. Moreover, the concentration range in which the effect of addition was observed was higher than that of oxalate. The optimum concentration of each dicarboxylic acid is considered to vary depending on the type and amplification size of the target DNA, the PCR buffer composition, and the like.

  The present invention provides novel compositions and methods useful in the field of molecular biology, particularly useful in generating DNA from template nucleic acids and further DNA amplification. In addition, novel compositions and methods useful for polymerase chain reaction (PCR) are provided.

It is a substitute drawing of the electrophoresis photograph which confirmed the addition effect of an oxalate ion in PCR using mixed enzyme (EX-Taq). It is a substitute drawing of the electrophoresis photograph which confirmed the kind of salt with an effect by PCR using mixed enzyme (EX-Taq). It is a substitute drawing of the electrophoretic photograph which confirmed the kind of carboxylate which has an effect in PCR using mixed enzyme (EX-Taq). It is a substitute drawing of the electrophoretic photograph which confirmed the addition effect of the oxalate ion in PolI type enzyme (Taq). It is a substitute drawing of the electrophoretic photograph which confirmed the addition effect of the oxalate ion in PolI type enzyme (Taq). Evaluation is performed by PCR of a target DNA different from that in FIG. It is a substitute drawing of the electrophoresis photograph which confirmed the addition effect of the oxalate ion in alpha type enzyme (native Pfu). It is a substitute drawing of the electrophoresis photograph which confirmed the kind of salt which has an effect in PCR using alpha type enzyme (native Pfu). It is a substitute drawing of the electrophoretic photograph which confirmed the addition effect of the oxalate ion in Hot start enzyme (KOD-plus). It is a substitute drawing of the electrophoretic photograph which confirmed the kind of salt with an effect by PCR using the Hot start enzyme (KOD-plus). It is a substitute drawing of the electrophoresis photograph which confirmed the addition effect of malonate ion and maleate ion in Hot start enzyme (KOD-plus).

Claims (24)

A reaction solution composition comprising at least one of an oxalate ion, a malonate ion, and a maleate ion as an anionic substance effective for promoting DNA synthesis in an enzymatic reaction for synthesizing DNA. 2. The reaction liquid composition according to claim 1, wherein oxalate ion, malonate ion, and maleate ion are added in the form of an inorganic salt. 3. The reaction liquid composition according to claim 2, wherein oxalate ion, malonate ion and maleate ion are added in the form of a salt with an alkali metal, an alkaline earth metal or ammonium. 4. The reaction liquid composition according to claim 3, wherein oxalate ion, malonate ion, and maleate ion are added in a form containing at least one of potassium salt or sodium salt. Oxalate ion, malonate ion, maleate ion are zinc oxalate, ammonium oxalate, potassium oxalate, calcium oxalate, diethyl oxalate, oxalate N, N'-disuccinimidyl, dimethyl oxalate, oxalate Tin oxide, cerium oxalate, iron oxalate, copper oxalate, sodium oxalate, nickel oxalate, bis oxalate, oxalic acid (2,4-dinitrophenyl), oxalic acid (2,4,6-trichlorophenyl) , Manganese oxalate, methyl oxalate, lanthanum oxalate, lithium oxalate, or isopropylidene malonate, ethyl malonate, diethyl malonate, dibenzyl malonate, dimethyl malonate, thallium malonate, disodium malonate, or , Monosodium maleate, diethyl maleate, maleic acid The chlorpheniramine, di-n-butyl maleate, and mono-n-butyl maleate are prepared and added from at least one of the group consisting of mono-n-butyl maleate. Reaction liquid composition. As a reaction solution component for performing DNA synthesis, at least one of the group consisting of a template nucleic acid, DNA synthase, one or more oligonucleotide primers, one or more nucleotides, or derivatives, buffers, and salts thereof is used. The composition according to claim 1, which is contained. The composition according to claim 6, wherein the template nucleic acid is RNA. The composition according to claim 6, wherein the template nucleic acid is DNA. The composition according to claim 6 or 7, wherein the DNA synthase is a reverse transcriptase. The reverse transcriptase is AMV-RT, M-MLV-RT, HIV-RT, EIAV-RT, RAV2-RT, C.I. 10. The composition of claim 9, selected from the group consisting of C. hydroformans DNA polymerase, SuperScript I, SuperScript II, and mutants, variants and derivatives thereof. object. The composition according to claim 6 or 8, wherein the DNA synthase is a DNA polymerase. 12. The composition according to claim 11, wherein the DNA synthase is a heat-resistant DNA polymerase. The composition according to claim 11, wherein the DNA synthase is a heat-resistant α-type DNA polymerase. 12. The composition according to claim 11, wherein the DNA synthase is a heat-resistant Pol I type DNA polymerase. 12. The composition according to claim 11, wherein the DNA synthase is a mixed enzyme obtained by mixing a heat-resistant α-type DNA polymerase and a heat-resistant Pol I-type DNA polymerase. The DNA polymerase is Taq, EX-Taq, LA-Taq, Expand series, Platinum series, Tbr, Tfl, Tru, Tth, T1i, Tac, Tne, Tma, Tih, Tfi, Pfu, Pfubo, Pyrobest, Pwo, KOD. 12. A composition according to claim 11 selected from the group consisting of: Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENT, DEEPVENT, and mutants, variants and derivatives thereof. The buffer is Tris (TRIS), Tolidine (TRICINE), Bis-Tricine (BIS-TRICINE), Hepes (HEPES), Mops (MOPS), Tes (TES), Tapes (TAPS), Pipes (PIPES), And a composition selected from the group consisting of Caps (CAPS). The salt is potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate, magnesium chloride, magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, manganese sulfate, sodium chloride, sodium acetate, lithium chloride, and acetic acid. The composition of claim 6 selected from the group consisting of lithium. Further, as additives useful for DNA synthesis, DMSO, glycerol, formamide, betaine, tetramethylammonium chloride, PEG, Tween 20, NP40, ectoine, polyols, E. coli SSB protein, phage T4 gene 32 protein, BSA The composition of claim 6 comprising at least one selected from the group consisting of: The composition according to claim 6, wherein the nucleotide is deoxyphosphonucleotide or a derivative thereof. 21. The composition of claim 20, wherein the deoxyphosphonucleotide is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dITP, dUTP, [alpha] -thio-dNTPs, biotin-dUTP, fluorescein-dUTP, and sigoxygenin-dUTP. object. A method for synthesizing DNA using the composition according to any one of claims 1 to 21. The method according to claim 22, wherein the method for synthesizing DNA comprises polymerase chain reaction (PCR). 24. The method of claim 23, wherein said PCR amplification comprises repeating at least 25 DNA denaturation, annealing, and DNA polymerization processes.

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