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WO2015019952A1 - Test d'amplification d'acide nucléique - Google Patents

Test d'amplification d'acide nucléique Download PDF

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WO2015019952A1
WO2015019952A1 PCT/JP2014/070323 JP2014070323W WO2015019952A1 WO 2015019952 A1 WO2015019952 A1 WO 2015019952A1 JP 2014070323 W JP2014070323 W JP 2014070323W WO 2015019952 A1 WO2015019952 A1 WO 2015019952A1
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pcna
amino acid
nucleic acid
dna polymerase
kod
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哲大 小林
弘嵩 松本
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Toyobo Co Ltd
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Toyobo Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • the present invention relates to a nucleic acid amplification method by PCR.
  • the present invention can be used not only for research but also for clinical diagnosis and environmental examination.
  • PCR polymerase chain reaction
  • DNA denaturation by heat treatment dissociation from double-stranded DNA to single-stranded DNA
  • primer annealing of primer to template single-stranded DNA
  • DNA polymerase This is a method of amplifying a target nucleic acid in a sample by repeating the cycle with three steps of extension of the primer using.
  • Target nucleic acids can be amplified hundreds of thousands of times even from a small amount of sample such as several copies. It has come to be used.
  • Non-patent Document 1 PCR is easily affected by contaminants such as pigments, proteins, and sugars, and the contaminants inhibit the reaction.
  • a gene in a biological sample is amplified, purification of DNA is required.
  • the purification operation is complicated and takes time, and there is a risk of causing contamination during the operation.
  • the target nucleic acid content in the sample is small, it may not be recovered. For these reasons, there has been a demand for a method for simply and efficiently amplifying a target nucleic acid in a biological sample.
  • an object of the present invention is to provide an efficient method for amplifying a target nucleic acid. More specifically, the present invention provides a nucleic acid amplification method that enables a target nucleic acid in a biological sample to be directly amplified without going through a purification step. Furthermore, the other object of this invention is to provide the reagent kit suitable for said objective. In summary, an object of the present invention is to provide an improved PCR method and a PCR reaction reagent suitable for amplification of genes present in animal and plant tissues, body fluids and excreta.
  • the nucleic acid amplification method of the present invention uses a DNA polymerase belonging to Family B and PCNA to amplify DNA in a biological sample without undergoing a purification step. It is.
  • the present inventor uses a DNA polymerase and PCNA belonging to Family B in a nucleic acid amplification method in which a biological sample itself and a gene amplification reaction solution are mixed and reacted, so that even if a large amount of biological contaminants exist. Furthermore, the present inventors have found that PCR is possible even if a biological sample such as blood or nail is directly added to the PCR reaction solution, and the present invention has been achieved.
  • PCNA proliferating nuclear antigen
  • PCNA acts under conditions containing a large amount of an inhibitor
  • addition of PCNA is known to increase the resistance of a polymerase to an inhibitor (crude resistance).
  • DNA in a biological sample is amplified without undergoing a purification step using DNA polymerase and PCNA belonging to Family B.
  • the representative invention of the present application is as follows.
  • [Claim 1] A method for amplifying a target nucleic acid in a biological sample by adding a biological sample that has not undergone a purification step to a nucleic acid amplification reaction solution, the DNA containing the family B and Proliferating Cell Nuclear Antigen (PCNA) included in the reaction solution
  • PCNA Proliferating Cell Nuclear Antigen
  • Item 2 A nucleic acid amplification method characterized by [Section 2] Item 2.
  • the DNA polymerase belonging to Family B is an archaea-derived DNA polymerase.
  • the DNA polymerase belonging to Family B is a DNA polymerase isolated from a bacterium of the genus Pyrococcus or Thermococcus.
  • the DNA polymerase belonging to Family B is a DNA polymerase isolated from a bacterium of the genus Pyrococcus or Thermococcus.
  • PCNA is a mutant that is loaded alone into DNA.
  • PCNA is at least one of (a) an N-terminal region consisting of amino acids corresponding to positions 82, 84 and 109 in sequence 21 or 22, and (b) a C-terminal region consisting of amino acids corresponding to positions 139, 143 and 147, Item 6.
  • the nucleic acid amplification method according to any one of Items 1 to 5, which is a mutant having one modification.
  • nucleic acid amplification method according to any one of Items 1 to 6, wherein the nucleic acid amplification method is a variant of any one of the above modified with a neutral amino acid, or a modified amino acid corresponding to both the 109th and 143rd amino acids with a neutral amino acid.
  • nucleic acid amplification method according to any one of Items 1 to 7, wherein the DNA polymerase belonging to Family B is a mutant having further reduced base analog detection activity.
  • variant 9 Item 9. The nucleic acid according to Item 8, wherein the variant of DNA polymerase belonging to Family B having reduced base analog detection activity is one of the following (a) to (c): Amplification method.
  • a variant of a DNA polymerase belonging to Family B having reduced base analog detection activity is at least one amino acid among amino acids corresponding to the 7, 36, and 93rd amino acid sequences of SEQ ID NO: 1 or SEQ ID NO: 2 Item 10.
  • the present invention eliminates the risk of loss and carryover during DNA purification, and further reduces time and cost. Moreover, a gene can be accurately amplified by using a polymerase belonging to Family B. This technique can be widely used not only in the research field, but also in the clinical field such as genetic diagnosis or the field of forensic medicine, or in the examination of microorganisms in food or the environment.
  • mutant type in the case of “mutant PCNA” means that it has an amino acid sequence different from the conventionally known PCNA, and it is due to artificial mutation or due to mutation in nature. There is no distinction.
  • the nucleic acid amplification method in the present invention is a method for amplifying a target nucleic acid in a biological sample by adding a biological sample that has not undergone a purification step to a nucleic acid amplification reaction solution, and comprises a polymerase belonging to Family B and a Proliferating Cell Nuclear Antigen (PCNA). ) In the reaction solution.
  • PCNA Proliferating Cell Nuclear Antigen
  • nucleic acid in a biological sample that has not undergone a purification step is amplified without purification.
  • Purification is a method for separating contaminants such as tissues and cell walls of biological samples from DNA in biological samples. Methods for separating DNA using phenol or phenol / chloroform, ion exchange resins, glass filters, etc. Alternatively, there is a method of separating DNA with a reagent having a protein aggregation action.
  • the nucleic acid amplification method in the present invention is a method in which a biological sample is added to a nucleic acid amplification reaction solution and amplified without taking these purification methods.
  • the “biological sample that has not undergone the purification step” means that the biological sample itself, or a liquid biological sample diluted with a solvent such as water, or a solid biological sample is added to a solvent such as water and heated. The thing crushed over is mentioned.
  • the biological sample applied to the nucleic acid amplification method of the present invention is not particularly limited as long as it is a sample collected from a living body.
  • it refers to animal and plant tissues, body fluids, excreta, cells, bacteria, viruses and the like.
  • Body fluid includes blood and saliva, and cells include, but are not limited to, leukocytes separated from blood.
  • the DNA polymerase used in the nucleic acid amplification method of the present invention is a DNA polymerase belonging to family B.
  • the DNA polymerase belonging to the family B is preferably an archaea-derived DNA polymerase.
  • DNA polymerases derived from archaea belonging to family B include DNA polymerases isolated from bacteria of the genera Pyrococcus and Thermococcus.
  • DNA polymerase derived from the genus Pyrococcus include Pyrococcus furiosus and Pyrococcus sp. Including, but not limited to, DNA polymerases isolated from GB-D, Pyrococcus Wosei, Pyrococcus abyssi, Pyrococcus horikoshii.
  • DNA polymerases derived from the genus Thermococcus include Thermococcus kodakaraensis, Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9 ° N-7), Thermococcus sp. Including, but not limited to, DNA polymerase isolated from KS-1, Thermococcus celler, or Thermococcus sicili.
  • PCR enzymes using these DNA polymerases are commercially available, Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England) , Tgo (Roche), Pwo (Roche).
  • KOD DNA polymerase excellent in extensibility and thermal stability is preferable.
  • a mutant obtained by modifying the DNA polymerase described later to modify the 3′-5 ′ exonuclease region and / or to have a decreased base analog detection activity may be used.
  • PCNA PCNA also referred to herein as Proliferating Cell Nuclear Antigen
  • the PCNA is preferably heat resistant to withstand the thermal cycle of PCR, and preferably remains active after PCR. More preferably, it is soluble even after heat treatment at 80 ° C. for 30 minutes, and the activity remains at 50% or more, more preferably at least 70%, more preferably at least 90%.
  • PCNA used in the nucleic acid amplification method of the present invention is more preferably PCNA isolated from bacteria belonging to the genus Pyrococcus and Thermococcus.
  • PCNAs derived from the genus Pyrococcus include Pyrococcus furiosus (SEQ ID NO: 22), Pyrococcus sp. Including, but not limited to, PCNA isolated from GB-D, Pyrococcus Wosei, Pyrococcus abyssi, Pyrococcus horikoshii.
  • PCNAs derived from the genus Thermococcus include Thermococcus kodakaraensis (SEQ ID NO: 21), Thermococcus gorgonaris, Thermococcus literalis, Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9 ° N-7), Thermococcus sp. Including, but not limited to, PCNA isolated from KS-1, Thermococcus celer, or Thermococcus siculi.
  • PCNA used in the nucleic acid amplification method of the present invention may be a modified methionine corresponding to position 73 of SEQ ID NO: 21 or SEQ ID NO: 22 in order to increase the expression level. More preferably, it is modified to M73L, but is not limited thereto.
  • the PCNA used in the nucleic acid amplification method of the present invention may be a mutant that is loaded alone into DNA.
  • PCNA usually forms a multimer and has a ring-like structure. Loading to DNA indicates that the DNA is allowed to pass inside the ring structure of the PCNA multimer, and PCNA can be loaded into DNA only in combination with a factor usually called RFC.
  • Mutants that load DNA alone are those that modify the sites involved in PCNA multimer formation and destabilize multimer formation, making it easier to pass DNA into PCNA multimers without RFC. Show.
  • PCNA The site where PCNA is related to multimer formation is PCNA (SEQ ID NO: 21) derived from Thermococcus kodakaraensis, and PCNA of Pyrococcus furiosus (SEQ ID NO: 22) is an N consisting of amino acids 82, 84 and 109. Examples thereof include a terminal region and a C-terminal region consisting of amino acids 139, 143 and 147. The N-terminal region is positively charged, the C-terminal region is negatively charged, and multimers are formed by interaction.
  • SEQ ID NO: 21 or SEQ ID NO: 22 as an example also applies to PCNAs other than the PCNA specifically presenting the sequences in this specification.
  • a region relating to multimer formation consisting of amino acids 82, 84, 109, 139, 143, and 147 of SEQ ID NO: 21 Indicates the corresponding area.
  • PCNA variants that load DNA alone are more preferably involved in PCNA multimer formation, (A) N-terminal region consisting of amino acids corresponding to positions 82, 84 and 109, or (b) C-terminal region consisting of amino acids corresponding to positions 139, 143 and 147, having at least one modification and no RFC Both include mutants that load into DNA and promote the elongation reaction of DNA polymerase.
  • the amino acid corresponding to the 143rd position of SEQ ID NO: 21 is changed to a basic amino acid
  • the 82nd and 143rd positions are both changed to neutral amino acids
  • the 147th position is changed to a neutral amino acid, or 109 And the like, in which both the 145th and 143rd are modified to neutral amino acids.
  • neutral amino acids of the present invention include glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, proline, serine, threonine, cysteine, methionine, asparagine, and glutamine.
  • alanine has the least influence on the three-dimensional structure of the peripheral site of the substitution site.
  • Examples of basic amino acids include arginine, histidine and lysine as long as they are natural. Arginine is preferable.
  • a sequence in which the 147th amino acid residue is replaced with alanine (D147A), the 82nd and 143rd amino acid residues are alanine.
  • Whether or not a PCNA mutant can be loaded into DNA alone can be evaluated by PCR.
  • a PCNA mutant is added to a normal PCR reaction solution containing DNA as a template, buffer material, magnesium, dNTPs, primers, and DNA polymerase belonging to Family B, with no PCNA added, or with wild-type PCNA added
  • the amount of amplification with that of DNA it can be confirmed whether it can be loaded into DNA alone.
  • the PCR amplification amount does not change, but rather the amplification amount tends to decrease.
  • a mutant that can be loaded into DNA alone can obtain an amplification amount superior to that without PCNA addition or with wild-type PCNA addition.
  • the evaluation of “whether a PCNA mutant can be loaded alone into DNA” (evaluation of amplification enhancing activity) follows the following method.
  • KOD -Plus- Ver. 2 manufactured by Toyobo
  • 10 ⁇ PCR Buffer attached 1 ⁇ PCR Buffer and 1.5 mM MgSO 4 , 0.2 mM dNTPs containing dUTP instead of dTTP (dATP, dUTP, dCTP, dGTP)
  • about 250 ng of PCNA to be evaluated was added to 50 ⁇ l of a reaction solution containing 15 pmol of the primer described in SEQ ID NOS: 13 and 14 for amplifying 1.3 kb, 10 ng of human genomic DNA (Roche) and 1U KOD DNA polymerase V93K mutant.
  • PCR is performed on a schedule of 98 ° C., 10 seconds ⁇ 65 ° C., 30 seconds ⁇ 68 ° C., 1 minute 30 seconds repeated 35 cycles.
  • 5 ⁇ l of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and compared with an amplified DNA fragment of about 1.3 kb under UV irradiation to which wild type PCNA was added or to which PCNA was not added. By doing so, it is possible to evaluate whether or not the PCNA can be loaded into DNA alone. The amount of amplification increases with the addition of PCNA that can be loaded into DNA alone.
  • the nucleic acid amplification method of the present invention is a method for amplifying a target nucleic acid in a biological sample by adding a biological sample that has not undergone a purification step to a nucleic acid amplification reaction solution, Is used for amplification, and is not particularly limited except that PCNA is contained in the reaction solution.
  • a typical example of a method that can be amplified by a DNA polymerase is PCR.
  • PCR DNA polymerase
  • DNA is used as a template, and one primer, dNTP (deoxyribonucleotide triphosphate) is reacted.
  • dNTP deoxyribonucleotide triphosphate
  • a primer extension method, a sequencing method, a conventional method that does not perform temperature cycling, and a cycle sequence method are included.
  • Amplification target DNA obtained from an unpurified biological sample, (A) DNA polymerase belonging to family B (b) a pair of primers in which one primer is complementary to the DNA extension product of the other primer (c) a DNA synthesis substrate (deoxynucleotide triphosphate (dNTP)) (D) a buffer solution containing magnesium ions, ammonium ions and / or potassium ions, and (E) PCNA Mixed,
  • A DNA polymerase belonging to family B
  • a pair of primers in which one primer is complementary to the DNA extension product of the other primer (c) a DNA synthesis substrate (deoxynucleotide triphosphate (dNTP)) (D) a buffer solution containing magnesium ions, ammonium ions and / or potassium ions, and (E) PCNA Mixed,
  • dNTP deoxynucleotide triphosphate
  • E PCNA Mixed
  • a PCR enhancing factor (described later), BSA, a nonionic surfactant, and the like may be further used as necessary.
  • a mutant having a modified base analog detection activity which will be described later, is used as a DNA polymerase
  • a base analog such as dUTP can be used as a DNA synthesis substrate.
  • an antibody having the activity of suppressing the polymerase activity and / or 3'-5 'exonuclease activity of the thermostable DNA polymerase may be used.
  • the antibody include a monoclonal antibody and a polyclonal antibody. This reaction composition is particularly effective for increasing the sensitivity of PCR and reducing nonspecific amplification.
  • the reagents and kits for carrying out the nucleic acid amplification method of the present invention contain DNA polymerase belonging to Family B and PCNA in the reaction solution, The configuration is not particularly limited.
  • the applied nucleic acid amplification method is not particularly limited.
  • examples of the reagent and kit of the present invention include the following configurations (a) to (e), but are not limited thereto.
  • A a variant of a DNA polymerase belonging to Family B having reduced base analog detection activity
  • b a pair of primers in which one primer is complementary to the DNA extension product of the other primer
  • c DNA synthesis Substrate (deoxynucleotide triphosphate (dNTP))
  • D a buffer solution containing magnesium ions, ammonium ions and / or potassium ions
  • PCNA PCNA
  • the reagents and kits may further use other reagents such as BSA and nonionic surfactant as required.
  • the modified DNA polymerase used in the nucleic acid amplification method of the present invention may further contain at least one amino acid modification in any of the amino acid sequences of the 3′-5 ′ exonuclease active region.
  • the 3′-5 ′ exoase activity refers to the ability to remove the incorporated nucleotide from the 3 ′ end of the DNA polymer, and the above 3′-5 ′ exonuclease region is a DNA polymerase belonging to family B and highly DNA polymerase (SEQ ID NO: 1) derived from Thermococcus kodakaraensis, DNA polymerase (SEQ ID NO: 2) derived from Pyrococcus furiosus, DNA derived from Thermococcus gorgonarius Polymerase (SEQ ID NO: 3), DNA polymerase derived from Thermococcus litoralis (SEQ ID NO: 4), DNA polymerase derived from Pyrococcus sp.
  • SEQ ID NO: 1 derived from Thermococcus kodakaraensis
  • SEQ ID NO: 2 DNA polymerase derived from Pyrococcus furiosus
  • SEQ ID NO: 3 DNA polymerase derived from Therm
  • GB-D (SEQ ID NO: 5), derived from Thermococcus sp. JDF-3 DNA polymerase (SEQ ID NO: 6), Thermoco DNA polymerase derived from Cass SP 9 ° N-7 (SEQ ID NO: 7), DNA polymerase derived from Thermococcus sp KS-1 (SEQ ID NO: 8), DNA polymerase derived from Thermococcus cellar (SEQ ID NO: 9)
  • SEQ ID NO: 10 the amino acids 137 to 147, 206 to 222, and 308 to 318 are used.
  • the present invention is also applicable to DNA polymerases other than the DNA polymerase specifically presenting the sequence.
  • the 3′- consisting of amino acids 137 to 147, 206 to 222, and 308 to 318 of SEQ ID NO: 1 The region corresponding to the 5 ′ exonuclease region is shown.
  • amino acids corresponding to positions 137 to 147, 206 to 222, and 308 to 318 shown in SEQ ID NO: 1 are DNA polymerases having an amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1.
  • An expression comprising amino acid sequences corresponding to positions 137 to 147, 206 to 222, and 308 to 318 of SEQ ID NO: 1.
  • the modification to the above 3'-5 'exonuclease region can consist of substitution, deletion, or addition. Modifications to amino acids corresponding to positions 137 to 147, 206 to 222, and 308 to 318 in SEQ ID NO: 1 are shown.
  • DNA polymerase in which the 3′-5 ′ exonuclease active region is modified one in which at least one of amino acids corresponding to positions 141, 142, 143, 210, 311 in SEQ ID NO: 1 or SEQ ID NO: 2 is modified is preferable.
  • These modified DNA polymerases are deficient in 3′-5 ′ exonuclease activity. More preferably, it is a DNA polymerase deficient in 3′-5 ′ exonuclease activity, wherein the amino acid modification is any one selected from D141A / E143A, I142R, N210D, or Y311F.
  • 3'-5 'exonuclease activity-deficient (exo (-)) DNA polymerase includes a complete lack of activity, for example, 0.03%, 0.05% compared to the parent enzyme , 0.1%, 1%, 5%, 10%, 20%, or a maximum of 50% or less exonuclease activity.
  • DNA polymerase in which the 3'-5 'exonuclease active region is modified is any one selected from H147E or H147D in SEQ ID NO: 1 or SEQ ID NO: 2. These modified DNA polymerases have improved PCR efficiency while maintaining exonuclease activity.
  • a method for producing a DNA polymerase in which the 3′-5 ′ exonuclease active region is modified and a method for analyzing the 3′-5 ′ exonuclease activity are known.
  • US Pat. No. 6,946,273 Patent Document 3
  • a DNA polymerase with improved PCR efficiency refers to a modified DNA polymerase in which the amount of PCR product is increased compared to the parent enzyme.
  • a method for analyzing whether the amount of the PCR product is increased as compared with the parent enzyme is described in Japanese Patent No. 3891330 (Patent Document 4).
  • the DNA polymerase belonging to family B used in the nucleic acid amplification method of the present invention may be a mutant having reduced base analog detection activity.
  • Base analogs refer to bases other than adenine, cytosine, guanine, and thymine, and include uracil and inosine.
  • a DNA polymerase belonging to Family B binds strongly when a base analog such as uracil or inosine is detected, and inhibits the polymerase function.
  • the base analog detection activity refers to an activity that strongly binds to a base analog and inhibits the polymerase function.
  • a DNA polymerase mutant belonging to family B having reduced base analog detection activity is a DNA polymerase mutant belonging to family B characterized by low binding ability to uracil and inosine.
  • An example of such a mutant having a decreased base analog detection activity of a DNA polymerase belonging to Family B is an archaeal DNA polymerase mutant.
  • the amino acid sequence (uracil-binding pocket) relating to the binding of uracil formed by amino acids 1 to 40 and amino acids 78 to 130 is modified, and compared to wild-type DNA polymerase, uracil and inosine An archaeal DNA polymerase variant characterized by low binding ability.
  • the amino acid sequence relating to the binding of uracil is highly conserved in DNA polymerase derived from Pyrococcus and DNA polymerase derived from Thermococcus.
  • DNA polymerase SEQ ID NO: 1 derived from Thermococcus kodakaraensis
  • SEQ ID NO: 2 derived from Thermococcus kodakaraensis
  • Pyrococcus furiosus SEQ ID NO: 2
  • Thermococcus gorgonarius SEQ ID NO: 3
  • Thermococcus litoralis (SEQ ID NO: 4), it is formed by amino acids 1 to 40 and amino acids 78 to 130.
  • Pyrococcus sp. GB-D (SEQ ID NO: 5), it is formed by amino acids 1 to 40 and amino acids 78 to 130.
  • Thermococcus sp. JDF-3 (SEQ ID NO: 6), it is formed by amino acids 1 to 40 and amino acids 78 to 130.
  • Thermococcus sp 9 ° N-7 (SEQ ID NO: 7), it is formed by amino acids 1 to 40 and amino acids 78 to 130.
  • KS-1 (SEQ ID NO: 8), it is formed by amino acids 1 to 40 and amino acids 78 to 130.
  • Thermococcus cellar (SEQ ID NO: 9), it is formed by amino acids 1-40 and amino acids 78-130.
  • Thermococcus cyclis (SEQ ID NO: 10), it is formed by amino acids 1 to 40 and amino acids 78 to 130.
  • DNA polymerase mutant for use in the nucleic acid amplification method of the present invention is the seventh, thirty-six, thirty-seven, thirty-nine, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-nineth, thirty-seventh, ninety-nineth, thirty-seventh, thirty-seventh, ninety-nineth, thirty-seventh, ninety-nineth, thirty-seventh, ninety-nineth, thirty-seventh, ninety-seventh, thirty-seventh, ninety-seventh, thirty-seventh, ninety-nine
  • the mutant of the DNA polymerase belonging to the above family B may be one represented by the following amino acid sequence (b).
  • BLAST Basic local alignment search tool
  • NCBI National Institute of Biotechnology Information
  • ncbi. nlm. nih The amino acid sequence identity is calculated by using default (initial setting) parameters in gov / BLAST /.
  • the mutant of the DNA polymerase belonging to the above family B may be one represented by the following amino acid sequence (c).
  • “several” is not limited as long as “decreased base analog detection activity” is maintained, but is, for example, a number corresponding to less than about 20% of all amino acids, preferably less than about 15%. It is a corresponding number, more preferably a number corresponding to less than about 10%, even more preferably a number corresponding to less than about 5%, and most preferably a number corresponding to less than about 1%. More specifically, the number of amino acid residues to be mutated is, for example, 2 to 160, preferably 2 to 120, more preferably 2 to 80, still more preferably 2 to 40, and even more. The number is preferably 2-5.
  • amino acids corresponding to positions 7, 36, 37, 90 to 97, and 112 to 119 in the amino acid sequence shown in SEQ ID NO: 1 are amino acid sequences that are not completely identical to the amino acid sequence shown in SEQ ID NO: 1.
  • a position (order) on SEQ ID NO: 1 and a corresponding position are when the primary structure of the sequence is compared (alignment) , A position corresponding to the position of SEQ ID NO: 1.
  • the DNA polymerase mutant belonging to Family B having reduced base analog detection activity used in the nucleic acid amplification method of the present invention more preferably corresponds to amino acids Y7, P36, or V93 in SEQ ID NO: 1 or SEQ ID NO: 2. It has at least one amino acid modification selected from amino acids.
  • Y7 means a tyrosine (Y) residue that is the seventh amino acid, and one letter of the alphabet represents an abbreviation of a commonly used amino acid.
  • the Y7 amino acid has tyrosine (Y) substituted with a nonpolar amino acid, specifically selected from the group consisting of Y7A, Y7G, Y7V, Y7L, Y7I, Y7P, Y7F, Y7M, Y7W, and Y7C.
  • Y tyrosine
  • the P36 amino acid is a proline (P) substituted with a positively charged polar amino acid, specifically a P36H, P36K, or P36R amino acid substitution.
  • the V93 amino acid is a valine (V) having a positive charge and substituted with a polar amine acid, specifically an amino acid substitution of V93H, V93K, or V93R.
  • the modification is modification of at least one amino acid selected from the group consisting of Y7A, P36H, P36K, P36R, V93Q, V93K, and V93R. More preferably, it is P36K, P36R or P36H. More preferably, it is P36H.
  • a variant of a DNA polymerase belonging to Family B having reduced base analog detection activity in the present invention is selected from two or more amino acids selected from amino acids corresponding to amino acids Y7, P36, or V93 in SEQ ID NO: 1 or SEQ ID NO: 2.
  • a modified amino acid may also be used. Specific examples include Y7A / V93K, Y7A / P36H, Y7A / P36R, Y7A / V93R, Y7A / V93Q or P36H / V93K, and preferred examples include Y7A / P36H or Y7A / V93K. It is not limited to.
  • Patent Document 1 or 2 any of the amino acids 7, 36, 37, 90 to 97, and 112 to 119 assumed to be directly related to the interaction with uracil is modified.
  • Several variants of DNA polymerase belonging to family B are exemplified, but not all of the variants have good properties that meet the subject of the present application, some of which have lost activity Can also be seen.
  • mutants Based on the modification of the DNA polymerase exemplified in the above (8) and (9), various mutants can be considered as the modified DNA polymerase used in the nucleic acid amplification method of the present invention. Examples of such mutants include, but are not limited to, mutants of DNA polymerase belonging to Family B having any of the following modifications (1) to (4).
  • the base analog detection activity in the present invention can be evaluated by PCR.
  • the base analog is typically uracil.
  • a dUTP solution is added at a final concentration of 0.5 ⁇ M to 200 ⁇ M to a normal PCR reaction solution containing DNA as a template, buffer material, magnesium, dNTPs, primers, and a DNA polymerase to be evaluated, and thermal cycling is performed. .
  • the presence or absence of a PCR product can be confirmed by ethidium bromide-stained agarose electrophoresis, and the detection activity of uracil can be evaluated by the allowable dUTP concentration.
  • a DNA polymerase having a high uracil detection activity inhibits the extension reaction by adding a little dUTP, and the PCR product cannot be confirmed.
  • DNA polymerase with low uracil detection activity can confirm gene amplification by PCR without problems even when a high concentration of dUTP is added.
  • a variant of DNA polymerase belonging to family B having reduced base analog detection activity was subjected to optimal thermal cycling using any primer and DNA as a template in an enzyme optimal reaction buffer.
  • it refers to a DNA polymerase that can confirm a PCR product without inhibiting the extension reaction even when a high concentration of dUTP is added compared to a wild type without mutation.
  • the DNA polymerase mutant belonging to Family B that can amplify PCR even when dUTP is added at a concentration of 0.5 ⁇ M is compared with the wild type. Thus, it is presumed to have reduced base analog detection activity.
  • the evaluation of the base analog detection activity in the present invention follows the following method. KOD -Plus- Ver. 2 (Toyobo) attached 10 ⁇ PCR Buffer or Pfu DNA Polymerase (Agilent) attached 10 ⁇ PCR Buffer, 1 ⁇ PCR Buffer, 1.5 mM MgSO 4 , 0.2 mM dNTPs (dATP, dTTP, dCTP, dGTP), 15 pmol of the primer according to SEQ ID NOS: 13 and 14, which amplifies about 1.3 kb, 10 ng of human genomic DNA (Roche), and 1 U of each enzyme in a 50 ⁇ l reaction solution containing dUTP ( Roche) to a final concentration of 0.5, 5, 50, 100, 200 ⁇ M.
  • PCR is performed with PCR system GeneAmp 9700 (Applied Biosystem) with a schedule of repeating 98 cycles of 10 ° C., 10 seconds ⁇ 65 ° C., 30 seconds ⁇ 68 ° C., 1 minute 30 seconds.
  • 5 ⁇ l of the reaction solution is subjected to agarose electrophoresis, ethidium bromide staining, and an amplified DNA fragment of about 1.3 kb is confirmed under ultraviolet irradiation to evaluate whether the base analog detection activity is reduced. .
  • a site-directed mutagenesis method based on the inverse PCR method can be used.
  • KOD-Plus-Mutageness Kit manufactured by Toyobo
  • the above-mentioned modified DNA polymerase gene is transferred to an expression vector as necessary.
  • E. coli as a host is transformed with the expression vector, and then applied to an agar medium containing a drug such as ampicillin to form a colony.
  • the colony is inoculated into a nutrient medium such as LB medium or 2 ⁇ YT medium and cultured at 37 ° C. for 12 to 20 hours, and then the cells are crushed and the crude enzyme solution is extracted.
  • a vector derived from pBluescript is preferable. Any known method may be used as a method for crushing bacterial cells.
  • ultrasonic treatment a physical crushing method such as French press or glass bead crushing, or a lytic enzyme such as lysozyme can be used.
  • the crude enzyme solution is heat-treated at 80 ° C. for 30 minutes to inactivate the host-derived polymerase, and the DNA polymerase activity is measured.
  • any method may be used as a method for obtaining purified DNA polymerase from the strain selected by the above method, for example, the following method.
  • the crude enzyme solution is obtained by crushing and extraction by enzymatic or physical crushing methods.
  • the obtained crude enzyme extract is heat-treated, for example, at 80 ° C. for 30 minutes, and then the DNA polymerase fraction is recovered by ammonium sulfate precipitation.
  • This crude enzyme solution can be desalted by a method such as gel filtration using Sephadex G-25 (manufactured by Amersham Pharmacia Biotech). After this operation, it can be separated and purified by heparin sepharose column chromatography to obtain a purified enzyme preparation.
  • the purified enzyme preparation is purified by SDS-PAGE to such an extent that it shows almost a single band.
  • the DNA polymerase used in the nucleic acid amplification method of the present invention measures the activity as follows. If the enzyme activity is strong, samples should be stored in storage buffer (50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol, 0.1% Tween 20, 0.1% Nonidet P40, 50% glycerin). Dilute and measure. (1) 25 ⁇ l of the following solution A, 5 ⁇ l of solution B, 5 ⁇ l of solution C, 10 ⁇ l of sterilized water, and 5 ⁇ l of enzyme solution are added to a microtube and reacted at 75 ° C. for 10 minutes.
  • storage buffer 50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol, 0.1% Tween 20, 0.1% Nonidet P40, 50% glycerin.
  • A 40 mM Tris-HCl buffer (pH 7.5) 16 mM magnesium chloride 15 mM dithiothreitol 100 ⁇ g / mL BSA (bovine serum albumin) B: 1.5 ⁇ g / ⁇ l activated calf thymus DNA C: 1.5 mM dNTP (250 cpm / pmol [3H] dTTP) D: 20% trichloroacetic acid (2 mM sodium pyrophosphate) E: 1 mg / mL calf thymus DNA
  • Example 1 Preparation of KOD-PCNA mutant A plasmid containing a modified thermostable PCNA gene derived from Thermococcus kodakaraensis KOD1 strain was prepared.
  • the DNA template used for mutagenesis was PCNA (SEQ ID NO: 23) (pKODPCNA) derived from Thermococcus kodakaraensis KOD1 strain cloned into pBluescript. Mutation was introduced using KOD-Plus-Mutageness Kit (manufactured by Toyobo) according to the instruction manual. The mutant was confirmed by decoding the base sequence. Escherichia coli DH5 ⁇ was transformed with the obtained plasmid and used for enzyme preparation.
  • Example 2 Preparation of Pfu-PCNA mutant A plasmid containing a modified thermostable PCNA gene derived from Pyrococcus furiosus was prepared.
  • PCNA SEQ ID NO: 24
  • pPfuPCNA derived from Pyrococcus furiosus cloned in pBluescript was used. Mutation was introduced using KOD-Plus-Mutageness Kit (manufactured by Toyobo) according to the instruction manual. The mutant was confirmed by decoding the base sequence. Escherichia coli DH5 ⁇ was transformed with the obtained plasmid and used for enzyme preparation.
  • Example 1 The plasmids prepared in Example 1 and Example 2 are shown in Table 1.
  • Example 3-1 Preparation of KOD DNA Polymerase Mutants Various mutants of KOD DNA polymerase were prepared by the following method for use in the evaluation of PCNA in Examples described later.
  • a modified thermostable DNA polymerase gene (SEQ ID NO: 11) (pKOD) derived from Thermococcus kodakaraensis KOD1 strain cloned in pBluescript was used.
  • mutants of Pfu DNA polymerase were prepared by the following method for use in the evaluation of PCNA in Examples described later.
  • a plasmid containing a modified thermostable DNA polymerase gene derived from Pyrococcus furiosus was prepared.
  • thermostable DNA polymerase gene SEQ ID NO: 12
  • pPfu modified thermostable DNA polymerase gene derived from Pyrococcus furiosus cloned in pBluescript
  • Mutation was introduced using KOD-Plus-Mutageness Kit (manufactured by Toyobo) according to the instruction manual. The mutant was confirmed by decoding the base sequence.
  • Escherichia coli JM109 was transformed with the obtained plasmid and used for enzyme preparation.
  • the plasmids prepared in Examples 3-1 and 3-2 are shown in Table 2 and Table 3.
  • Example 4 Production of modified heat-resistant PCNA
  • the cells obtained in Examples 1 and 2 were cultured as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing sterilized 100 ⁇ g / mL ampicillin was dispensed into a 500 mL Sakaguchi flask. Escherichia cultivated for 16 hours at 37 ° C. in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco) containing 100 ⁇ g / mL ampicillin in advance in this medium. E. coli DH5 ⁇ (plasmid transformant) (using a test tube) was inoculated and cultured at 37 ° C.
  • TB medium Molecular cloning 2nd edition, p.A.2
  • LB medium 1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco
  • the bacterial cells are collected from the culture solution by centrifugation, suspended in 50 mL of disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then subjected to sonication. By crushing, a cell lysate was obtained. Next, the cell lysate was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, the nucleic acid treatment using polyethyleneimine, ammonium sulfate precipitation, and Q sepharose chromatography were performed.
  • a storage buffer 50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0 .1% Tween 20, 0.1% nonidet P40, 50% glycerin
  • a storage buffer 50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0 .1% Tween 20, 0.1% nonidet P40, 50% glycerin
  • Example 5 Production of modified thermostable DNA polymerase The cells obtained in Examples 3-1 and 3-2 were cultured as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing sterilized 100 ⁇ g / mL ampicillin was dispensed into a 500 mL Sakaguchi flask. Escherichia cultivated for 16 hours at 37 ° C. in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco) containing 100 ⁇ g / mL ampicillin in advance in this medium.
  • TB medium Molecular cloning 2nd edition, p.A.2
  • LB medium 1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco
  • Coli JM109 (plasmid transformant) (using a test tube) was inoculated and cultured at 37 ° C. for 16 hours with aeration.
  • the bacterial cells are collected from the culture solution by centrifugation, suspended in 50 mL of disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then subjected to sonication. By crushing, a cell lysate was obtained. Next, the cell lysate was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation.
  • thermostable DNA polymerase 50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0 1% Tween 20, 0.1% nonidet P40, 50% glycerin
  • the DNA polymerase activity measurement in the purification step was performed according to the [DNA polymerase activity measurement method] described in (13) above. When the enzyme activity was high, the sample was diluted for measurement.
  • Example 6 Evaluation of PCNA
  • KOD-PCNA mutants prepared in Example 1 (M73L, M73L / E143R, M73L / R109A / E143A, M73L / D147A, M73L / R82A / E143A) M73L / E143F and M73L / E143A) are used to amplify 1.3 kb of human ⁇ -globin in accordance with the method for measuring the amplification enhancing activity shown in (6) above using the difference in amplification amount in the presence of dUTP. Compared. At this time, the KOD V93K mutant prepared in Example 3-1 was used as the DNA polymerase.
  • the M73L mutation is considered to correspond to the wild type.
  • KOD-Plus-Ver. 2 manufactured by Toyobo Co., Ltd.
  • MgSO 4 1 ⁇ PCR Buffer
  • dNTPs dATP, dUTP, dCTP, dGTP
  • 0.2 mM dTTP is replaced with dUTP
  • 1.5 mM MgSO 4 15 pmol Primer (SEQ ID NOs: 13 and 14)
  • 10 ng of human genomic DNA (Roche), and 50 ⁇ l of a reaction solution containing 1 U of enzyme mixed with an antibody was reacted at 94 ° C.
  • FIG. 1 shows the results of electrophoresis of the products obtained by adding 250 ng of various PCNA mutants and performing PCR reaction.
  • the PCNA mutants used were 7 types in total: M73L, M73L / E143R, M73L / R109A / E143A, M73L / D147A, M73L / R82A / E143A, M73L / E143F, and M73L / E143A.
  • the KOD V93K mutant used this time has a small amount of amplification due to dUTP inhibition. Although no band was confirmed in the case of no PCNA addition or in the M73L mutant, a firm band was confirmed by the addition of other KOD-PCNA mutants.
  • PCNA forms a multimer and promotes a nucleic acid synthesis reaction. Usually, however, the reaction cannot proceed without loading into DNA without the action of RFC.
  • the modifications of M73L / E143R, M73L / R109A / E143A, M73L / D147A, M73L / R82A / E143A, M73L / E143F, and M73L / E143A are modifications to sites involved in multimer formation and moderately reduce multimer formation. Therefore, it is conceivable that PCNA can be loaded onto DNA and the amount of PCR amplification has been improved.
  • Example 7 Crude (blood) resistance was evaluated by adding various PCNA mutants by changing the amount of blood added to the amplification reaction solution from blood by adding PCNA .
  • the difference in the amount of amplification of 3.6 kb of HBg was compared using no PCNA addition and KOD-PCNA mutants (M73L / E143R, M73L, M73L / D147A, M73L / R82A / E143A).
  • KOD-Plus- was used for the DNA polymerase.
  • PCR was performed using KOD-Plus-Ver. 2 (Toyobo) attached Buffer, dNTPs, MgSO 4 , enzyme solution (KOD-Plus-), 1 ⁇ PCR Buffer, and 1.0 mM MgSO 4 , 15 pmol primer (SEQ ID NOs: 17 and 18), 1U
  • a 50 ⁇ l reaction solution containing 1 ⁇ g of KOD-Plus- blood was added to 8% of the reaction solution, and no PCNA and various concentrations of PCNA variants (0.5 ⁇ g, 1 ⁇ g, 2 ⁇ g, 4 ⁇ g) Were compared.
  • the reaction was performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 94 cycles at 94 ° C.
  • FIG. 2 blood is used as a sample, a reaction solution is prepared so that the proportion of blood in the reaction solution is 8%, and various PCNA mutants are added at 0.5 ⁇ g, 1 ⁇ g, 2 ⁇ g, and 4 ⁇ g to perform PCR reaction. The results of electrophoresis of the product obtained are shown.
  • Example 8 Amplification from blood by adding commercially available enzymes (derived from archaebacteria) and PCNA KOD It was evaluated whether the addition of PCNA also improved the crude (blood) resistance to enzymes belonging to Family B. For comparison, polymerases of KOD-Plus- (manufactured by Toyobo), PrimeSTAR HS (manufactured by Takara Bio), MightyAmp (manufactured by Takara Bio), and PrimeSTAR GXL (manufactured by Takara Bio) were used. The difference in the amount of amplification of 3.6 kb of HBg in the presence was compared. For PCNA, KOD-PCNA M73L / D147A was used.
  • PCR was performed using KOD-Plus-Ver. 2 using (Toyobo Co., Ltd.) attached Buffer, dNTPs, MgSO 4, 1 ⁇ PCR Buffer, and 1.0 mM MgSO 4, 15 pmol of primer (SEQ ID NO: 17 and 18), the reaction solution 50 ⁇ l containing each enzyme 1U
  • the blood was added to 16% of the reaction solution, and no PCNA and 250 ng of KOD-PCNA M73L / D147A were compared.
  • the reaction was performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 94 cycles at 94 ° C. for 2 minutes followed by 30 cycles of 98 ° C., 10 seconds ⁇ 68 ° C. and 4 minutes. After completion of the reaction, 5 ⁇ l of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.
  • FIG. 3 shows the results obtained by using blood as a sample, preparing a reaction solution so that the proportion of blood in the reaction solution is 16%, adding KOD-PCNA M73L / D147A to various enzymes, and performing a PCR reaction.
  • the result of electrophoresis of the product was shown.
  • the total number of enzymes used was KOD-Plus-, PrimeSTAR HS, MightyAmp, and PrimeSTAR GXL.
  • 1 indicates KOD-Plus-
  • 2 indicates PrimeSTAR HS
  • 3 indicates the result of NightyAmp
  • 4 indicates the result of PrimeSTAR GXL
  • ⁇ indicates no PCNA
  • + indicates that PCNA was added.
  • Example 9 Amplification from plant lysate by addition of PCNA It was evaluated whether or not the addition of PCNA to the KOD DNA polymerase mutant improved the crude (plant) resistance. For comparison, KOD H147E mutant, KOD Y7A / V93K mutant, KOD Y7A / P36H / N210D mutant, and Taq polymerase were used, and the difference in amplification amount of rbcL 1.3 kb was compared. For PCNA, KOD-PCNA M73L / D147A was used.
  • a rice leaf 3 mm square was added to 100 ⁇ l of Buffer A (100 mM Tris-HCl (pH 9.5), 1 M KCl, 10 mM EDTA) and heat-treated at 95 ° C. for 10 minutes, and used as a lysate.
  • Buffer A 100 mM Tris-HCl (pH 9.5), 1 M KCl, 10 mM EDTA
  • PCR for KOD mutants was performed using KOD-Plus-Ver.
  • Taq DNA polymerase was manufactured by Toyobo and mixed with Anti-Taq High (manufactured by Toyobo). The reaction was performed by adding 2% of plant lysate to 50 ⁇ l of reaction solution containing Buffer, 2 mM dNTPs attached to 1 ⁇ BlendTaq, 10 pmol of primer (same as above), and 2.5 U of enzyme mixed with antibody. In addition, 4%, 8% and 16% were added, and no PCNA and 250 ng of KOD-PCNA M73L / D147A were compared. The reaction was performed at 94 ° C.
  • FIG. 4 shows the case where plant lysate was used as a sample, the reaction solution was prepared so that the proportion of lysate in the reaction solution was 2, 4, 8, 16%, and KOD-PCNA M73L / D147A was added to various enzymes. The result of carrying out PCR reaction and electrophoresis of the obtained product is shown.
  • the enzymes used were KOD H147E mutant, KOD Y7A / V93K mutant, KOD Y7A / P36H / N210D mutant, and Taq polymerase in total.
  • 1, 2, 8, and 16 indicate the percentage of plant lysate added,-indicates no PCNA, and + indicates that PCNA was added.
  • Example 10 Amplification from plant lysate by addition of commercially available family B enzyme and PCNA It was evaluated whether crude (plant) resistance was improved by addition of PCNA to various commercially available family B enzymes. For comparison, KOD-Plus-, KOD Dash, and PrimeSTAR GXL were used, and the difference in amplification amount of rbcL 1.3 kb was compared. For PCNA, KOD-PCNA M73L / D147A was used.
  • a rice leaf 3 mm square was added to 100 ⁇ l of Buffer A (100 mM Tris-HCl (pH 9.5), 1 M KCl, 10 mM EDTA) and heat-treated at 95 ° C. for 10 minutes, and used as a lysate.
  • Buffer A 100 mM Tris-HCl (pH 9.5), 1 M KCl, 10 mM EDTA
  • each attached buffer is used, and 15 pmol of the primer (SEQ ID NOs: 15 and 16) and the plant lysate in the recommended reaction solution are 1%, 2%, 4%, and 6% of the reaction solution.
  • no PCNA and 250 ng of KOD-PCNA M73L / D147A were compared.
  • the reaction was carried out using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 94 cycles at 94 ° C. for 2 minutes followed by 35 cycles of 98 ° C., 10 seconds ⁇ 65 ° C., 30 seconds ⁇ 68 ° C., 1.5 minutes. It was. After completion of the reaction, 5 ⁇ l of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.
  • FIG. 5 shows the use of plant lysate as a sample, preparing reaction solutions such that the proportion of lysate in the reaction solution is 1, 2, 4, 6%, and adding KOD-PCNA M73L / D147A to various enzymes. The result of carrying out PCR reaction and electrophoresis of the obtained product is shown.
  • the enzymes used were a total of three types: KOD-Plus-, KOD Dash, and primeSTAR GXL. In each photograph, 1, 2, 4, and 6 indicate the percentage of the plant lysate added,-indicates no PCNA, and + indicates that PCNA was added.
  • a 10% fecal suspension was heat-treated at 95 ° C. for 10 minutes.
  • PCR was performed using KOD-Plus-Ver. 2 (Toyobo) attached Buffer, dNTPs, MgSO 4 , enzyme (KOD-PLUS-), 1 ⁇ PCR Buffer, and 1.0 mM MgSO 4 , 15 pmol primer (SEQ ID NO: 17 and 18), 5 ng human genome
  • feces were added to 0.2%, 0.4%, 0.8% and 1.6% of the reaction solution, and no PCNA and KOD PCNA What added 250 ng of M73L / D147A was compared.
  • the reaction was carried out using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 94 cycles of 94 ° C., 2 minutes prior reaction, and 35 cycles of 98 ° C., 10 seconds ⁇ 68 ° C., 4 minutes. After completion of the reaction, 5 ⁇ l of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.
  • FIG. 6 shows that stool, an inhibitor, was added to the reaction solution for amplifying HBg3.6k from the human genome so that the ratio was 0%, 0.2%, 0.4%, 0.8%, 1.6%.
  • the results of electrophoresis of the product obtained by performing PCR reaction with and without KOD-PCNA M73L / D147A are shown.
  • the enzyme used is KOD-Plus-.
  • 0, 0.2, 0.4, 0.8, and 1.6 indicate the percentage of feces added,-indicates no PCNA, and + indicates that PCNA was added.
  • KOD-Plus without PCNA addition when stool was added in an amount of 0.2% or more, inhibition was not observed and amplification was not confirmed.
  • PCNA mutant was added, the band even if 0.4% stool was contained Was confirmed (FIG. 6). It was shown that the PCNA mutant also has the effect of improving the resistance to faecal inhibition.
  • Example 12 Amplification from blood by addition of PCNA using a reaction solution containing dUTP
  • a reaction solution containing dUTP As the enzyme, the KOD Y7A / V93K mutant was used, and the difference in the amount of HBg 482 bp was compared.
  • KOD-PCNA M73L / D147A was used for PCNA.
  • PCR was performed using KOD-Plus-Ver. 2 (manufactured by Toyobo), using Buffer, MgSO 4 attached, 1 ⁇ PCR Buffer, and 1.5 mM MgSO 4 , 15 pmol primer (SEQ ID NOS: 19 and 20), dNTPs (dATP, dUTP) in which 2 mM dTTP was replaced with dUTP , DCTP, dGTP), in a 50 ⁇ l reaction solution containing 1 U enzyme mixed with KOD antibody, 0.002%, 0.02%, 0.2%, 2%, 5% A comparison was made between 10% and PCNA-free and KOD-PCNA M73L / D147A added at 250 ng.
  • the reaction was carried out using PCR system GeneAmp 9700 (Applied Biosystem) with a schedule of 94 cycles at 94 ° C. for 2 minutes followed by 35 cycles of 98 ° C., 10 seconds ⁇ 65 ° C., 30 seconds ⁇ 68 ° C., 1 minute. After completion of the reaction, 5 ⁇ l of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.
  • FIG. 7 blood is used as a sample, and blood is added to a reaction solution containing dUTP at a ratio of 0.002%, 0.02%, 0.2%, 2%, 5%, 10%,
  • the result of carrying out PCR reaction with and without addition of KOD-PCNA M73L / D147A and electrophoresis of the obtained product is shown.
  • -indicates no PCNA, and + indicates that PCNA was added.
  • KOD Y7A / V93K without PCNA addition when 10% of blood was added, inhibition was not observed and amplification was not confirmed.
  • PCNA mutant was added, a firm band was confirmed even if 10% of blood was contained.
  • the PCNA mutant was also shown to be effective in PCR in the presence of dUTP.
  • Example 13 Amplification from blood by addition of PCNA using reaction solution containing dUTP As in FIG. 7, whether or not crude (blood) resistance is improved by addition of PCNA was also evaluated in a PCR reaction system containing dUTP.
  • As the enzyme KOD Y7A / V93K mutant, KOD Y7A / P36H / N210D was used, and the difference in the amount of 1.3 kb amplification of HBg was compared.
  • Pfu-PCNA M73L / D147A was used as PCNA.
  • PCR was performed using KOD-Plus-Ver. 2 (manufactured by Toyobo), using Buffer, MgSO 4 attached, 1 ⁇ PCR Buffer, and 1.5 mM MgSO 4 , 15 pmol primer (SEQ ID NO: 13 and 14), dNTPs (dATP, dUTP in which 2 mM dTTP was replaced with dUTP) , DCTP, dGTP), and 1U of each enzyme mixed with KOD antibody, in a 50 ⁇ l reaction solution, 0.02%, 0.2%, 0.5%, 2%, 5% %, 10%, and PCNA-free and Pfu-PCNA M73L / D147A added with 250 ng were compared.
  • KOD-Plus-Ver. 2 manufactured by Toyobo
  • the reaction was carried out using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 94 cycles at 94 ° C. for 2 minutes followed by 35 cycles of 98 ° C., 10 seconds ⁇ 65 ° C., 30 seconds ⁇ 68 ° C., 1.5 minutes. It was. After completion of the reaction, 5 ⁇ l of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.
  • FIG. 8 shows the use of blood as a sample, and the reaction solution was prepared so that the proportion of blood in the reaction solution was 0.02%, 0.2%, 0.5%, 2%, 5%, 10%.
  • 1 shows the result of electrophoresis of the product obtained by adding Pfu-PCNA M73L / D147A to KOD Y7A / V93K mutant and KOD Y7A / P36H / N210D and performing PCR reaction.
  • -indicates no PCNA, and + indicates that PCNA was added.
  • inhibition was not performed without addition of PCNA, and amplification was not confirmed from blood.
  • a solid band was confirmed even when 10% of blood was contained in the PCNA mutant added (FIG. 8).
  • the PCNA mutant was shown to be effective in PCR in the presence of dUTP.
  • Example 14 It was also evaluated whether the crude (plant) tolerance was improved by adding PCNA even in a PCR reaction system containing amplified dUTP from plant lysate by adding PCNA. For comparison, KOD Y7A / P36H / N210D mutant and Taq polymerase were used, and the difference in amplification amount of rbcL 1.3 kb was compared. For PCNA, KOD-PCNA M73L / D147A was used.
  • a rice leaf 3 mm square was added to 100 ⁇ l of Buffer A (100 mM Tris-HCl (pH 9.5), 1 M KCl, 10 mM EDTA) and heat-treated at 95 ° C. for 10 minutes, and used as a lysate.
  • Buffer A 100 mM Tris-HCl (pH 9.5), 1 M KCl, 10 mM EDTA
  • the PCR of KOD Y7A / P36H / N210D was performed using KOD-Plus-Ver.
  • the reaction was carried out using PCR system GeneAmp 9700 (Applied Biosystem) with a schedule of repeating 94 cycles at 94 ° C. for 2 minutes followed by 35 cycles of 98 ° C., 10 seconds ⁇ 65 ° C. 30 seconds ⁇ 68 ° C., 1.5 minutes. .
  • Taq DNA polymerase was manufactured by Toyobo and mixed with Anti-Taq High (manufactured by Toyobo).
  • the reaction was performed by adding 50 ⁇ l of Buffer attached to 1 ⁇ BlendTaq, 10 pmol primer (same as above), dNTPs (dATP, dUTP, dCTP, dGTP) in which 2 mM dTTP was replaced with dUTP, and 2.5 U enzyme mixed with the antibody.
  • dNTPs dATP, dUTP, dCTP, dGTP
  • 2 mM dTTP was replaced with dUTP
  • 2.5 U enzyme mixed with the antibody In the reaction solution, plant lysate was added at 2%, 4%, 8% and 16% to the reaction solution, and PCNA-free and KOD-PCNA M73L / D147A added 250 ng were compared.
  • the reaction was performed at 94 ° C.
  • plant lysate was used as a sample, and the reaction solution was prepared so that the proportion of lysate in the reaction solution was 2, 4, 8, 16%, and KOD-PCNA M73L / D147A was added to various enzymes.
  • the result of carrying out PCR reaction and electrophoresis of the obtained product is shown.
  • the enzymes used were KOD Y7A / P36H / N210D mutant and Taq polymerase in total.
  • 1, 2, 8, and 16 indicate the percentage of plant lysate added,-indicates no PCNA, and + indicates that PCNA was added.
  • PCR from body tissues we examined whether PCR is possible using nails, hair, and oral mucosa as a template.
  • a piece of nail cut with a nail clipper and one piece of hair were added to 180 ⁇ l of 50 mM NaOH, crushed by heat treatment at 95 ° C. for 10 minutes, and then neutralized by adding 20 ⁇ l of 1M Tris-HCl (pH 8.0). The supernatant was used as a template.
  • the oral mucosa was obtained by suspending mucosa collected with a cotton swab in 200 ⁇ l of water as a template.
  • PCR for HBg 482 bp was performed and the difference in amplification was compared. did. As above, PCR was performed using KOD-Plus-Ver.
  • Each reaction solution was also added with 250 ng of KOD-PCNA M73L / E143R mutant, which is a PCR enhancing factor.
  • PCR was performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 94 cycles of 94 ° C., 2 minutes and 98 cycles of 10 ° C., 10 seconds ⁇ 65 ° C., 10 seconds ⁇ 68 ° C., 1 minute.
  • 5 ⁇ l of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.
  • FIG. 10 shows results obtained by performing PCR reaction with various DNA polymerases in the presence of KOD-derived PCNA mutant (M73L / E143R) using nail, hair and oral mucus as samples, and electrophoresis of the obtained products. Indicates.
  • the DNA polymerase used was a total of three types: KOD (wild type) and two KOD mutants (Y7A / V93K, Y7A / P36H / N210D). The left side of each photograph uses dTTP, and the right side uses dUTP. When 1 lane is a nail sample, 2 lane is hair, 3 lane is oral mucosa.
  • the respective “+” lanes are in the presence of KOD-derived PCNA mutant (M73L / E143R), and “ ⁇ ” indicates no PCNA.
  • KOD-derived PCNA mutant M73L / E143R
  • ⁇ indicates no PCNA.
  • wild-type KOD DNA polymerase was not amplified in the presence of dUTP, but a band was confirmed even in the presence of dUTP in the KOD Y7A / V93K and KOD Y7A / P36H / N210D mutants having decreased base analog detection activity. (FIG. 10).
  • As a result of adding PCNA it was confirmed that the amplification amount was improved as compared with the case where PCNA was not added.
  • KOD KOD
  • Example 16 It was evaluated whether amplified dUTP from stool and gene amplification can be performed in the presence of stool.
  • As the enzyme KOD Y7A / P36H / N210D mutant and Taq polymerase were used, and the difference in amplification of about 700 bp of Salmonella invA gene was compared by real-time PCR using SYBR GREEN I and melting curves.
  • the KOD Y7A / P36H / N210D mutant was also added with KOD-PCNA M73L / D147A added as a PCR enhancing factor.
  • a 10% fecal suspension was heat-treated at 95 ° C. for 10 minutes.
  • a buffer attached to KOD Dash manufactured by Toyobo
  • 1 ⁇ PCR Buffer 50 copies of Salmonella genome, 4 pmol primer (SEQ ID NO: 25 and 26), 2 mM dTTP to dUTP
  • 20 ⁇ l of a reaction solution containing 0.4 U of enzyme mixed with substituted dNTPs (dATP, dUTP, dCTP, dGTP), 1/30000 SYBR GREEN I, and KOD antibody feces were added to the reaction solution at 0, 0, 0.
  • the reaction is as follows: Buffer attached to 1 ⁇ Taq (Mg attachment type), 50 copies of Salmonella genome, 4 pmol of primer (same as above), 2 mM dTTP substituted with dUTP (dATP, dUTP, dCTP, dGTP), 4 mM In 20 ⁇ l of a reaction solution containing 1 U of enzyme mixed with MgSO 4 , 1/30000 SYBR GREEN I, antibody, feces were added to the reaction solution at 0, 0.1, 0.25, 0.5, 1.0. , 1.5, 2.0, 2.5%, 95 ° C., 30 seconds pre-reaction, 98 ° C., 10 seconds ⁇ 60 ° C. 10 seconds ⁇ 68 ° C., 30 seconds repeated 50 cycles
  • the schedule was performed using LightCycler 2.0 (Roche). After completion of the reaction, a target peak appearing in the latter half of 80 ° C. was confirmed by melting curve analysis.
  • Table 4 shows Cq values of Example 16.
  • Table 4 shows Cq values (default settings of LightCycler 2.0) of real-time PCR performed in the presence of dUTP and feces.
  • N. D. Indicates that no amplification was observed and no Cq value was obtained. As a result, amplification was not observed when 0.5% stool was added to Taq polymerase, but amplification was confirmed even when 2.5% stool was added to KOD Y7A / P36H / N210D. Further, comparing the presence or absence of PCNA, it was found that the one with PCNA added had a smaller Cq value and showed excellent PCR efficiency.
  • FIG. 12 shows the results of melting curve analysis of the obtained amplification product using PCR in the presence of dUTP and feces.
  • the total number of polymerases used was KOD Y7A / P36H / N210D mutant, KOD Y7A / P36H / N210D mutant with KOD-PCNA M73L / D147A added, and Taq polymerase.
  • 1 shows the result of KOD Y7A / P36H / N210D
  • 2 shows the result of adding KOD-PCNA M73L / D147A to KOD Y7A / P36H / N210D
  • 3 shows the result of Taq polymerase.
  • KOD Y7A / V93K, P36H, P36K, P36R, V93K, V93R, Y7A / P36H, Y7A / P36R, Y7A / V93R, P36H / H147E, P36K / H147E, P36R / H147E, V93K / H147E, V93K / R147E / H147E, Y7A / P36H / H147E, Y7A / P36R / H147E, Y7A / V93K / H147E, Y7A / V93R / H147E, P36H / N210D, P36K / N210D, P36R / N210D, V93K / N210D, V93R7 / N210D, Y7A / V93K / N210D, Y7A / V93R / N210D, P36H / N
  • PCNA is also a variant of KOD-PCNA M73L / E143R, M73L / R82A / E143A, M73L / R109A / E143A, Pfu-PCNA M73L / D143R, M73L / D147A, M73L / R82A / D143A, M73L / R109A / D143A.
  • the same reaction was performed, and it was confirmed that the PCR efficiency was improved as compared with the case where PCNA was not added.
  • the present invention eliminates the risk of loss and carryover during DNA purification, and further reduces time and cost. Moreover, a gene can be accurately amplified by using a polymerase belonging to Family B. This technique can be widely used not only in the research field, but also in the clinical field such as genetic diagnosis or the field of forensic medicine, or in the examination of microorganisms in food or the environment.

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Abstract

L'invention fournit un test d'amplification d'acide nucléique permettant une amplification directe sans processus de purification préalable, d'un acide nucléique cible dans un échantillon biologique. Plus précisément, l'invention concerne un procédé selon lequel un échantillon biologique est ajouté à un liquide de réaction d'amplification d'acide nucléique sans processus de purification préalable, et un acide nucléique cible est amplifié dans l'échantillon biologique. Ce test d'amplification d'acide nucléique est caractéristique en ce qu'une polymérase appartenant à la famille B et un antigène nucléaire de prolifération cellulaire (PCNA), sont inclus dans le liquide de réaction.
PCT/JP2014/070323 2013-08-06 2014-08-01 Test d'amplification d'acide nucléique Ceased WO2015019952A1 (fr)

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WO2015122432A1 (fr) * 2014-02-17 2015-08-20 東洋紡株式会社 Procédé permettant d'améliorer la précision de l'amplification de l'acide nucléique

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WO2015122432A1 (fr) * 2014-02-17 2015-08-20 東洋紡株式会社 Procédé permettant d'améliorer la précision de l'amplification de l'acide nucléique

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