JP2004141105A - Method for promoting dna synthesis reaction and composition therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new composition useful for carrying out production of DNA from a template nucleic acid and further DNA proliferation and a method therefor, and particularly to provide the new composition useful for polymerase chain reaction (PCR) or the like, and the method therefor. <P>SOLUTION: The reaction solution composition comprises (1) an anionic substance having effect for promoting DNA synthesis in an enzymatic reaction synthesizing the DNA based on the template nucleic acid and (2) at least one of a reagent having glycine as a base unit and DMSO. The present invention enables DNA proliferation to the object DNA in which proliferation has been difficult hitherto. <P>COPYRIGHT: (C)2004,JPO

Description

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【図面の簡単な説明】
【図１】混合型酵素（ＥＸ−Ｔａｑ）を用いたＰＣＲにおいてシュウ酸カリウムと他試薬の相乗効果を確認した電気泳動写真の代用図面である。
【図２】α型酵素（Ｐｆｕ）を用いたＰＣＲにおいてシュウ酸カリウムと他試薬の相乗効果を確認した電気泳動写真の代用図面である。
【図３】Ｈｏｔ　ｓｔａｒｔ酵素（ＫＯＤ−Ｐｌｕｓ）を用いたＰＣＲにおいてシュウ酸カリウムと他試薬の相乗効果を確認した電気泳動写真の代用図面１である。
【図４】Ｈｏｔ　ｓｔａｒｔ酵素（ＫＯＤ−Ｐｌｕｓ）を用いたＰＣＲにおいてシュウ酸カリウムと他試薬の相乗効果を確認した電気泳動写真の代用図面２である。
【図５】α型酵素（ＫＯＤ）を用いたＰＣＲにおいてシュウ酸カリウムとベタインのｌｏｎｇ
ＰＣＲに対する相乗効果を確認した電気泳動写真の代用図面である。
【図６】α型酵素（ＫＯＤ）を用いたＰＣＲにおいてベタインとシュウ酸カリウムのｌｏｎｇ
ＰＣＲに対する相乗効果を確認した電気泳動写真の代用図面である。
【図７】Ｌｏｎｇ　ＰＣＲにおけるＨｏｔ　ｓｔａｒｔ　ＰＣＲの効果を確認した電気泳動写真の代用図面である。
【図８】ＧＣ　ｒｉｃｈ　ｔａｒｇｅｔのＰＣＲに効果的な試薬のｌｏｎｇ　ＰＣＲへの効果を確認した電気泳動写真の代用図面である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention belongs to the field of molecular biology. The present invention relates to novel compositions and methods useful in generating DNA from template nucleic acids and performing further DNA amplification. The present invention relates specifically to novel compositions and methods useful for the polymerase chain reaction (PCR).
[0002]
[Prior art]
DNA amplification technology represented by PCR (for example, see Non-patent Document 4) is a well-known technology in the art. Detection, analysis, transcription and amplification of nucleic acids by PCR technology is one of the most important manipulation methods in modern molecular biology, especially for studying gene expression, infectious agents or genetic diseases. It is important in application to diagnosis of DNA, production of cDNA, analysis of retroviruses, and the like.
[0003]
Since the performance of DNA amplification depends on the performance of the DNA synthase to be used, various DNA synthases have been searched or improved from nature so far. For example, PCR was initially performed using a DNA synthetase derived from a normal-temperature bacterium such as Escherichia coli and having insufficient heat resistance. The PCR reaction involves thermocycling (between about 23 ° C. to about 100 ° C., preferably between about 37 ° C. to about 95 ° C.), multiple cyclings (preferably in the range of about 5 to about 99, more The success rate (the probability of actually amplifying the target DNA) is low due to the extremely harsh conditions that require more than about 20 cycles, most preferably about 25-40 cycles. However, at present, it is common knowledge to use a DNA synthase derived from a thermophilic bacterium and having excellent heat resistance.
[0004]
One of the important performances required for DNA synthase is "synthesis rate". The method of measuring the DNA synthesis rate is not particularly limited as long as it can be known by those skilled in the art. One of the typical methods is a single-stranded DNA (1.6 μg) of M13 and a primer complementary thereto. Using (16 pmol) ア as a substrate, various DNA polymerases, KOD, Pfu, Deep Vent, Taq, etc. (5 U) are reacted in respective buffers, and the reaction time and the size of synthesized DNA are determined. It can be calculated from the relationship.
For example, the DNA synthesis rates of each polymerase are KOD polymerase {105-130} bases / second, Pfu polymerase {24.8} bases / second, Deep Vent polymerase @ 23.3 bases / second, and Taq polymerase {61.0} bases / second (Takagi). , M. et al .: Characterization of DNA DNA polymerase from Pyrococcus sp. Strain KOD1 and applications applications to PCR. Appl. Environ. 450, 63, 450-63.
[0005]
One of the important performances required for DNA synthase is "accuracy". The method for measuring the accuracy of the DNA polymerase in the DNA synthesis reaction is not particularly limited as long as it can be known by those skilled in the art, but in the present invention, it was calculated from the mutation occurrence rate when the plasmid pMol # 21 was amplified by PCR.
[0006]
As an example of a method for evaluating the accuracy at the time of DNA synthesis, a method using a ribosomal protein S12 (rpsL) gene involved in streptomycin resistance can be mentioned. Streptomycin is an antibiotic that inhibits prokaryotic protein synthesis and binds to bacterial 30S ribosomal RNA (rRNA) to inhibit the initiation complex formation reaction of protein synthesis and causes misreading of the genetic code. In the streptomycin resistant mutant, a mutation is found in ribosomal protein S12. This mutation is known to exhibit a pleiotropic effect, such as suppressing the termination codon reading by the suppressor tRNA, in order to increase the translation fidelity of the ribosome. Therefore, when amplification is performed by PCR using the rpsL gene as a template, a mutation is introduced at a certain probability, and if it is a mutation at the amino acid level, the structure of the rpsL protein changes, and streptomycin cannot act on 30S ribosomal RNA. Something like that happens. Therefore, when Escherichia coli is transformed with the amplified plasmid DNA, the more mutations are introduced, the more frequently the streptomycin resistant bacteria appear.
[0007]
Plasmid pMol @ 21 (described in Journal of Molecular Biology (1999) 289, 835-850) is a plasmid containing the rpsL gene and the ampicillin resistance gene. A primer set for PCR amplification (one of which was biotinylated and an MluI restriction enzyme site was introduced) was designed on the ampicillin resistance gene of this plasmid, and the entire length of the plasmid was PCR-amplified with a heat-resistant DNA polymerase. The obtained PCR product was purified using streptavidin beads, cut out using the restriction enzyme MluI, and then ligated using DNA ligase to transform Escherichia coli. Accuracy during DNA synthesis can be determined by inoculating two types of plates containing ampicillin and ampicillin and streptomycin and calculating the ratio of colonies that appeared on each plate.
[0008]
When the accuracy of the PCR product of TaqΔ DNA polymerase was examined by the method for determining the accuracy at the time of DNA synthesis described above, the mutation insertion rate was 4% or more. On the other hand, the enzyme having 3'-5 'exonuclease activity alone has a mutation insertion rate of 0.05 to 1%, and the enzyme having no 3'-5' exonuclease activity, such as Taq DNA polymerase, has a 3'-5 'exonuclease activity. The mixed enzyme prepared by mixing an enzyme having -5 'exonuclease activity had a mutation insertion rate of 2 to 4%. Incidentally, the mutation insertion rate of KOD @ DNA polymerase is 0.1% or less, which is the most suitable enzyme for obtaining a highly accurate PCR product.
[0009]
One of the important performances required for a DNA synthase is a performance indicating the length of a target DNA that can be amplified (hereinafter referred to as “long-PCR performance”).
[0010]
In general, DNA polymerases, which are one type of thermostable DNA synthase, are broadly classified into Pol I-type enzymes typified by Taq DNA polymerase and Tth DNA polymerase, and α-type enzymes typified by Pfu DNA polymerase. In general, PolI type enzymes have a high DNA synthesis rate, but are inferior in accuracy due to lack of 3'-5 'exonuclease activity. On the other hand, the α-type enzyme has excellent 3′-5 ′ exonuclease activity and therefore has high accuracy, but has a low DNA synthesis rate. As described above, the above two types have excellent characteristics in terms of "synthesis speed" and "accuracy", but do not have both.
[0011]
For the purpose of combining the advantages of both types, a technique (mixed type enzyme) (mixed type enzyme) in which two types of thermostable enzymes are mixed and used has been disclosed (for example, see Non-Patent Document 1). It is said that this enzyme is mainly composed of a PolI type enzyme, and that DNA synthesis is mainly performed by a PolI type enzyme, and correction of an incorrect base is performed only by an α type enzyme.
[0012]
In addition, KOD DNA polymerase, which is a single enzyme (α-type enzyme) and has both “synthesis rate” and “accuracy”, is disclosed (for example, see Patent Document 1).
[0013]
The method for measuring the 3'-5 'exonuclease activity is not particularly limited as long as it can be known by those skilled in the art. One of the typical methods is to label the 3' end of the HindIII degradation product of λDNA with 3H. This is used as a substrate, and it is confirmed by examining the rate of elimination of 3H under the optimal conditions of each polymerase. More specifically, for example, a buffer solution (20 mM Tris-HCl pH 6.5, 10 mM KCl, 6 mM (NH4 ) Leave in 2 {SO4}, 2 mM {MgCl2}, 0.1% Triton {X-100, 10 μg / ml BSA)} and examine the ratio of [3H] TTP released. First, 0.2 mM of dATP, dGTP, dCTP and [3H] TTP were added to {HindIII digest (10 μg) of λ DNA, and the 3 ′ end was extended with Klenow polymerase. The DNA fragments are recovered by extraction and ethanol precipitation, and free mononucleotides are further removed by a span column (Clontech) to prepare.
[0014]
In addition, various studies have been made to improve the composition of a DNA synthesis reaction solution. Buffers include, for example, Tris (TRIS), Tricine (TRICINE), Bis-Tricine (BIS-TRICINE), Hepes (HEPES), Mops (MOPS), TES (TES), Tapes (TAPS), Pipes (PIPES), Caps (CAPS) and others were tried. In the salt, 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 acetic acid Lithium was tried. Examples of the additive include DMSO, glycerol, formamide, betaine, tetramethylammonium chloride, PEG, Tween 20, NP40, ectoine, polyols, Escherichia coli (E.coli) SSB protein, and phage T4 gene 32 A protein, BSA, has been attempted (see, for example, Patent Documents 2 to 6, Non-Patent Documents 2 and 3). The above reagents are not effective for all DNA synthases, and each enzyme is not suitable for its orientation.A unique reaction solution composition is examined for each enzyme, and an optimal reaction buffer is recommended for each enzyme. Therefore, it was considered impossible to achieve higher performance than the attached buffer.
[0015]
In addition, as a result of intensive studies, we have filed an application with the finding that anionic substances are also important for 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 the PCR reaction system. In addition, they have found that oxalate ion, malonate ion and maleate ion are particularly effective among divalent carboxyl ions.
[Patent Document 1]
Japanese Patent No. 312148
[Patent Document 2]
JP-T-09-511133 (Claim 1 etc.)
[Patent Document 3]
U.S. Pat. No. 5,545,539 (claim 1, etc.)
[Patent Document 4]
WO 96/12041 pamphlet (claim 1, etc.)
[Patent Document 5]
US Pat. No. 6,114,150 (Claim 1 etc.)
[Patent Document 6]
WO 99/46400 pamphlet (claim 1, etc.)
[Non-patent document 1]
Barns, @W. M. Author, @Proc. {Natl. {Acad. {Sci. USA, 91, 1994, p. 2216-2220
[Non-patent document 2]
Chevet, @E. Author, Nucleic Acids Research, Volume 23, 1995, p. 3343-3344
[Non-Patent Document 3]
Kovarova, @M. Author, Nucleic Acids Research, 28 volumes, 2000, p70
[Non-patent document 4]
Nature, {324} (6093), 1986, p. 13-19
[Non-Patent Document 5]
Kunkel, Journal of Biological Chemistry, 260, 1985, p. 5787-5796
[0016]
[Problems to be solved by the invention]
With respect to DNA amplification, particularly the required performance such as “synthesis rate”, “accuracy”, and “long-PCR performance” in PCR, the required level is increasingly higher with the development of biotechnology in recent years. In particular, as the entire sequence of the human genome has been elucidated, the focus of research is not only on detecting genes and analyzing differences, but also on analyzing the functions of the genes themselves or the proteins encoded by the genes. The importance of accuracy in DNA amplification is becoming more important in view of the fact that
Therefore, it is desired to establish a DNA amplification method that has both of these. However, in any of the above prior arts, the performance of all of "synthesis speed", "accuracy", and "long-PCR performance" is at the highest level. I can't say.
[0017]
[Means for Solving the Problems]
As a result of diligent research, we have found that the DNA amplification performance is further improved by combining a divalent carboxyl ion composed of a dicarboxylate and at least one of a reagent having glycine as a basic unit and DMSO. In addition, the inventors have found that oxalate ions are particularly effective among divalent carboxyl ions, and trimethylglycine is particularly effective among reagents having glycine as a basic unit. Further, they have found that the use of oxalate ion and DMSO also has a synergistic effect, and thus completed the present invention.
Improvement of DNA amplification performance by the synergistic addition effect includes, specifically, successful amplification of a PCR target having a high GC content, successful amplification of a long-chain target which has been impossible so far, and the like. Further, improvement in the amount of PCR amplification is one of the synergistic effects of the present patent.
[0018]
That is, the present invention has the following configuration.
Item 1. (1) In an enzyme reaction for synthesizing DNA based on a nucleic acid serving as a template, at least one of an anionic substance effective for promoting DNA synthesis and (2) a reagent having glycine as a basic unit or DMSO is used. A reaction solution composition comprising:
Item 2. Item 3. The reaction solution composition according to Item 1, wherein the reagent having glycine as a basic unit is trimethylglycine (betaine).
Item 3. Item 3. The reaction solution composition according to Item 1 or 2, wherein the anionic substance effective in promoting DNA synthesis is a substance having a carboxyl group.
Item 4. Item 4. The reaction solution composition according to Item 3, wherein the substance having a carboxyl group is a dicarboxylate.
Item 5. The dicarboxylate is zinc oxalate, ammonium oxalate, potassium oxalate, calcium oxalate, diethyl oxalate, N, N'-disuccinimidyl oxalate, dimethyl oxalate, tin oxalate, cerium oxalate, oxalate Iron oxide, 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, maleic acid Diethyl, chlorpheniramine maleate, maleate di n- butyl, is selected from the group consisting of maleic acid mono -n- butyl ester A composition according to claim 4.
Item 6. Item 6. The composition according to items 2 to 5, wherein the concentration of trimethylglycine (betaine) is 0.5 to 1.5 M and / or the concentration of DMSO is 2 to 5%.
Item 7. [7] The composition according to [1] to [6], further comprising one or more enzymes having DNA polymerase activity.
Item 8. Item 8. The composition according to Item 7, wherein the enzyme having DNA polymerase activity is selected from the group consisting of DNA polymerase and reverse transcriptase.
Item 9. The DNA polymerase is Taq, EX-Taq, LA-Taq, Expand series, Plutinum series, Tbr, Tfl, Tru, Tth, T1i, Tac, Tne, Tma, Tih, Tfi, Pfu, Pfutubo, Pyrobe, Pwo, KOD. Item 9. The composition according to Item 8, which is selected from the group consisting of Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENT, DEEPVENT, KOD, and mutants, variants and derivatives thereof.
Item 10. Item 9. The composition according to Item 8, wherein the DNA polymerase is a thermostable DNA polymerase having a DNA synthesis rate of at least 30 bases / second and having 3'-5 'exonuclease activity.
Item 11. (11) The DNA polymerase according to (10), wherein the DNA polymerase is a thermostable DNA polymerase having a DNA synthesis rate of at least 30 bases / second and a probability of an error occurring in PCR using pMol # 21 as a template of 4% or less. Composition.
Item 12. {The reverse transcriptase is AMV-RT, M-MLV-RT, HIV-RT, EIAV-RT, RAV2-RT, C.I. Item 8. The composition according to Item 8, which is selected from the group consisting of hydrogenoformans DNA polymerase, SuperScript I, SuperScript II, and mutants, variants and derivatives thereof. object.
Item 13. Item 13. The composition according to Item 8 or 12, wherein the RNase H activity of the reverse transcriptase is substantially reduced.
Item 14. Items containing at least one of a group consisting of a nucleic acid serving as a template for DNA synthesis, a DNA synthase, one or more oligonucleotide primers, one or more nucleotides or derivatives thereof, a buffer, and a salt. Composition.
Item 15. 15. The composition according to item 14, wherein the nucleotide or a derivative thereof is a deoxyphosphonucleotide or a derivative thereof.
Item 16. 16. The composition according to claim 15, wherein the deoxyphosphonucleotide or a derivative thereof is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dITP, dUTP, α-thio-dNTPs, biotin-dUTP, fluorescein-dUTP, and sigoxigenin-dUTP. .
Item 17. A method for synthesizing DNA, the method comprising: (a) mixing a nucleic acid template with one or more compositions according to items 1 to 16 to form a mixture; and (b) the template The DNA synthesis rate is at least 30 bases / second and the probability of occurrence of an error in PCR using pMol @ 21 as a template is 4% to produce a first nucleic acid molecule complementary to all or part of Incubating the mixture under the following conditions:
Item 18. In order to prepare a second nucleic acid molecule complementary to the whole or part of the first nucleic acid molecule, an error occurs in PCR using a DNA synthesis rate of at least 30 bases / second and pMol @ 21 as a template. Item 18. The method according to Item 17, further comprising a step of incubating the first nucleic acid molecule under a condition that the probability of the occurrence is 4% or less.
Item 19.核酸 A nucleic acid molecule produced according to the method of item 17.
Item 20. A method for amplifying a DNA molecule, the method comprising: (a) mixing a nucleic acid template with one or more compositions according to items 1 to 16 to form a mixture; and (B) In order to amplify a nucleic acid molecule complementary to the whole or a part of the template, the DNA synthesis rate is at least 30 bases / second, and the probability that an error occurs in PCR using pMol @ 21 as a template is low. Incubating the mixture under conditions that are 4% or less.
Item 21. A method for sequencing a DNA molecule, the method comprising: (a) adding one or more primers to the nucleic acid molecule to be sequenced, one or more compositions according to items 1-16. Mixing with one or more nucleotides and one or more termination factors to form a mixture; (b) synthesizing a population of molecules complementary to all or a portion of the molecule to be sequenced. Incubating the mixture under conditions where the DNA synthesis rate is at least 30 bases / second and the probability of error occurring in PCR using pMol @ 21 as a template is 4% or less; and (c) Separating the population to determine the nucleotide sequence of all or part of the molecule to be sequenced.
Item 22. Further, the method of {17, 18, 20, 21} employing Hot \ start \ PCR.
Item 23. 17. A kit for use in synthesizing a DNA molecule, the kit comprising one or more compounds or components according to any one of [1] to [16].
Item 24. A kit for use in synthesizing a DNA molecule consisting of two or more parts, wherein one or more compounds or components according to any one of Items 1 to 16 are any one of the kits. A kit characterized by being included as a component of a part, being dispersed in several parts or concentrated in one part.
[0019]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the additive for promoting the DNA synthesis reaction of the present invention will be specifically described.
The present invention provides at least (1) an anionic substance which is effective in promoting DNA synthesis in an enzymatic reaction for synthesizing DNA based on a nucleic acid serving as a template, and (2) a reagent having glycine as a basic unit or DMSO. A reaction solution composition comprising:
[0020]
In the present invention, “promoting a DNA synthesis reaction” means that “synthesis rate” and / or “long-PCR performance” are improved.
[0021]
The anionic substance which is effective in promoting DNA synthesis and which is contained in the reaction solution composition of the present invention is not particularly limited, and examples thereof include substances having a carboxyl group. Preferably, a dicarboxylate is used. The dicarboxylate is added, for example, in the form of an inorganic salt. It preferably contains at least one of oxalate ion, malonate ion and maleate ion. More preferably, these inorganic salts are in the form of salts with alkali metals (preferably in the form of potassium or sodium salts) or with alkaline earth metals or ammonium.
[0022]
Specifically, zinc oxalate, ammonium oxalate, potassium oxalate, calcium oxalate, diethyl oxalate, N, N'-disuccinimidyl oxalate, dimethyl oxalate, tin oxalate, cerium oxalate, oxalate Iron oxide, 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, maleic acid Diethyl, chlorpheniramine maleate, di-n-maleate Chill, maleic acid mono -n- butyl ester and the like, both, or the like can be used commercially available products.
[0023]
The above-mentioned anionic substance is usually used in a concentration range of 0.1 to 20 mM. Preferably it is 0.1 to 10 mM, more preferably 1 to 10 mM.
[0024]
The reagent having glycine as a basic unit, which is contained in the reaction solution composition of the present invention, is not particularly limited, but preferably includes trimethylglycine (betaine), and a commercially available product can be used. The effective concentration of trimethylglycine (betaine) is 0.5 to 2M, preferably 0.5 to 1.5M, and more preferably 1 to 1.5M. On the other hand, a commercially available product can be used as DMSO. The effective concentration of DMSO is 0.1 to 15%, preferably 2 to 10%, and more preferably 5 to 10%.
[0025]
In the reaction solution composition of the present invention, the effect can be expected to be further enhanced by adding both the anionic substance and a reagent having glycine as a basic unit or DMSO to form an optimal combination. Determining the optimal combination of oxalate ion concentration and betaine concentration may require consideration of the type and amplification size of the target DNA, the composition of the PCR buffer, and the like.
[0026]
The reaction solution composition of the present invention can be used for an enzyme reaction for synthesizing DNA. The DNA synthase contained in the reaction solution composition of the present invention is not particularly limited, and examples thereof include DNA polymerase and reverse transcriptase. The DNA synthase is preferably heat-resistant. Commercially available products can be used for these enzymes. Further, the reaction solution composition of the present invention can also include a plurality of enzymes for synthesizing these DNAs having various different properties.
[0027]
The DNA polymerase contained in the reaction solution composition of the present invention is roughly classified into α-type DNA polymerase and PolI-type DNA polymerase. Generally, it is said that the PolI type enzyme has a high DNA synthesis rate, but is inferior in accuracy due to lack of 3'-5 'exonuclease activity. On the other hand, the α-type enzyme is said to have excellent 3′-5 ′ exonuclease activity and thus has high accuracy, but is said to have a low DNA synthesis rate. An α-type DNA polymerase and a Pol I-type DNA polymerase can be mixed to form a mixed enzyme.
[0028]
PolI type enzymes are represented by Taq @ DNA polymerase and Tth @ DNA polymerase. In addition, EX-Taq, LA-Taq, Expand series, Plutinum series, Tbr, Tfl, Tru, T1i, Tac, Tne, Tma, Tih, Tfi and mutants, variants and derivatives thereof, and the like. The α-type enzyme is represented by Pfu, and in addition, Pfutubo, Pyrobest, Pwo, KOD, Bst, Sac, Sso, Poc, Pub, Mth, Pho, ES4, VENT, DEEPVENT, and mutants thereof , Variants and derivatives.
[0029]
The DNA polymerase used in the present application is a thermostable DNA polymerase having a DNA synthesis rate of at least 30 bases / second and a probability that an error occurs in PCR using pMol # 21 as a template is 4% or less. The DNA polymerase used in the present application can more preferably amplify a target DNA having an error probability of 1% or less and / or 20 kb or more in PCR using pMol # 21 as a template.
The selection of the DNA polymerase can be appropriately selected in consideration of performance such as “synthesis rate”, “accuracy”, “long-PCR performance”, and “heat resistance”. Several enzymes can be combined for selection.
From the viewpoint of accuracy, since the accuracy mainly depends on the presence or absence of 3′-5 ′ exonuclease activity which is a characteristic of the enzyme, an enzyme having 3′-5 ′ exonuclease activity (for example, α-type enzyme) is used. It is preferable to select.
Among them, KOD is particularly preferable because it is excellent in performance other than accuracy. KOD is a thermostable DNA having a DNA synthesis rate of at least 30 bases / second, having a 3'-5 'exonuclease activity, and having an error probability of 1% or less in PCR using pMol @ 21 as a template. It is a polymerase. Furthermore, a target DNA of 20 kb or more can be amplified.
[0030]
The reaction solution composition of the present invention may contain a reverse transcriptase. Examples of reverse transcriptase include AMV-RT, M-MLV-RT, HIV-RT, EIAV-RT, RAV2-RT, C.I. Hydrogenformans DNA polymerase, SuperScript I, SuperScript II, and mutants, variants and derivatives thereof. The RNase H activity of the reverse transcriptase may be substantially reduced by modification or the like.
[0031]
The reaction solution composition of the present invention is useful for DNA synthesis, as well as a nucleic acid serving as a template, one or more oligonucleotide primers, one or more nucleotides or derivatives thereof, a buffer, a salt, and DNA synthesis. At least one of the group consisting of various additives.
[0032]
Specifically, the reaction solution composition of the present invention further comprises, in addition to DNA synthase, forward and reverse primers having a base sequence complementary to the target nucleic acid, four types of dNTPs, divalent cations, A buffer and the like can be included. Examples of the divalent cation include a magnesium ion. The concentration is preferably about 1 to 3 mM. Examples of the buffer include Tris buffer {(pH 6.5, 75 ° C.)} and Tricine buffer {(pH 6.5, 75 ° C.)}.
One specific composition is as follows. 20 mM {Tris-HCl} (pH 6.5, 75 ° C) 10 mM {KCl 6 mM} (NH 4) 2 SO 41 to 3 mM Mg 2 Cl 20.1% Triton X-10010 μg / ml BSA 20 to 200 μM dNTPs 0.1 pM to 1 μM Primer 0.1 to 250 ng template DNA.
[0033]
Alternatively, the reaction solution composition of the present invention further comprises, in addition to DNA synthase, forward and reverse primers having a base sequence complementary to the target nucleic acid, four dNTPs, a divalent cation, a buffer, It may contain a nucleic acid probe, a coloring reagent, a luminescent reagent, and the like.
[0034]
One or more nucleotides or derivatives thereof contained in the reaction solution composition of the present invention are not particularly limited, and include deoxyphosphonucleotides or derivatives thereof. Specific preferred examples of the composition include substances selected from the group consisting of dATP, dCTP, dGTP, dTTP, dITP, dUTP, α-thio-dNTPs, biotin-dUTP, fluorescein-dUTP, and sigoxigenin-dUTP.
[0035]
Salts contained in the reaction solution composition of the present invention are not particularly limited, but 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. These salts can be easily obtained as commercial products.
[0036]
The reaction solution composition of the present invention is further added with an antibody of DNA synthase to completely suppress the enzymatic activity at room temperature, suppress the production of by-products such as primer dimers, and protect the degradation of primer. A hot start antibody for a method for increasing the PCR success rate (Hot start PCR method), a known enhancer, and the like can be included.
[0037]
The present invention also provides a method for synthesizing DNA, the method comprising: (a) using a nucleic acid template as an effect of promoting DNA synthesis in an enzymatic reaction for synthesizing DNA based on (1) a nucleic acid serving as a template; (2) mixing with a reaction solution composition characterized by containing an anionic substance having the following properties and (2) at least one of a reagent having glycine as a basic unit or DMSO; and (b) ) Probability of generating an error in PCR using pMol @ 21 as a template when the rate of DNA synthesis is at least 30 bases / second to prepare a first nucleic acid molecule complementary to all or a part of the template. Incubating the mixture under conditions where is less than or equal to 4%. Further, the invention includes a nucleic acid molecule made according to the method.
[0038]
In the above method (b), the condition that the probability of occurrence of an error in PCR using pMol @ 21 as a template is 1% or less and / or the condition under which a target DNA of 20 kb or more can be amplified. preferable.
[0039]
The present invention further provides the method for synthesizing DNA, wherein a second nucleic acid molecule complementary to the whole or a part of the first nucleic acid molecule is produced at a DNA synthesis rate of at least 30 bases / second, In addition, the method further comprises a step of incubating the first nucleic acid molecule under a condition that a probability that an error occurs in PCR using pMol # 21 as a template is 4% or less.
[0040]
In the above method (b), the condition that the probability of occurrence of an error in PCR using pMol @ 21 as a template is 1% or less and / or the condition under which a target DNA of 20 kb or more can be amplified. preferable.
[0041]
The present invention further provides a method for amplifying a DNA molecule, comprising the steps of: (a) converting a nucleic acid template to an anionic substance effective in promoting DNA synthesis; and (2) glycine. Mixing with a reaction solution composition containing at least one of the reagent or DMSO in the basic unit to form a mixture; and (b) a nucleic acid complementary to all or a part of the template Incubating the mixture under conditions where the rate of DNA synthesis is at least 30 bases / second and the probability of error occurring in PCR using pMol # 21 as a template is 4% or less for amplifying the molecule. And a method comprising:
[0042]
In the above method (b), the condition that the probability of occurrence of an error in PCR using pMol @ 21 as a template is 1% or less and / or the condition under which a target DNA of 20 kb or more can be amplified. preferable.
[0043]
Examples of the enzyme reaction for synthesizing DNA used in the method of the present invention include a reaction with a DNA polymerase and a reaction with a reverse transcriptase. Among them, it is particularly effective in a reaction by a DNA polymerase, especially in a gene amplification method based on PCR. Further, the PCR amplification is more effective in a method in which DNA denaturation, annealing, and DNA polymerization processes are repeated at least 25 times.
[0044]
The gene amplification method based on the PCR method comprises repeating the three-step cycle 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 region of a target nucleic acid sandwiched between a pair of primers (Nature, {324} (6093), 13-13-19 (1986)). That is, the nucleic acid of the sample is denatured in the denaturation step, each primer is hybridized to a region on the single-stranded target nucleic acid complementary to each primer in the subsequent annealing step, and in the subsequent elongation step, the DNA is obtained starting from each primer. The DNA strand complementary to each single-stranded target nucleic acid serving as a template is extended by the action of the polymerase to obtain a 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 theoretically becomes 2ⁿIt is amplified twice.
[0045]
Preferred reaction conditions for amplification are thermocycling, ie, changing the temperature of the reaction mixture to achieve each step of the PCR cycle. Thermal cycling is performed in a temperature range between about 23C and about 100C, preferably between about 37C and about 95C. Nucleic acid denaturation is usually performed at 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. Extension of the DNA is usually carried out at between about 55C and about 80C, preferably at about 68-72C. Cycling numbers will vary greatly depending on the amount of DNA product desired. The number of PCR cycles preferably ranges from about 5 to about 99, more preferably more than about 20 cycles, and most preferably about 25-40 cycles.
[0046]
In the present invention, a target nucleic acid can be detected from an amplification product generated by the above amplification reaction using, for example, a labeled probe. The labeled probe is an oligonucleotide having a base sequence complementary to the labeled nucleic acid, and binds a labeled substance or a labeled binding substance. Examples of the labeling substance include enzymes such as alkaline phosphatase, peroxidase and galactosidase, fluorescent substances and radioactive substances, and examples of the label binding substance include biotin and digoxigenin. The labeling substance may be bound via biotin, digoxigenin or avidin. As a method for introducing these labels into the probe, the oligonucleotide is synthesized by using, as a component of dNTP, dNTP that binds these labeling substances or label binding substances at the time of oligonucleotide synthesis.
[0047]
For detecting a nucleic acid bound to a labeled probe, a conventionally known method, for example, a Southern hybridization or Northern hybridization method can be used. These methods take advantage of the formation of hybrids when single-stranded DNAs and RNAs have complementarity with each other, to separate unknown nucleic acid fragment groups by, for example, agarose electrophoresis, and then to perform gel separation. Is made into a single strand by, for example, alkali treatment, and then transferred to a filter, immobilized, and then hybridized with a labeled probe. For the detection of a label, for example, when alkaline phosphatase is used as a labeling substance, when a chemiluminescent substrate, for example, a 1,2-dioxetane compound (PPD) is reacted, only the nucleic acid that has formed a hybrid emits light. By exposing this to an X-ray film, the size of the target nucleic acid and its position on electrophoresis can be confirmed.
[0048]
Another embodiment of the present invention is a method for sequencing a DNA molecule, comprising the steps of: (a) using the nucleic acid molecule to be sequenced with one or more primers to enhance DNA synthesis. , A reaction solution composition comprising at least one of a reagent having glycine as a basic unit or DMSO, and one or more nucleotides, and one or more nucleotides. (B) mixing at least 30 bases / second to synthesize a population of molecules complementary to all or a portion of the molecule to be sequenced. And incubating the mixture under conditions where the probability of an error occurring in PCR using pMol # 21 as a template is 4% or less; and (c) separating the population and separating the sequence Determining the entire or part of the nucleotide sequence of the molecule to be constant include, a method.
[0049]
In the above method (b), the condition that the probability of occurrence of an error in PCR using pMol @ 21 as a template is 1% or less and / or the condition under which a target DNA of 20 kb or more can be amplified. preferable.
[0050]
As a method for sequencing nucleic acids, the Maxam-Gilbert method (Maxam, AM and Gilbert, W., Proc. Natl. Acad. Sci. USA # 74: 560-564, 1997) and the Sanger method ( Sanger, F. {and} Coulson, AR, J. Mol. Biol. 94: 444-448, 1975) are widely known. In the Maxam-Gilbert method, DNA is radiolabeled, split into four samples, and treated with chemicals that selectively destroy specific nucleotide bases in the DNA and cleave the molecule at the site of damage. The sequence of the original DNA molecules can then be read from the film by separating the resulting fragments into different bands by gel electrophoresis and exposing the gel to X-ray film. The Sanger method, on the other hand, uses the DNA-synthesizing activity of DNA polymerase, which is based on the reaction terminating dideoxynucleoside triphosphate (Sanger, F. et al., Proc. Natl. Acad. Sci. USA # 74: 5463-5467, 1977). Combination with a mixture of short primers, any of which can be detectably labeled, yields a series of newly synthesized DNA fragments specifically terminated with one of the four dideoxy bases. These fragments are then analyzed by gel electrophoresis to determine the sequence. By performing four separate reactions (once for each ddNTP), the sequence of fairly complex DNA molecules can be rapidly determined (Sanger, F. et al., Nature 265: 678-695, 1977; Barnes, 1987). W., Meth.Enzymol. 152: 538-556, 1987).
[0051]
The present invention relates to a kit for use in synthesizing a DNA molecule, the kit having (1) an anionic substance effective in promoting DNA synthesis, and (2) glycine as a basic unit. A kit containing at least one of a reagent and DMSO.
[0052]
The kit may be composed of two or more parts, and the compound or component may be dispersed in several parts or concentrated in one part as a component of any part of the kit. Can be included.
[0053]
The kit of the present invention can further contain, in addition to the DNA synthase, various components that can be specifically included in the above-mentioned reaction solution composition.
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.
[0054]
Embodiment 1 FIG.
Mixed enzyme (EX-Taq) Effect of oxalate ion and betaine in cereal (Fig. 1 )
Normally, a synergistic effect of oxalate ion and betaine was confirmed on a target that could not be amplified by PCR using EX-Taq DNA polymerase (Takara Shuzo). PCR buffer was added to EX-Taq, potassium oxalate was added to a final concentration of 2 mM, and 0.2 mM dNTPs, a pair of primers described in SEQ ID NOS: 1 and 2 and 0.3 μM were used. Using 1.0 U of EX-Taq DNA polymerase and 20 ng of Genomic DNA derived from human cultured cell K562 as a template, the effect of different amounts of betaine on the PCR results was examined. After the pre-reaction at 94 ° C. for 2 minutes, PCR was performed using GeneAmp2400 (PE Applied Biosystems) on a schedule of repeating 35 cycles of 94 ° C., 15 seconds-60 ° C., 30 seconds-68 ° C., 8.5 minutes. . The results are shown in FIG.
[0055]
When betaine was not added, amplification of the target DNA was slightly observed, but when 0.5 M betaine was added, an increase in the amount of amplification was observed. From the above results, a synergistic effect of oxalate ion and betaine in the mixed enzyme was confirmed.
[0056]
Embodiment 2. FIG.
α-type enzyme (Native Pfu) Effect of oxalate ion and betaine in cereal (Fig. 2 )
In general, a synergistic effect of oxalate ion and betaine was confirmed on a target that could not be amplified by PCR using nPfu polymerase. PCR buffer is supplied with nPfu (manufactured by Stratagene) (20 mM Tris-HCl (pH 8.75), {10 mM KCl, {10 mM} (NH₄)₂SO₄, {2 mM} MgSO₄, {0.1%} Triton X-100, {100 ug / ml} BSA), potassium oxalate was added to a final concentration of 2 mM, and 0.2 mM @ dNTPs, a primer pair described in SEQ ID NOS: 5 and 6, 0.3 μM was used. . Using 2.5 U of nPfu DNA polymerase and 20 ng of Genomic DNA derived from human cultured cell K562 as a template, the effect of different amounts of betaine on the PCR results was examined. After a pre-reaction at 94 ° C. for 2 minutes, PCR was performed using GeneAmp2400 (PE Applied Biosystems) on a schedule of repeating 35 cycles of 94 ° C., 15 seconds-60 ° C., 30 seconds-68 ° C., 8 minutes. FIG. 2 shows the results.
[0057]
When no betaine was added, only a smear was observed, but amplification of the target DNA was confirmed when 0.5 M betaine was added. From the above results, a synergistic effect of oxalate ion and betaine was confirmed also in the α-type enzyme.
[0058]
Embodiment 3 FIG.
Hot start enzyme (KOD-plus) Effect of oxalate ion and betaine in cereal (Fig. 3 )
In order to confirm the synergistic effect of oxalate ion and betaine in the hot start enzyme, using hot start enzyme and KOD-plus DNA polymerase obtained by adding two kinds of KOD antibodies to KOD (α-type enzyme), ordinary PCR was performed. The study was performed on a target that was difficult to amplify. PCR buffer was added to KOD-plus (manufactured by Toyobo Co., Ltd.) to which potassium oxalate was added to a final concentration of 2 mM. Using 1.0 U of KOD-plus DNA polymerase and 20 ng of Genomic DNA derived from human cultured cell K562 as a template, the effect of different amounts of betaine on the PCR results was examined. After the pre-reaction at 94 ° C. for 2 minutes, PCR was performed using GeneAmp2400 (PE Applied Biosystems) on a schedule of repeating 35 cycles of 94 ° C., 15 seconds-60 ° C., 30 seconds-68 ° C., 8.5 minutes. . The results are shown in FIG.
[0059]
When betaine was not added, amplification of the target DNA was slightly observed, but an increase in the amount of amplification was observed when 0.5 to 1.5 M betaine was added. The amount of amplification reached the maximum when 1.5 M betaine was added. From the above results, a synergistic effect of oxalate ion and betaine was confirmed in the Hot Start enzyme.
[0060]
Embodiment 4. FIG.
Hot start enzyme (KOD-plus) The synergistic effect of oxalate ion and DMSO on 4 )
In order to confirm the synergistic effect of oxalate ion and DMSO on the hot start enzyme, a target that was difficult to amplify by ordinary PCR was examined using the hot start enzyme and KOD-plus DNA polymerase. PCR buffer was added to KOD-plus (manufactured by Toyobo Co., Ltd.), potassium oxalate was added to a final concentration of 2 mM, and 0.2 mM dNTPs, a primer pair described in SEQ ID NOS: 5 and 6 and 0.3 μM were used. As a template, 0.5 μL of cDNA obtained by reverse transcription of 500 ng of total RNA from human cultured cell K562 using Thermoscript (manufactured by Invitrogen) was used. Using 1.0 U of KOD-plus DNA polymerase, the effect of differences in the amount of DMSO added on the PCR results was examined. After a pre-reaction at 94 ° C. for 2 minutes, PCR was performed using GeneAmp2400 (PE Applied Biosystems) on a schedule of repeating 40 cycles of 94 ° C., 15 seconds-60 ° C., 30 seconds-68 ° C., 9 minutes. FIG. 4 shows the results.
[0061]
When no DMSO was added, amplification of the target DNA was slightly confirmed, but the amount of amplification was increased by the addition of DMSO, and sufficient amplification was confirmed when 5% DMSO was added. From the above results, a synergistic effect of oxalate ion and DMSO in the Hot start enzyme was confirmed.
[0062]
Embodiment 5 FIG.
α-type enzyme (KOD) Using PCR Of potassium oxalate and betaine long PCR The synergistic effect on 5 )
In order to confirm the synergistic effect of oxalate ion and betaine on long PCR, KOD DNA polymerase was used to examine a target exceeding 20 kb derived from the human genome, which was not reported to be amplified by α-type enzyme alone. PCR buffer was added to KOD-plus (manufactured by Toyobo Co., Ltd.) to which potassium oxalate was added to a final concentration of 2 mM, and 0.4 mM dNTPs, a primer pair described in SEQ ID NOS: 7 and 8 and 0.3 μM were used. Using 2.0 U of KOD DNA polymerase and 200 ng of Genomic DNA derived from human cultured cell K562 as a template, the effect of different amounts of betaine on the PCR results was examined. After pre-reaction at 94 ° C for 2 minutes, 98 ° C, 10 seconds-74 ° C, 18 minutes (5 cycles)-98 ° C, 10 seconds-72 ° C, 18 minutes (5 cycles) -98 ° C, 10 seconds-70 GeneAmp2400 (PE Applied Biosystems) on a schedule of repeating a cycle of 18 ° C, 18 minutes (5 cycles)-98 ° C, 10 seconds-68 ° C, 18 minutes (25 cycles), and finally an additional reaction at 68 ° C, 7 minutes. Was used to perform PCR. The results are shown in FIG.
[0063]
At a betaine concentration of 1.2 M or less, amplification of the target DNA was not observed, but sufficient amplification was confirmed when using 1.4 M and # 1.6 M. The optimal concentration of betaine differs depending on the enzyme used, and the amount of betaine used is not limited to the above concentration.
[0064]
Embodiment 6 FIG.
α-type enzyme (KOD) Using PCR Of betaine and potassium oxalate long PCR The synergistic effect on 6 )
In order to confirm the synergistic effect of oxalate ion and betaine on long PCR, KOD DNA polymerase was used to examine a target exceeding 20 kb derived from the human genome, which was not reported to be amplified by α-type enzyme alone. PCR buffer was added to KOD-plus (manufactured by Toyobo Co., Ltd.) to which betaine was added to a final concentration of 1.5 M, and 0.4 mM ΔdNTPs and a primer pair 0.3 μM were used. The primer pairs described in SEQ ID NOS: 9 and 10 were used for PCR of tPA22 kb, and the primer pairs described in SEQ ID NOS: 9 and 11 were used for PCR of tPA24 kb. Using 2.0 U of KOD DNA polymerase and 200 ng of Genomic DNA derived from human cultured cell K562 as a template, the effect of different amounts of potassium oxalate on the PCR results was examined. After pre-reaction at 94 ° C for 2 minutes, 98 ° C, 10 seconds-74 ° C, 18 minutes (5 cycles)-98 ° C, 10 seconds-72 ° C, 18 minutes (5 cycles) -98 ° C, 10 seconds-70 GeneAmp2400 (PE Applied Biosystems) on a schedule of repeating a cycle of 18 ° C, 18 minutes (5 cycles)-98 ° C, 10 seconds-68 ° C, 18 minutes (25 cycles), and finally an additional reaction at 68 ° C, 7 minutes. Was used to perform PCR. FIG. 6 shows the results.
[0065]
When tPA22kb was targeted, amplification of the target was confirmed only when 2 mM potassium oxalate was added. When tPA24 kb was used as a target, amplification of the target was slightly confirmed when 1.5 mM or 2.0 mM potassium oxalate was added. Since the optimal concentration of potassium oxalate varies depending on the enzyme used, the amount of potassium oxalate used is not limited to the above concentration.
[0066]
Embodiment 7 FIG.
Long PCR In Hot start PCR Confirm the effect of 7 )
Under the above reaction conditions in which oxalate ions and betaine coexist, in order to confirm the effect of hot start PCR using an antibody, long PCR using KOD DNA polymerase was examined. PCR @ buffer was added to KOD-plus (manufactured by Toyobo Co., Ltd.) to which a final concentration of 2 mM potassium oxalate and 1.5 M betaine were added, and 0.4 mM @dNTPs and a primer pair 0.3 μM were used. The tPA12 kb PCR had the primer pairs described in SEQ ID NOS: 9 and 12, the tPA15 kb PCR had the primer pairs described in SEQ ID NOs. 9 and 13, the tPA18 kb PCR had the primer pairs described in SEQ ID NOs. Primer pairs described in SEQ ID NOS: 9 and 10 for PCR of tPA22 kb, primer pairs described in SEQ ID NOS: 9 and 11 for PCR of tPA24 kb, and primer pairs described in SEQ ID NOS: 15 and 16 for PCR of β-globin17.5 kb It was used. Using 2.0 U of KOD DNA polymerase and 200 ng of Genomic DNA derived from human cultured cell K562 as a template, the effect of Hot start PCR was examined with and without an antibody. After pre-reaction at 94 ° C for 2 minutes, 98 ° C, 10 seconds-74 ° C, 18 minutes (5 cycles)-98 ° C, 10 seconds-72 ° C, 18 minutes (5 cycles) -98 ° C, 10 seconds-70 GeneAmp2400 (PE Applied Biosystems) on a schedule of repeating a cycle of 18 ° C, 18 minutes (5 cycles)-98 ° C, 10 seconds-68 ° C, 18 minutes (25 cycles), and finally an additional reaction at 68 ° C, 7 minutes. Was used to perform PCR. FIG. 7 shows the results.
[0067]
When evaluation was performed with six types of targets, reduction of smearing and expansion of amplification amount were generally observed by using the antibody. In particular, in the case of the tPA22 kb target, when no antibody was used, amplification of the target was not observed, but amplification was confirmed by using the antibody.
Hot \ start \ PCR was expected to be effective also for long \ PCR, but there is no clear example, and it seems that this is the first report with a target of 20 kb or more in the human genome.
[0068]
Embodiment 8 FIG.
GC rich target of PCR Of effective reagents long PCR Confirm the effect on 8 )
Similar to betaine, a reagent "PCRx" (manufactured by Invitrogen), which is considered to work effectively on a target having a high GC content, was similarly examined for its effect on long @ PCR. PCR buffer was added to KOD-plus (manufactured by Toyobo Co., Ltd.) and potassium oxalate was added to a final concentration of 2 mM, and 0.4 mM dNTPs and a primer pair 0.3 μM were used. The primer pairs described in SEQ ID NOS: 17 and 18 were used for PCR of tPA9 kb, and the primer pairs described in SEQ ID NOS: 9 and 10 were used for PCR of tPA12 kb. Using 1.0 U of KOD DNA polymerase and 200 ng of Genomic DNA derived from human cultured cell K562 as a template, the effects of adding betaine and PCRx were compared. After the pre-reaction at 94 ° C for 2 minutes, 98 ° C, 10 seconds-74 ° C, 12 minutes (5 cycles) -98 ° C, 10 seconds-72 ° C, 12 minutes (5 cycles) -98 ° C, 10 seconds-70 GeneAmp2400 (PE Applied Biosystems, Inc.) on a schedule in which a cycle of 98 ° C., 12 minutes (5 cycles) -98 ° C., 10 seconds-68 ° C., 12 minutes (25 cycles) is repeated, and finally an additional reaction at 68 ° C., 7 minutes. Was used to perform PCR. FIG. 8 shows the results.
[0069]
With any of the tPA 9 kb and $ 12 kb targets, little amplification of the target was observed when no additives were added. However, addition of 1M or 1.5M betaine resulted in sufficient amplification of both targets. On the other hand, when PCRx was used at a 1x concentration, a 2x concentration, and a 3x concentration according to the instructions for use, no amplification was observed. From the above results, it can be said that a reagent that is effective for the GC 必 ず し も rich target is not necessarily effective for long PCR, and that the effectiveness for long に 対 す る PCR is a property specific to betaine. The effect of betaine on long イ ン PCR has been confirmed not only for α-type DNA polymerase but also for all thermostable DNA polymerases.
[0070]
【The invention's effect】
As described above, according to the present invention, PCR can be performed even on a target DNA which has been impossible so far, and the success rate of PCR can be increased. Further, the synergistic effect of the divalent carboxylate, betaine and DMSO could be confirmed with a plurality of DNA synthases, and was effective not only in a simple DNA synthesis reaction but also in a PCR method. In particular, even with α-type DNA polymerase which has been considered unsuitable for long に は PCR, long PCR can be performed by the synergistic effect of oxalate ion and betaine, and the effect on long PCR can be stabilized by using Hot start PCR. I found out.
[0071]
[Sequence list]

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[0087]

[0088]

[Brief description of the drawings]
FIG. 1 is a substitute drawing of an electrophoresis photograph confirming a synergistic effect of potassium oxalate and other reagents in PCR using a mixed enzyme (EX-Taq).
FIG. 2 is a substitute drawing of an electrophoresis photograph confirming a synergistic effect of potassium oxalate and other reagents in PCR using an α-type enzyme (Pfu).
FIG. 3 is a substitute drawing 1 of an electrophoresis photograph confirming a synergistic effect of potassium oxalate and other reagents in PCR using Hot @ start enzyme (KOD-Plus).
FIG. 4 is a substitute drawing 2 of an electrophoresis photograph confirming a synergistic effect of potassium oxalate and other reagents in PCR using Hot @ start enzyme (KOD-Plus).
FIG. 5 shows the long time of potassium oxalate and betaine in PCR using α-type enzyme (KOD).
It is a substitute drawing of the electrophoresis photograph which confirmed the synergistic effect on PCR.
FIG. 6 shows a long relationship between betaine and potassium oxalate in PCR using α-type enzyme (KOD).
It is a substitute drawing of the electrophoresis photograph which confirmed the synergistic effect on PCR.
FIG. 7 is a substitute drawing of an electrophoresis photograph confirming the effect of Hot Start PCR on Long PCR.
FIG. 8 is a substitute drawing of an electrophoresis photograph confirming the effect of GC 試 薬 rich target on PCR on long PCR of a reagent effective for PCR.

Claims (24)

(1) In an enzyme reaction for synthesizing DNA based on a nucleic acid serving as a template, at least one of an anionic substance effective for promoting DNA synthesis and (2) a reagent having glycine as a basic unit or DMSO is used. A reaction solution composition comprising: The reaction solution composition according to claim 1, wherein the reagent having glycine as a basic unit is trimethylglycine (betaine). 3. The reaction solution composition according to claim 1, wherein the anionic substance effective in promoting DNA synthesis is a substance having a carboxyl group. The reaction solution composition according to claim 3, wherein the substance having a carboxyl group is a dicarboxylate. The dicarboxylate is zinc oxalate, ammonium oxalate, potassium oxalate, calcium oxalate, diethyl oxalate, N, N'-disuccinimidyl oxalate, dimethyl oxalate, tin oxalate, cerium oxalate, oxalate Iron oxide, 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, maleic acid Diethyl, chlorpheniramine maleate, maleate di n- butyl, is selected from the group consisting of maleic acid mono -n- butyl ester The composition of claim 4. The composition according to claims 2 to 5, wherein the concentration of trimethylglycine (betaine) is 0.5-1.5M and / or the concentration of DMSO is 2-5%. The composition according to claims 1 to 6, further comprising one or more enzymes having DNA polymerase activity. The composition according to claim 7, wherein the enzyme having DNA polymerase activity is selected from the group consisting of DNA polymerase and reverse transcriptase. The DNA polymerase is Taq, EX-Taq, LA-Taq, Expand series, Plutinum series, Tbr, Tfl, Tru, Tth, T1i, Tac, Tne, Tma, Tih, Tfi, Pfu, Pfutubo, Pyrobe, Pwo, KOD. 9. The composition according to claim 8, wherein the composition is selected from the group consisting of Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENT, DEEPVENT, KOD, and mutants, variants and derivatives thereof. 9. The composition according to claim 8, wherein the DNA polymerase is a thermostable DNA polymerase having a DNA synthesis rate of at least 30 bases / sec and having 3'-5 'exonuclease activity. The DNA polymerase according to claim 10, wherein the DNA polymerase is a thermostable DNA polymerase having a DNA synthesis rate of at least 30 bases / second and a probability of an error occurring in PCR using pMol # 21 as a template of 4% or less. A composition as described. The reverse transcriptase is AMV-RT, M-MLV-RT, HIV-RT, EIAV-RT, RAV2-RT, C.I. 9. The method according to claim 8, which is selected from the group consisting of C. hydroformans DNA polymerase, SuperScript I, SuperScript II, and mutants, variants and derivatives thereof. Composition. 13. The composition according to claim 8 or 12, wherein the RNase H activity of the reverse transcriptase is substantially reduced. 14. The method according to claim 1, which comprises at least one of the group consisting of a nucleic acid serving as a template for DNA synthesis, a DNA synthetase, one or more oligonucleotide primers, one or more nucleotides or a derivative thereof, a buffer, and a salt. A composition as described. The composition according to claim 14, wherein the nucleotide or a derivative thereof is a deoxyphosphonucleotide or a derivative thereof. The composition according to claim 15, wherein the deoxyphosphonucleotide or a derivative thereof is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dITP, dUTP, α-thio-dNTPs, biotin-dUTP, fluorescein-dUTP, and sigoxigenin-dUTP. object. A method of synthesizing DNA, comprising: (a) mixing a nucleic acid template with one or more compositions of claims 1-16 to form a mixture; In order to produce a first nucleic acid molecule complementary to the whole or a part of the template, the DNA synthesis rate is at least 30 bases / second and the probability that an error will occur in PCR using pMol @ 21 as a template is 4%. Incubating the mixture under conditions that are less than or equal to%. In order to prepare a second nucleic acid molecule complementary to the whole or part of the first nucleic acid molecule, an error occurs in PCR using a DNA synthesis rate of at least 30 bases / sec and pMol @ 21 as a template. 18. The method of claim 17, further comprising incubating the first nucleic acid molecule under conditions where the probability of the first nucleic acid molecule is 4% or less. A nucleic acid molecule produced according to the method of claim 17. A method for amplifying a DNA molecule, comprising: (a) mixing a nucleic acid template with one or more compositions of claims 1 to 16 to form a mixture; And (b) the probability that an error will occur in PCR using pMol @ 21 as a template when the DNA synthesis rate is at least 30 bases / second and amplifying a nucleic acid molecule complementary to all or a part of the template. Incubating the mixture under conditions where is less than or equal to 4%. 17. A method for sequencing a DNA molecule, the method comprising: (a) providing the nucleic acid molecule to be sequenced with one or more primers, one or more compositions according to claims 1-16. Mixing with the compound, one or more nucleotides, and one or more terminators to form a mixture; (b) synthesizing a population of molecules complementary to all or a portion of the molecule to be sequenced. Incubating the mixture under conditions where the DNA synthesis rate is at least 30 bases / second and the probability of error occurring in PCR using pMol @ 21 as a template is 4% or less; and (c) C) separating said population and determining the nucleotide sequence of all or part of said molecule to be sequenced. 22. The method of claims 17, 18, 20, 21 further employing Hot \ start \ PCR. A kit for use in synthesizing a DNA molecule, the kit comprising one or more compounds or components according to any of claims 1 to 16. A kit for use in synthesizing a DNA molecule consisting of two or more parts, wherein one or more compounds or components according to any one of claims 1 to 16 are contained in any of the kits. A kit characterized in that it is contained as a constituent component in several parts or concentrated in one part.

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