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WO2024190829A1 - Nouveau procédé de préparation de banques à partir d'une réaction de ligature d'oligo adn et d'une réaction continue d'amplification de l'adn due à l'oligo adn ligaturé - Google Patents

Nouveau procédé de préparation de banques à partir d'une réaction de ligature d'oligo adn et d'une réaction continue d'amplification de l'adn due à l'oligo adn ligaturé Download PDF

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WO2024190829A1
WO2024190829A1 PCT/JP2024/009803 JP2024009803W WO2024190829A1 WO 2024190829 A1 WO2024190829 A1 WO 2024190829A1 JP 2024009803 W JP2024009803 W JP 2024009803W WO 2024190829 A1 WO2024190829 A1 WO 2024190829A1
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oligo
sequence
reaction
dna
target product
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Japanese (ja)
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信也 蔵田
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Nippon Steel Eco Tech Corp
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Nippon Steel Eco Tech Corp
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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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • 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
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the present invention relates primarily to assays for detecting or sequencing a specific group of nucleic acid sequences.
  • the present invention provides an amplification method in which one or more artificially designed nucleic acid sequences (hereinafter, "artificial nucleic acid sequences"), including barcode sequences, which are artificial nucleic acid sequences for identifying a sample, are added to a target nucleic acid sequence.
  • artificial nucleic acid sequences including barcode sequences, which are artificial nucleic acid sequences for identifying a sample
  • Detecting specific nucleic acid sequences is used in many fields, including genetic diagnosis in medicine, food hygiene inspections, and environmental monitoring.
  • nucleic acid sequencing has become much easier and cheaper than conventional sequencing methods, and is therefore spreading as a new analytical method.
  • NGS next-generation sequencers
  • specific artificial nucleic acid sequences including barcode sequences for identifying the sample must be added to both ends of the nucleic acid fragment to be sequenced, and a nucleic acid amplification reaction step for this purpose is necessary in addition to the step of amplifying the target nucleic acid sequence.
  • nucleic acid fragments to be subjected to NGS by using a primer that has a specific artificial nucleic acid sequence containing a barcode sequence added to the 5' end of the primer specific to the target nucleic acid sequence (prior art [1]).
  • this method requires the preparation of primers with different barcode sequences for each type of nucleic acid sequence, for the number of samples, which is extremely uneconomical ( Figure 1).
  • Non-Patent Document 1 A method of performing two-stage PCR (prior art [2]) is commonly used as a means of economically obtaining an amplified product to which an artificial nucleic acid sequence has been added (Non-Patent Document 1).
  • a first PCR is performed using a primer to which a part of the 3' end of the artificial nucleic acid sequence has been added to the 5' end of a specific sequence (hereinafter, artificially added specific primer) to obtain a product derived from the target nucleic acid (hereinafter, specific product).
  • a second PCR is performed using this as a template and a primer to which an artificial nucleic acid sequence such as a barcode sequence has been added to the 3' end (hereinafter, artificial sequence primer).
  • Prior art [4] discloses a method in which, in addition to an artificial addition specific primer, in a reaction for adding an artificial nucleic acid sequence to a target gene sequence, two pairs of artificial sequence primers, a long artificial sequence primer (hereinafter, long artificial sequence primer; "primer pair Y” in FIG. 4) and a short artificial sequence primer (hereinafter, short artificial sequence primer; "primer pair Z” in FIG. 4), are used to suppress the amplification of non-specific sequences derived from the primers and to efficiently amplify the target product.
  • a long artificial sequence primer hereinafter, long artificial sequence primer; "primer pair Y" in FIG. 4
  • short artificial sequence primer hereinafter, short artificial sequence primer; "primer pair Z” in FIG. 4
  • the target product can be amplified more efficiently by lowering the concentration of the artificial addition specific primer and the artificial sequence primer (primer pair Y) containing a barcode sequence, etc., and increasing the concentration of the short primer (primer pair Z).
  • the prior art [4] has the following problems: (1) strict optimization of reaction conditions is required for each target nucleic acid, which requires an excessive amount of time and effort; and (2) sensitivity is lower than that of conventional PCR, which uses only one primer pair, and more initial templates (target genes) are required.
  • Patent Documents 4 and 5 A method (prior art [5]) that can solve the problems of the above prior art [4] has also been proposed (Patent Documents 4 and 5), but this prior art also has the following problems.
  • NGS analysis it is essential to quantify the target product so that the amount of the target product added falls within the range specified by each NGS device manufacturer.
  • simple and low-cost methods such as electrophoresis, fluorometry, and absorbance measurement, as well as quantitative PCR (hereinafter, qPCR), which is a relatively complicated and expensive method, are commonly used.
  • qPCR quantitative PCR
  • the former simple and low-cost method cannot accurately distinguish the target product from the non-target product
  • the ratio of the target product in the specific products is extremely high, the difference between the quantitative value obtained assuming that all specific products are the target product and the true amount of the target product can be ignored, so it can be used without any problems.
  • the ratio of the target product in the specific products is low, it is necessary to use the qPCR method, which is a complicated and expensive method but can distinguish the presence or absence of an artificial nucleic acid sequence and quantify only the target product.
  • the qPCR method which is a complicated and expensive method but can distinguish the presence or absence of an artificial nucleic acid sequence and quantify only the target product.
  • not only the desired product but also many specific products to which no artificial nucleic acid sequence has been added are synthesized, making quantification using cumbersome and costly qPCR necessary.
  • the adapter sequence is added not only to the target product but also to the non-target product, which is then sequenced. Since the non-target product does not have a barcode sequence added, it is not possible to identify which sample the gene sequence comes from, so unusable gene sequence information is output from the NGS depending on the proportion of the non-target product.
  • the amount of usable gene sequence information decreases in proportion to the increase in unusable gene sequence information. Therefore, in the case of the NGS made by ONT, it is very important to reduce the non-target product as much as possible and increase the proportion of the target product in order to reduce costs. Under normal operating conditions in the prior art [5], the ratio of the target product to the specific products is around 50%, which means that 50% of the output gene sequence information cannot be used, and the cost of obtaining the amount of target gene sequence information is doubled compared to samples that do not contain non-target products.
  • oligo DNA artificially added specific SL primer and SL oligo with artificial 3' sequence
  • a substance blocking DNA elongation
  • SD polymerase Bioron
  • the former oligo DNA is lengthened because a sequence needs to be added for the purpose of forming an intramolecular secondary structure, and the synthesis cost is high because a blocker needs to be inserted, and the latter SD polymerase needs to be added in an amount 10 times that specified in the manufacturer's protocol to obtain a sufficient effect, leading to high reagent costs.
  • (1a) A step of reacting three types of oligo DNA pairs (ia) to (iiia) below in the presence of a ligase to ligate the oligo DNA (iia) and the oligo DNA (iiia) below by a nick repair reaction using DNA ligase to synthesize a linked primer pair (linked primer (F) and linked primer (R) in FIG. 5 ).
  • (ia) A linking oligo pair (linking oligo (F) and linking oligo (R) in FIG. 5) having a complementary sequence to a sequence required for linking oligo DNAs (hereinafter, linking sequence) in a nick repair reaction by ligase.
  • (2a) A step of carrying out gene amplification using the linked primer pair synthesized in the above step (1).
  • the unpurified reaction solution obtained in the above step (1a) is directly added to the reaction solution (2a) to carry out the above two types of reactions (1a) and (2a), or in a reaction solution to which all necessary components (DNA polymerase, DNA ligase, dNTPs, etc.) have been added, the above two types of reactions (1a) and (2a) are carried out successively in the order of (1a) and (2a) without opening the reaction tube, thereby making it possible to prepare a library containing a high proportion of the target product in an economical and simple manner.
  • (1b) A step of synthesizing a linked primer pair (linked primer (F) and linked primer (R) in FIG. 6 ) by linking two types of oligo DNA pairs of the following (ib) and (iib) by a click chemistry reaction: (ib) A pair of 3'CC oligos having a sequence specific to the gene to be analyzed and whose 5' ends are modified with a click chemistry functional group (3'CC oligo (F) and 3'CC oligo (R) in FIG. 6).
  • the unpurified reaction solution obtained in the above step (1b) is directly added to the reaction solution (2b) to carry out the above two types of reactions (1b) and (2b), or, in a reaction solution to which all necessary components (DNA polymerase, dNTPs, etc.) have been added, the above two types of reactions (1b) and (2b) are carried out successively in the order of (1b) and (2b) without opening the reaction tube, thereby making it possible to prepare a library containing a high proportion of the target product in an economical and simple manner.
  • Such a technology can be applied to analyses for various purposes. For example, genes that may be the cause of cancer can be comprehensively analyzed in order to identify the location of gene mutations.
  • this technology can be suitably used when using NGS in various SNP analyses, gene mutation analyses, gene expression analyses, and microbiota analyses, but the scope of application is not limited as long as the test subject is a nucleic acid amplification product and the method involves analyzing multiple target genes by adding an artificial nucleic acid sequence and identifying the sample.
  • the gist of the present invention is as follows.
  • a method for preparing a target product in which an artificial nucleic acid sequence is added to a target nucleic acid sequence comprising the steps of (1a) and (2a) below:
  • linking oligo pair (linking oligo (F) and linking oligo (R) in FIG. 5) having a complementary sequence to a sequence required for linking oligo DNAs (hereinafter, linking sequence) in a nick repair reaction by ligase.
  • linking sequence a sequence required for linking oligo DNAs in a nick repair reaction by ligase.
  • a 3'LG oligo pair having a part of the 3'-terminal sequence of the linking sequence at the 5'-terminal side, a sequence specific to the gene to be analyzed at the 3'-terminal side, and a phosphorylated 5'-terminal (3'LG oligo(F) and 3'LG oligo(R) in FIG. 5 ).
  • the kit according to [3] which comprises a ligation oligo having a 3'-end modified to prevent DNA elongation.
  • [6] A method for preparing a target product in which an artificial nucleic acid sequence is added to a target nucleic acid sequence, the method comprising the steps of (1b) and (2b) below: (1b) A step of synthesizing a linked primer pair (linked primer (F) and linked primer (R) in FIG. 6 ) by linking two types of oligo DNA pairs of the following (ib) and (iib) by a click chemistry reaction: (ib) A pair of 3'CC oligos having a sequence specific to the gene to be analyzed and whose 5' ends are modified with a click chemistry functional group (3'CC oligo (F) and 3'CC oligo (R) in FIG. 6).
  • the gene amplification method is selected from the group consisting of PCR (Polymerase Chain Reaction) method, LCR (Liagase Chain Reaction) method, RPA (Recombinase Polymerase Amplification) method, SIBA (Strand-Invasion Based Amplification) method, LAMP (Loop-Mediated Iso-thermal Amplification) method, SDa (Strand Displacement Amplification) method, and NEAR (Nicking Endonuclease Amplification) method.
  • PCR Polymerase Chain Reaction
  • LCR Liagase Chain Reaction
  • RPA Recombinase Polymerase Amplification
  • SIBA Strand-Invasion Based Amplification
  • LAMP Long-Mediated Iso-thermal Amplification
  • SDa Strand Displacement Amplification
  • NEAR NEAR
  • the gene amplification method is a PCR method using at least one DNA polymerase selected from the group consisting of PCR enzymes having an extension rate faster than 10 sec/kb.
  • PCR enzyme used is at least one DNA polymerase selected from the group consisting of repliQa HiFi ToughMix and KOD ONE PCR Master Mix.
  • a method for preparing a mixture containing two or more target products having different barcode sequences comprising: adding a dsDNA-specific dye to each gene amplification reaction solution containing the target products; quantifying the concentration of each target product from the fluorescence value of the dye; and mixing each gene amplification reaction solution so that the target products have an arbitrary concentration ratio based on the quantification results.
  • purification is carried out on a mixture of target products.
  • Dissociation corrected fluorescence intensity value Fn ,TT /Fn ,DN F n,TT : The temperature at which only the desired product exists as dsDNA in any cycle (n) (TT; Temperature at which only the (Abbreviation for Targeted product exists as dsDNA) F n,DN : Fluorescence intensity measured at a temperature at which all DNA dissociates in an arbitrary cycle (n) (DN; short for De Nature ) [24]
  • F CBFI,DN Fluorescence intensity measured at a temperature where all DNA dissociates in the cycle before an increase in fluorescence due to gene amplification is confirmed (CBFI).
  • a method for modifying a target product comprising: adding a single base, A, to the 3' end of the target product amplified using a primer whose 5' end is phosphorylated in the method according to [1] or [2], and then adding an adapter sequence to the product using a ligase.
  • the method according to [31] comprising amplifying a target product using a PCR enzyme having proofreading activity, and then adding a single base A to the 3' end of the target product by adding an inactivator of the PCR enzyme and a DNA polymerase having an activity of adding A to the 3' end to the reaction solution.
  • the present invention relates to (ia) A linking oligo pair (linking oligo (F) and linking oligo (R) in FIG. 5) having a complementary sequence to a sequence required for linking oligo DNAs (hereinafter, linking sequence) in a nick repair reaction by ligase. (iia) A 3'LG oligo pair having a part of the 3'-terminal sequence of the linking sequence at the 5'-terminal side, a sequence specific to the gene to be analyzed at the 3'-terminal side, and a phosphorylated 5'-terminal (3'LG oligo(F) and 3'LG oligo(R) in FIG. 5 ).
  • the present invention provides a method for obtaining a desired product derived from one or more target nucleic acids, using an oligo-DNA set comprising the above-mentioned (iia) and (iiia), a reaction reagent such as a DNA polymerase for carrying out an amplification reaction, and a reaction reagent for carrying out a DNA ligase reaction for linking the oligo-DNAs described in the above (iia) and (iiia).
  • the present invention provides a method for amplifying, for example, one type of nucleic acid sequence in a sample and obtaining a target product in which an artificial nucleic acid sequence containing a barcode sequence is added to both ends of the amplified product.
  • the details are described below (see Figures 5 to 7).
  • the reaction solution used in the embodiment described in FIG. 5 contains the oligo DNA set (ia) to (iiia), amplification reagents including DNA polymerase, and reagents including DNA ligase for carrying out a ligase reaction. Using these, the following reaction steps (1a) and (2a) are carried out to obtain the desired product.
  • Reaction step (1a) By mixing the three types of oligo DNA pairs (ia) to (iiia) in a reaction solution, the 5'LG oligo (F or R) and the 3'LG oligo (F or R) are bound to the linking oligo (F or R) in a form adjacent to each other, and a ligase recognizes the linking site and links the 5'LG oligo (F or R) and the 3'LG oligo (F or R) by a nick repair reaction.
  • Reaction step (2a) A reaction step for obtaining a target product by gene amplification using the linking primer pair (F and R) obtained in the step (1a). The reaction solution used in the embodiment described in FIG.
  • reaction step (1b) A reaction in which the oligo DNAs (F or R) described in (ib) and (iib) above are mixed in a reaction solution to link the 5' CC oligo (F or R) and the 3' CC oligo (F or R), each modified with a click chemistry functional group, by click chemistry.
  • reaction step (2b) A reaction step to obtain a target product by gene amplification using the linked primer pair (F and R) obtained in the step (1b).
  • this suppression method utilizes the fact that the linked primer is longer than the unlinked oligo, and as a result, the dissociation temperature of the linked primer is higher than that of the unlinked oligo, and after the target product derived from the linked primer is first synthesized (for example, in the case of PCR, the target product is first synthesized in the second cycle, and assuming a synthesis efficiency of 100%, the amount of the target product in the second cycle is the same as that of the initial template), there is no problem in binding of the linked primer to the target product, and the annealing temperature is raised to a temperature at which the binding of the unlinked oligo can be sufficiently suppressed, thereby suppressing the amplification of non-target products and making the amplification of the target product dominant.
  • FIG. 7 shows the reaction step (2a) as an example, the same can be said for the reaction step (2b), and the method shown in FIG. 7 can suppress the amplification of non-target products and make the amplification of the target product dominant.
  • the present invention has various variations as described below.
  • Two or more pairs of 3'LG oligo pairs or 3'CC oligo pairs containing a target gene-specific sequence are used, and the reaction steps (1a), (2a), or (2a), (2b) are carried out to obtain two or more target products.
  • the above variation is effective when introducing different barcode sequences into each end and performing sequence separation (demultiplexing) including not only barcode sequences but also combinations thereof.
  • reaction steps (1a), (2a) or (2a), (2b) can be carried out independently, and the reaction steps can be arbitrarily separated and used depending on the purpose. However, an embodiment in which all necessary materials are added to the same reaction solution before the start of the reaction and the reaction steps are continuously carried out in a single reaction is more advantageous in terms of economy and simplicity.
  • the linking oligo which serves as a scaffold for linking the 3'LG oligo and the 5'LG oligo, functions only in the reaction step (1a) and does not function in the reaction step (2a), which is a gene amplification reaction. For this reason, it is desirable to label the 3' end of the linking oligo so that it is not extended by DNA polymerase.
  • the label is generally modified with phosphorylation, fluorescent dyes, amino linkers, biotin, etc., but any modification method that can prevent the extension of the 3' end by DNA polymerase may be used.
  • the ligase used in the reaction step (1a) can be any type of ligase as long as it has the activity of repairing a nick in double-stranded DNA. However, since the ligase does not contribute to the reaction step (2a), which is a gene amplification step, it is preferable to use a ligase that is completely inactivated in the step. For example, in the case of a gene amplification method in which the reaction temperature is high, such as the PCR method, a non-thermostable ligase is inactivated.
  • non-thermostable ligases that have the activity of repairing a nick in double-stranded DNA include T3 DNA Ligase, T4 DNA Ligase, T7 DNA Ligase, PBCV-1 DNA Ligase, Chlorella virus DNA Ligase, and E. coli DNA Ligase.
  • examples of heat-resistant ligases having the activity of repairing nicks in double-stranded DNA include Taq DNA Ligase and 9 o N DNA Ligase. Even when a gene amplification reaction with a high reaction temperature is adopted, these heat-resistant ligases maintain their activity to a certain extent in the reaction step (2a) (gene amplification step), and may affect the reaction step (2a).
  • the functional group of click chemistry used to modify the oligo-DNA used in the reaction step (1b) may be any functional group that can modify the oligo-DNA and can link the 5' CC oligo and the 3' CC oligo.
  • the binding between the azide group and the alkyne group, which is a typical reaction of click chemistry requires copper ions as a catalyst, and the ions cause DNA damage and DNA strand cleavage, so it is not very preferable as a click chemistry reaction used in the present invention.
  • DBCO dibenzocyclooctyne
  • ADIBO azadibenzocyclooctyne
  • BCN bicyclononyne
  • DBN 4,8-diazacyclononyne
  • the DNA polymerase used in the reaction steps (2a) and (2b) may be any polymerase that can be used for gene amplification.
  • the reaction steps (2a) and (2b) of ligating oligo DNAs if an activated DNA polymerase is present, the ligation reaction of the oligo DNAs may be inhibited.
  • the reaction step (2a) when the 5'LG oligo is bound to the ligation oligo, if the DNA polymerase is present in an activated state, the 5'LG oligo will extend using the ligation oligo as a template, and the 3'LG oligo will not be able to bind to the ligation oligo, which may result in inhibition of the ligation of the 5'LG oligo and the 3'LG oligo by the ligase.
  • the gene amplification reactions used in the reaction steps (2a) and (2b) may be any gene amplification reactions using primers.
  • Specific gene amplification methods include PCR (Polymerase Chain Reaction) method, LCR (Liagase Chain Reaction) method, RPA (Recombinase Polymerase Amplification) method, SIBA (Strand-Invasion Based Amplification) method, LAMP (Loop-Mediated Iso-thermal Amplification) method, SDA (Strand Displacement Amplification) method, and NEAR (Nicking Endonuclease Amplification) method.
  • Examples of the amplification method include the Helicase-dependent Amplification (HDA) method, the Nucleic Acid Sequence-Based Amplification (NASBA) method, the ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method, the Rolling Cycle method, and the Smart Amplification Process (SMAP) method.
  • HDA Helicase-dependent Amplification
  • NASBA Nucleic Acid Sequence-Based Amplification
  • ICAN Isothermal and Chimeric primer-initiated Amplification of Nucleic acids
  • Rolling Cycle method the Rolling Cycle method
  • SMAP Smart Amplification Process
  • the present invention is expected to provide the following effects. (1) In a single-step process, a library containing a high percentage of the desired product having an artificial nucleic acid sequence added to its end can be obtained. (2) In the present invention, after the target product is initially synthesized, the annealing temperature is increased, making it possible to limit the primers that contribute to the gene amplification reaction to only the ligated primer, eliminating the need to use a primer for amplification using the target nucleic acid as a template and a primer for amplification using an amplified product derived from the target nucleic acid as a template in the same reaction solution as in the prior art methods [3] and [4], and thus making it possible to suppress the generation of non-specific amplification products such as primer dimers.
  • the minimum required amount of initial template can be kept lower than in the prior art described above, and the same sensitivity as that of general gene amplification using only primers specific to the target nucleic acid can be ensured.
  • reaction steps (1a) and (1b) even in a one-step closed reaction system, by changing the temperature conditions, it is possible to completely separate the reaction of linking two oligo DNAs (reaction steps (1a) and (1b)) and the amplification reaction (reaction steps (2a) and (2b)), and therefore it is possible to carry out both reactions under optimal temperature conditions.
  • the oligo DNA sequence that contributes to the reaction of linking two oligo DNAs can react under the same conditions regardless of the target nucleic acid, the optimization of the reaction conditions in the method of the present invention is limited to the amplification using the amplification product derived from the target nucleic acid as a template, and therefore a reaction system can be constructed simply and quickly, similar to general gene amplification.
  • 10 types of forward primers and 10 types of reverse primers are required for each target nucleic acid, for a total of 20 types of primers, and since the set is required for each type of target nucleic acid, it is necessary to prepare 2,000 types of primers.
  • the amplification of the target nucleic acid is performed by a 3'LG oligo or a 3'CC oligo
  • the addition of the artificial nucleic acid sequence including the barcode sequence is performed by a 5'LG oligo or a 5'CC oligo, so the type of oligo DNA required for each depends on the type of target nucleic acid for the former and the number of samples to be analyzed for the latter.
  • 3'LG oligo or 3'CC oligo is 200 types including forward / reverse primers
  • 5'LG oligo or 5'CC oligo is 200 types including forward / reverse primers
  • the linking oligo is one type, so a total of 401 types including the oligo, and even if the artificial nucleic acid sequence added to both ends is different, only two types of linking oligo are required, so a total of 402 types of oligo DNA including the oligo are sufficient.
  • linking oligo is not required, so 400 types are sufficient.
  • the sequence of 5'LG oligo, 5'CC oligo, and linking oligo depending on the difference in target nucleic acid they can be commonly used regardless of the target nucleic acid, and can be used without waste. (5) Since the concentration of the target product to be subjected to NGS needs to be constant, it is necessary to adjust the concentration after the target product is created.
  • the present invention it is possible to prepare a library in which the target product accounts for the majority (95% or more) of the specific products with the standard manufacturer-recommended enzyme usage amount without using a special labeling substance or oligo-DNA having a structure, so that the target product can be accurately quantified even with the low-cost and simple method described above.
  • the target product can be quantified more simply and at a lower cost than the prior art [5].
  • (6) In the case of analysis that requires the addition of adapters required for analysis to both ends of the target product using ligase or the like, such as ONT's NGS, adapters are added not only to the target product but also to non-target products, and sequence analysis is performed.
  • the ratio of the target product under normal implementation conditions in the prior art [5] is around 50%, but the ratio of the target product can be increased to a maximum of about 90% by using the above-mentioned 3' artificial sequence-introduced SL oligo or artificially added specific SL primer, or by adding a high concentration of SD polymerase.
  • oligo-DNA modified with special substances is not required, and the proportion of the target product can be increased up to 95% by adding DNA polymerase at the concentration recommended by the manufacturer. This makes it possible to increase the proportion of the target product more simply and at lower cost than in the prior art [5]. As a result, this method can be suitably used as a library preparation method for the above-mentioned ONT NGS and the like.
  • the present invention makes it possible to prepare libraries containing a high percentage of the desired product economically and with simple operations.
  • primers with different barcode sequences must be prepared for each type of nucleic acid sequence, the number of samples.
  • a first amplification is performed using an artificially added specific primer in which a portion of the 3' end of an artificial nucleic acid sequence is added to the 5' end of a specific sequence, to obtain a specific product derived from the target nucleic acid.
  • a second amplification is performed using the product as a template, using an artificial sequence primer that has an artificial nucleic acid sequence such as a barcode sequence on the 3' end.
  • an artificial sequence primer that has an artificial nucleic acid sequence such as a barcode sequence on the 3' end.
  • reaction scheme of the novel ligation-based library preparation method Reaction scheme of the novel library preparation method based on click chemistry. A diagram showing the principle of the amplification of non-target products using unlinked oligos, the problems associated with this, and the suppression of the amplification of non-target products by increasing the reaction temperature during gene amplification.
  • FIG. 1 is a diagram showing the amount of a specific product under each reaction condition carried out in Example 1.
  • Electrophoresis (PAGE) photograph after ligase reaction in Example 2.
  • Electrophoresis photograph after click chemistry reaction. Comparison of the theoretical composition ratio of DNA-Mock and the actual composition ratio of the target product in Example 6. Comparison of the actual composition ratio of the target product and the actual composition ratio of the non-target product in Example 6. Graph showing the number of reads for each target gene in Example 7.
  • (b) Relationship between the concentration of double-stranded DNA after purification and the final corrected fluorescence intensity The flow of the new process and the time-saving effect achieved by applying the items described in Examples 8 to 11 are shown below.
  • the present invention provides a method for preparing a target product in which an artificial nucleic acid sequence is added to a target nucleic acid sequence, the method comprising the following steps (1a) and (2a): (1a) A step of reacting three types of oligo DNA pairs (ia) to (iiia) below in the presence of a ligase to ligate the oligo DNA (iia) and the oligo DNA (iiia) below by a nick repair reaction using DNA ligase to synthesize a linked primer pair (linked primer (F) and linked primer (R) in FIG. 5 ). (ia) A linking oligo pair (linking oligo (F) and linking oligo (R) in FIG.
  • linking sequence a sequence required for linking oligo DNAs (hereinafter, linking sequence) in a nick repair reaction by ligase.
  • linking sequence a sequence required for linking oligo DNAs (hereinafter, linking sequence) in a nick repair reaction by ligase.
  • a 3'LG oligo pair having a part of the 3'-terminal sequence of the linking sequence at the 5'-terminal side, a sequence specific to the gene to be analyzed at the 3'-terminal side, and a phosphorylated 5'-terminal (3'LG oligo(F) and 3'LG oligo(R) in FIG. 5 ).
  • the linking oligo has a sequence at its 5'-end side that is complementary to a portion of the 5'-end sequence of the 3'LG oligo, and a sequence at its 3'-end side that is complementary to a portion of the 3'-end sequence of the 5'LG oligo, and both of the former and latter sequences are preferably 4 to 30 bases long, preferably 6 to 20 bases long, and more preferably 8 to 12 bases long.
  • the 3'LG oligo pair has a sequence at its 5' end that is complementary to the sequence at the 5' end of the linking oligo, and a sequence at its 3' end that is specific to the gene to be analyzed.
  • the former 5' end sequence may be 4-30 bases long, preferably 6-20 bases long, and more preferably 8-12 bases long, while the latter 3' end sequence may be set to a sequence length generally preferred for gene amplification primers, and no special setting dependent on the method of the present invention is necessary.
  • the 5' end of the 3'LG oligo needs to be phosphorylated in order to enable linking with the 5'LG oligo by ligase.
  • the 5'LG oligo pair has an artificial nucleic acid sequence at its 5' end, such as a barcode sequence for identifying the sample and an adapter for binding to the oligo DNA present on the NGS flow cell, and a sequence at its 3' end that is complementary to the sequence at the 3' end of the linking oligo.
  • the sequence and length of the former at the 5' end are determined by the NGS used, so they can be set according to the sequence recommended by the NGS manufacturer, and no special setting dependent on the method of the present invention is necessary.
  • the latter sequence at the 3' end should be 4 to 30 bases long, preferably 6 to 20 bases long, and more preferably 8 to 12 bases long.
  • linking oligo sequence at one end different from the linking oligo sequence at the other end, and designing the sequence at the 5' end of the 3'LG oligo that binds to the linking oligo pair and the sequence at the 3' end of the 5'LG oligo so that they are complementary to the sequence in question.
  • the dissociation temperature is preferably ⁇ 15°C relative to the ligase reaction temperature, preferably ⁇ 10°C, and more preferably ⁇ 5°C. Furthermore, it is desirable to make the dissociation temperatures of the 3'LG oligo and the 5'LG oligo relative to the linking oligo as consistent as possible so that the 3'LG oligo and the 5'LG oligo are reliably bound to the linking oligo at the ligase reaction temperature.
  • any type of polymerase may be used as long as it is usable for gene amplification.
  • the step of linking the oligo-DNAs if the DNA polymerase is present in an activated state, the ligation reaction of the oligo-DNAs may be inhibited.
  • reaction step (1a) ligation reaction by ligase
  • the DNA polymerase is present in an activated state when the 5'LG oligo is bound to the ligation oligo, the 3' end of the 5'LG oligo will be extended using the ligation oligo as a template, and the 3'LG oligo will not be able to bind to the ligation oligo, and as a result, the ligation of the 3'LG oligo and the 5'LG oligo by the ligase may be inhibited.
  • the target product obtained by the present invention is subjected to NGS analysis, it is desirable for the product to have as few base incorporation errors resulting from gene amplification as possible, and therefore it is desirable to use a polymerase that has proofreading activity (3' ⁇ 5' exonuclease activity) that can suppress base incorporation errors.
  • DNA polymerases having the above characteristics include PrimeSTAR (registered trademark) HS DNA Polymerase (Takara Bio Inc.), KOD plus (Toyobo Co., Ltd.), Exact Polymerase (Five Prime Co., Ltd.), repliQa HiFi ToughMix (Quantabio Co., Ltd.), and KOD ONE PCR Master Mix (Toyobo Co., Ltd.).
  • an effective method for suppressing the amplification of non-target products that arise as a result of the 3'LG oligo that was unable to be linked to the 5'LG oligo functioning as a primer is to raise the reaction temperature immediately after the target product derived from the linked primer is first synthesized in a manner that does not interfere with the annealing of the linked primer and that suppresses the annealing of the unlinked oligo (3'LG oligo).
  • the annealing temperature of any cycle from the cycle following the second cycle (the third cycle) in which the target product derived from the linked primer is first synthesized can be raised to a temperature at which the annealing of the linked primer to the target product is not hindered and the annealing of the unlinked oligo (3'LG oligo) can be suppressed, thereby suppressing the amplification of the non-target product and making the amplification of the target product dominant.
  • the cycle in which the annealing temperature is raised is preferably the third to eleventh cycles, preferably the third to fifth cycles, and more preferably the third cycle.
  • the annealing temperature is set based on the dissociation temperature of the target gene-specific sequence by 3 to 10 cycles, it is possible to ensure a sufficient amount of the product derived from the linked primer in the initial cycle with a low annealing temperature, and to ensure the final required amount of the target product even if the annealing temperature is subsequently increased.
  • the annealing temperature after temperature increase may be set within a temperature range in which the amplification efficiency of the linked primer is sufficiently higher than that of the unlinked 3'LG oligo, and the extension activity of the DNA polymerase does not decrease to a state in which gene amplification does not proceed normally, using the ratio of the target product to the specific product targeted by each practitioner (for example, the ratio of the target product to the specific product is 95%) as an index.
  • the annealing temperature is preferably 60 to 90°C, more preferably 65 to 85°C, and even more preferably 68 to 80°C.
  • PCR When PCR is used as the gene amplification method, if the annealing temperature after heating set as described above is close to the optimal extension temperature of DNA polymerase, two-step PCR may be performed in which annealing and extension are performed in the same step.
  • the annealing temperature in the initial cycle of gene amplification of the present invention is a value that should be determined from the dissociation temperature of the sequence region specific to the target gene, as in normal gene amplification, and is generally set to a temperature 2 to 5°C lower than the dissociation temperature, but in the present invention, it may be set according to a generally known method.
  • the gene amplification method is an isothermal gene amplification method such as the SIBA method, the SDA method, or the NEAR method
  • the reaction temperature is constant, so by raising the reaction temperature after the time has elapsed in which the target product is first obtained, it is possible to suppress the amplification of non-target products as described above and to make the amplification of the target product predominant.
  • any type of ligase can be used as long as it has the activity of repairing nicks in double-stranded DNA.
  • ligase activity is not necessary during gene amplification reaction (except for the LCR method)
  • Specific examples of non-thermostable ligases having the activity of repairing nicks in double-stranded DNA include T3 DNA Ligase, T4 DNA Ligase, T7 DNA Ligase, PBCV-1 DNA Ligase, Chlorella virus DNA Ligase, E. coli DNA Ligase, etc.
  • examples of heat-resistant ligases having the activity of repairing nicks in double-stranded DNA include Taq DNA Ligase and 9 o N DNA Ligase.
  • heat-resistant ligases maintain their activity even in gene amplification reactions and may affect gene amplification reactions in some way, the ligase will not act if the dissociation temperature between the linked oligo and the unlinked oligo (3'LG oligo and 5'LG oligo) is sufficiently lower than the annealing temperature of gene amplification. In such a situation, the use of a heat-resistant ligase will not affect the gene amplification reaction and can be used without problems.
  • the LCR method uses a heat-resistant DNA ligase for gene amplification, so that when the LCR method is adopted, if the oligo ligation reaction in the reaction step (1a) proceeds with the same ligase as the LCR method, it is possible to proceed with all reactions with one type of ligase, which is reasonable.
  • Taq DNA Ligase and E. coli DNA Ligase are NAD + -dependent enzymes, and the others are ATP-dependent enzymes, it is advisable to add each of the above substances to the reaction solution.
  • the present invention further provides a method for preparing a target product in which an artificial nucleic acid sequence is added to a target nucleic acid sequence, the method comprising the following steps (1b) and (2b): (1b) A step of synthesizing a linked primer pair (linked primer (F) and linked primer (R) in FIG. 6 ) by linking two types of oligo DNA pairs of the following (ib) and (iib) by a click chemistry reaction: (ib) A pair of 3'CC oligos having a sequence specific to the gene to be analyzed and whose 5' ends are modified with a click chemistry functional group (3'CC oligo (F) and 3'CC oligo (R) in FIG. 6).
  • (iib) A pair of 5' CC oligos having an artificial nucleic acid sequence such as a barcode sequence and modified with a functional group at the 3' end that undergoes a click chemistry reaction with the functional group modified in (ib) above (5' CC oligo (F) and 5' CC oligo (R) in FIG. 6).
  • (2b) A step of carrying out gene amplification using the linked primer pair synthesized in the above step (1b)
  • the 3' CC oligo pair has a sequence specific to the gene to be analyzed, but the sequence may be set to a sequence number generally preferred as a gene amplification primer, and no special consideration specific to the method of the present invention is required.
  • the 5' end of the oligo is modified with a functional group that generates click chemistry between the 3' end of the 5' CC oligo and the functional group modified to the 3' end of the 5' CC oligo.
  • the 5'CC oligo pair, barcode sequences for identifying samples and artificial nucleic acid sequences such as adapters for binding to oligo DNA present on the NGS flow cell are specified for each NGS device, so they can be set according to the sequences recommended by the NGS manufacturer to be used, and no special consideration is required depending on the method of the present invention.
  • the 3' end of the oligo is modified with a functional group that generates click chemistry between the functional group modified at the 5' end of the 3'CC oligo.
  • Azide groups and alkyne groups are typical functional groups for click chemistry, but these reactions require copper ions as a catalyst, and since the ions cause DNA damage and DNA strand cleavage, they are not very preferable for the click chemistry reaction used in the present invention.
  • dibenzocyclooctyne (DBCO), azadibenzocyclooctyne (ADIBO), bicyclononyne (BCN), 4,8-diazacyclononyne (DACN), etc. are known as functional groups that undergo click chemistry reactions with azide groups in the absence of copper ions, and these functional groups can be suitably used as functional groups to modify the oligo-DNA used in reaction step (1b).
  • an effective method is to raise the reaction temperature immediately after the initial synthesis of the target product derived from the linked primer to a temperature at which the annealing of the linked primer is not impeded and the annealing of the unlinked oligo (the 3' CC oligo in reaction step (2b)) can be suppressed, as in the reaction step (2a).
  • this step it is possible to suppress the amplification of non-target products and allow the amplification of the target product to predominate.
  • the timing of increasing the reaction temperature in this embodiment, and the reaction temperature after the increase, are the same as those described in the method including reaction steps (1a) and (2a) above.
  • the DNA polymerases that can be used in the method including reaction steps (1b3) and (2b4) are the same as those described in the method including reaction steps (1a) and (2a) above.
  • reaction steps (1a), (2a) and reaction steps (1b), (2b) can obtain two or more types of target products in a single reaction by using oligo pairs containing the same number of target gene-specific sequences as the target nucleic acid (3'LG oligo pairs in reaction steps (1a) and (2a), and 3'CC oligo pairs in reaction steps (1b) and (2b)).
  • reaction steps (1a), (2a), and reaction steps (1b3), (2b4) can all be carried out continuously in a single reaction without opening the reaction tube.
  • the gene amplification methods in the above-mentioned reaction step (2a) and reaction step (2b) include the PCR (Polymerase Chain Reaction) method, the LCR (Liagase Chain Reaction) method, the RPA (Recombinase Polymerase Amplification) method, the SIBA (Strand-Invasion Based Amplification) method, the LAMP (Loop-Mediated Iso-thermal Amplification) method, the SDa (Strand Displacement Amplification) method, the NEAR (N This can be done using the icking endonuclease amplification reaction method, the HDA (helicase-dependent amplification) method, the NASBA (nucleic acid sequence-based amplification) method, the ICAN (isothermal and chimeric primer-initiated amplification of nucleic acids) method, the rolling cycle method, the SMAP (SMART amplification process) method, etc.
  • PCR Polymerase Chain Reaction
  • LCR Liagase Chain Reaction
  • the present invention also provides a kit for preparing a target product, which comprises at least one pair of each of the following three types of oligo DNA pairs (ia) to (iiia).
  • the method for preparing the target product is described above (FIG. 5).
  • the ligation oligo (ia) in the kit of the present invention may be modified at the 3' end to prevent DNA elongation.
  • the kit of the present invention may further include a DNA polymerase and a DNA ligase.
  • the present invention also provides a kit for preparing a target product, which comprises at least one pair of each of the following two types of oligo DNA pairs (ib) and (iib).
  • the method for preparing the target product is described above (FIG. 6).
  • (ib) A pair of 3'CC oligos having a sequence specific to the gene to be analyzed and whose 5' ends are modified with a click chemistry functional group (3'CC oligo (F) and 3'CC oligo (R) in FIG. 6).
  • kits of the present invention may further comprise a DNA polymerase.
  • kits of the present invention such as the oligo DNA and DNA polymerase, are as described above.
  • kits of the present invention may also include reaction vessels, reaction buffers, instructions for use, target nucleic acids for positive control tests, etc.
  • this can be achieved by (1) using a PCR enzyme with a high extension speed (hereinafter, high-speed PCR enzyme), (2) changing from 3-step PCR to 2-step PCR during the gene amplification (PCR) reaction, or (3) increasing the annealing (/extension) temperature in the 2-step PCR described in (2) above.
  • high-speed PCR enzyme a PCR enzyme with a high extension speed
  • PCR gene amplification
  • the high-speed PCR enzyme described in (1) can shorten the time required for extension by using this enzyme, which makes it possible to shorten the gene amplification reaction time.
  • Rapid PCR enzymes that can be used in the present invention include repliQa HiFi ToughMix (manufactured by Quantabio) and KOD ONE PCR Master Mix (manufactured by Toyobo Co., Ltd.), but any rapid PCR enzyme that can be suitably applied to the present invention will suffice, and the type of enzyme does not limit the scope of application of the present invention.
  • this change narrows the range of temperature change (the difference between the dissociation temperature and the annealing temperature), which in turn reduces the time required for temperature change, thereby making it possible to shorten the gene amplification reaction time.
  • dsDNA-specific dye a fluorescent dye that emits fluorescence by specifically binding to double-stranded DNA
  • low-inhibitory dsDNA dye a fluorescent dye that emits fluorescence by specifically binding to double-stranded DNA
  • low inhibition dsDNA dyes examples include EvaGreen (manufactured by Biotium) and LC GREEN PLUS (manufactured by BioFire Defense), but any low inhibition dsDNA dye that is suitable for use in the present invention will suffice, and the type of dye does not limit the scope of application of the present invention.
  • the low inhibition dsDNA dye described above has low PCR inhibition, so that the target product can be amplified even when the substance is added to the reaction solution at a high concentration.
  • the concentration of the target product increases, the amount of dye bound per molecule of the target product can be maintained at saturation, so that even in the latter half of the amplification reaction when the concentration of the target product increases, there is a positive correlation between the concentration of the target product and the fluorescence intensity.
  • the library can be quantified simply and quickly by adding a high concentration of low inhibition dsDNA dye to the reaction solution of the present invention and directly measuring the fluorescence intensity without opening the reaction tube after the reaction is completed.
  • the length of the target product is often longer than that of the non-specific products, and therefore the dissociation temperature of the target product is generally higher than that of the non-specific products.
  • a real-time PCR device to carry out gene amplification according to the present invention, it is possible to simultaneously measure fluorescence at any timing and temperature, and therefore, as soon as amplification according to the present invention is completed, quantification of the target product can be completed from the fluorescence intensity value obtained above. In this way, by using a real-time PCR device, it is possible to carry out quantification of the target product more quickly and easily than by separately measuring fluorescence after gene amplification, making it more suitable as a gene amplification device for use in the present invention.
  • the temperature of the denaturation step in the gene amplification process is set to the temperature at which all double-stranded DNA contained in the reaction tube is denatured, so that in theory, the fluorescence intensity (fluorescence intensity from free low-inhibitory dsDNA dye not bound to dsDNA) is constant regardless of the degree of gene amplification.
  • This characteristic was used for the purpose of correcting the difference in optical properties between reaction tubes. Specifically, the fluorescence intensity measured at the temperature at which only the target product of each cycle exists as dsDNA is divided by the fluorescence intensity of the denaturation step of the same cycle, making it possible to roughly correct the difference in optical properties of the reaction tubes.
  • the specific calculation formula is shown below.
  • Dissociation corrected fluorescence intensity value Fn ,TT /Fn ,DN Fn ,TT : Fluorescence intensity measured at the temperature at which only the target product exists as dsDNA in any cycle (n) (TT; abbreviation for Temperature at which only the Targeted Product Exists as dsDNA)
  • Fn ,DN Fluorescence intensity measured at the temperature at which all DNA dissociates in any cycle (n) (DN; abbreviation for De Nature )
  • dissociation-corrected fluorescence intensity value should show the same value regardless of the difference in the reaction tube in the cycle before the increase in fluorescence due to gene amplification is confirmed, but in reality, the dissociation-corrected fluorescence intensity value in that cycle was confirmed to be slightly different between reaction tubes.
  • the dissociation-corrected fluorescence intensity value of each cycle was divided by the dissociation-corrected fluorescence intensity value of an arbitrary cycle in which no increase in fluorescence intensity due to gene amplification was observed in any reaction tube, so that the dissociation-corrected fluorescence intensity values in all reaction tubes were made uniform in an arbitrary cycle.
  • Final corrected fluorescence intensity value (Ffc ,TT / Ffc,DN ) / ( FCBFI,TT / FCBFI,DN )
  • F fc,TT Fluorescence intensity measured value at the temperature at which only the target product exists as dsDNA in the final cycle (fc) (TT; Temperature at which only the target product exists as dsDNA)
  • F fc,DN Fluorescence intensity measured value at the dissociation step (DN) in the final cycle
  • F CBFI,TT Fluorescence intensity measured value at the temperature at which only the target product exists as dsDNA in the cycle before the increase in fluorescence associated with gene amplification is confirmed (CBFI; Cycle Before Fluorescence Increase )
  • F CBFI,DN Fluorescence intensity measured value at the dissociation step in the cycle before the increase in fluorescence associated with gene amplification is confirmed
  • the quantitative value of dsDNA obtained by this calculation method based on the corrected fluorescence intensity was more accurate than the quantitative value of dsDNA quantified based on uncorrected fluorescence intensity (see Example 9 below), so the above calculation method can be said to be suitable as an embodiment for correcting differences in the optical properties of reaction tubes.
  • the above-mentioned embodiment is expected to have the following effects: (1) library quantification can be performed quickly and easily just by measuring the fluorescence of the reaction tube directly, (2) library purification, which previously had to be performed for each reaction tube in order to accurately quantify the DNA concentration, can now be performed for the mixture, and therefore the labor required for library purification can be significantly reduced, leading to a reduction in the time and cost of the entire process (FIG. 9).
  • the present invention also provides a method for preparing a mixture of target products using one of the above methods or any combination of two or more of the above methods.
  • the present invention also provides a kit for performing NGS analysis, comprising a dsDNA-specific dye, which prepares a mixture of target products for NGS analysis by the above method.
  • the present invention further provides a method for preparing a mixture containing two or more target products having different barcode sequences, the method comprising adding a dsDNA-specific dye to each gene amplification reaction solution containing the target products, quantifying the concentration of each target product from the fluorescence value of the dye, and mixing the gene amplification reaction solutions so that the target products have any concentration ratio based on the quantification results ( FIG. 9 ).
  • This reagent contains an anti-KOD antibody that inactivates KOD (a PCR enzyme manufactured by Toyobo Co., Ltd.) and a DNA polymerase that has A-attachment activity to the 3' end, and is capable of A-attachment to the 3' end of the target product simply by adding the reagent directly to the unpurified reaction solution after the amplification of the target product.
  • KOD a PCR enzyme manufactured by Toyobo Co., Ltd.
  • DNA polymerase that has A-attachment activity to the 3' end
  • the use of KOD is a prerequisite, if the expected effect can be confirmed, the use of this reagent can greatly simplify the A-attachment process to the 3' end (two steps: adding the reagent and keeping it warm) and reduce the time.
  • the present invention also provides a method for modifying a target product (FIG. 11), which comprises adding a single base A to the 3' end of a target product amplified using a primer phosphorylated at the 5' end in the above-mentioned method for preparing a target product, and then adding an adapter sequence to the product using a ligase.
  • the present invention also provides a method for modifying a target product, which comprises adding an adapter sequence to a mixture of target products prepared by the above-mentioned method or kit using a ligase.
  • oligo DNA shown in Table 2 was used, and ligase reaction and PCR were carried out continuously in one reaction tube without opening the tube, and the above-mentioned effects were evaluated from the quantitative value of the specific product obtained. All of the oligo DNA used in the examples described in this specification were synthesized by Nihon Gene Research Institute Co., Ltd.
  • the target gene in this example was the 16S rRNA gene derived from eubacteria, and the template used was DNA-Mock (manufactured by NBRC (National Institute of Technology and Evaluation, Biotechnology Center)), a mixture of 15 types of eubacterial genomes at known concentrations.
  • DNA-Mock manufactured by NBRC (National Institute of Technology and Evaluation, Biotechnology Center)
  • the reaction solution composition for each condition is shown in Table 4.
  • the cells shown in gray are the areas where the settings differ between conditions.
  • the reaction solution in Table 4 was subjected to a reaction under the temperature conditions in Table 3 using a PCR amplification device (Life touch (manufactured by Nippon Genetics Co., Ltd.)).
  • reaction solution was diluted 10,000-fold with TE buffer.
  • diluted reaction solution was used as a sample to quantify the specific product by real-time qPCR targeting the gene-specific sequence.
  • the gene-specific sequence was the same as the gene-specific sequence used in carrying out the present invention and described in Table 2 above.
  • the real-time PCR device used was Roter-Gene Q (Qiagen).
  • the composition of the reaction solution (including the sequences of the primers used) and the qPCR conditions are shown in Tables 5 and 6, respectively.
  • the real-time PCR device used was Roter-Gene Q (Qiagen).
  • the ligase reaction buffer contains substances that inhibit PCR for both heat-stable and non-heat-stable ligases, and that when added at the concentration recommended by the manufacturer, no specific product is obtained.
  • the reaction buffer is added at 1/3 the concentration recommended by the manufacturer, a specific product is confirmed (conditions [2] and [6]), and when added at 1/4 or less of the concentration, good amplification of the specific product occurs (conditions [3], [4], [7], [8]).
  • the reaction buffer for ligase was added at 1/4 the concentration recommended by the manufacturer in the following Example 3 and onwards.
  • Ampligase Thermostable DNA Ligase which is a heat-resistant ligase
  • Hi-T4 DNA Ligase which is a non-heat-resistant ligase
  • the buffer compositions differ, as mentioned above, they showed the same tendency in PCR inhibition.
  • composition ratio of the target product (ligase reaction system)
  • the composition ratio of the target product in the specific product in the present invention was confirmed by NGS manufactured by Nanopore.
  • reaction condition B does not change the temperature conditions during the reaction
  • reaction condition A is a three-step PCR like reaction condition B only for the first two cycles of PCR, and the subsequent cycles are two-step PCR in which annealing and extension are performed in the same step (72°C).
  • reaction condition A the temperature is raised to the annealing temperature at which the unlinked 3'LG oligo, which has a low dissociation temperature, does not function as a primer during the gene amplification reaction, suppressing the amplification of non-target products, while allowing the linked primer, which has a high dissociation temperature, to function preferentially, thereby improving the composition ratio of the target product.
  • the target gene in this example was the 16S rRNA gene derived from eubacteria, as in Example 1, and DNA-Mock (NBRC) was used as the template, as in Example 1.
  • the reaction product was first purified using magnetic beads for DNA purification (AMPure XP, Beckman Coulter, Inc.) according to the manufacturer's protocol.
  • the purified product was then quantified using Quant-iT PicoGreen dsDNA Assay Kits (Invitrogen, Inc.) according to the kit's protocol, and measurements were performed using a fluorescent plate reader (DTX800, Beckman Coulter, Inc.).
  • the purified product was processed using Nanopore's library preparation kit (Ligation Sequencing Kit [SQK-LSK110]) according to the kit's protocol.
  • This kit repairs and phosphorylates both ends of the purified product, and then adds double-stranded DNA (adapter) containing sequences and molecules (motor proteins) required for analysis to both ends of the target product using ligase.
  • the final product obtained above was quantified using the same Quant-iT PicoGreen dsDNA Assay Kits as above, and then sequence analysis was performed on the product using a Nanopore NGS (MinION Mk1B).
  • the flow cell used for the NGS analysis was a Nanopore Flongle, with one flow cell used per condition. The reason for this is that non-target products do not have barcode sequences, so when multiple samples are analyzed simultaneously using one flow cell, it is not possible to identify which sample the non-target product came from, and therefore it is not possible to determine the composition ratio of the target product for each condition.
  • base calling is the process of converting the raw sequence data obtained by the flow cell into a genetic sequence. This process was performed in SUP mode, which offers the highest accuracy), and demultiplexing (the process of separating sequence information for each sample using the barcode sequence as a marker) was performed. The obtained sequence data was then converted into base sequence information, after which the bacteria were identified based on the genetic sequence information using EPI2ME, a bioinformatics platform provided by Nanopore.
  • composition ratio of each product under each condition is shown in Table 10. Note that, although gene sequence information was obtained, gene information that was not recognized as a 16S rRNA gene by EPI2ME was determined to be gene information derived from a non-specific product. Next, gene information recognized as a 16S rRNA gene by EPI2ME was determined to be gene information derived from a specific product, and among them, the sequence information in which the barcode sequence was recognized by MinKNOW and multiplexed was determined to be gene sequence information derived from the target product, and the one to which the barcode sequence was not added was determined to be gene sequence information derived from a non-target product.
  • Table 10 shows the composition ratios of the target product, non-target product, and non-specific product under each condition.
  • the composition ratios of each product were close to each other, and it was confirmed that the effect of differences in ligase on the composition ratios of each product was relatively small.
  • the difference between conditions [1] and [2] and between conditions [3] and [4] is the reaction temperature conditions.
  • the PCR process is carried out in the first two cycles of three-step PCR, followed by two-step PCR (reaction condition A in Table 9).
  • reaction condition B in Table 9
  • the PCR is completed without changing the temperature conditions (reaction condition B in Table 9) under the same conditions as the three-step PCR in conditions [1] and [3]. Due to the difference in reaction temperature conditions, the composition ratio of the target product was significantly higher under conditions [1] and [3] (reaction condition A) than under conditions [2] and [4] (reaction condition B).
  • Table 11 shows the theoretical composition ratios of the 16S rRNA genes derived from 15 types of bacteria contained in DNA-Mock and the actual composition ratios under each condition.
  • FIG. 14 a graph showing the relationship between the theoretical composition ratio and the measured composition ratio is shown as FIG. 14.
  • the linear curve in FIG. 14 is an approximation function, and the R 2 value of the function is in the range of 0.67 to 0.68, and the slope of the function is in the range of 1.45 to 1.56. From this result, it was confirmed that there is a certain positive correlation between the theoretical composition ratio and the measured composition ratio, and that the correlation is almost the same regardless of the conditions. From the above results, it was confirmed that the same NGS analysis results were obtained under the condition (reaction condition A) in which the annealing temperature was changed during PCR as under the condition (reaction condition B) in which the annealing temperature was not changed as usual. Therefore, it was assumed that the effect of the above-mentioned difference in reaction conditions on the composition ratio of the target gene is low.
  • FIG. 15 shows the correlation between the measured composition ratio of the target product and the measured composition ratio of the non-target product.
  • the linear function is an approximation function
  • the R2 value of the function is in the range of 0.96 to 0.98
  • the slope of the function is in the range of 0.81 to 0.99.
  • ligase reaction system ⁇ Simultaneous analysis of multiple gene items (ligase reaction system)>
  • multiple target nucleic acids were targeted, and oligos were ligated using ligase and the target products were amplified using the ligated oligos in succession without opening the reaction tube, and then NGS analysis was performed on the obtained target products, thereby investigating the possibility of simultaneous analysis of multiple genes according to the present invention.
  • the oligo DNAs used in this example are shown in Table 12.
  • the target genes in this example were five human genes, and five pairs of 3'LG oligos corresponding to each target gene were prepared.
  • the preparation of the target product was carried out five times, and the target products obtained from the five reactions were analyzed in a single NGS analysis. Therefore, in order to separate the gene sequence information for each reaction, five 5'LG oligos with different barcode sequences were prepared, and a different 5'LG oligo was used for each reaction.
  • the linking oligo used was the same as that used in Example 3 above. Note that in this example, since the same artificial nucleic acid sequence is added to both ends of the target product, one 5'LG oligo and one linking oligo were used per reaction.
  • the target gene was a human gene
  • the additive template was commercially available human genomic DNA (Roche), and 100 ng of this was added per reaction tube.
  • the target product was prepared using the reaction solution composition shown in Table 13, based on the reaction temperature conditions shown as reaction condition A in Table 9.
  • the reaction products obtained were first purified using magnetic beads for DNA purification (AMPure XP, Beckman Coulter) according to the manufacturer's protocol.
  • the purified products obtained were then quantified using Quant-iT PicoGreen dsDNA Assay Kits (Invitrogen) according to the kit's protocol, and measurements were performed using a fluorescent plate reader (DTX800, Beckman Coulter). Based on the quantitative values, the concentrations of the target products were adjusted so that each reaction contained equimolar target products.
  • Example 3 the purified product was processed using a Nanopore library preparation kit (Ligation Sequencing Kit [SQK-LSK110]) according to the kit's protocol.
  • a Nanopore library preparation kit Ligation Sequencing Kit [SQK-LSK110]
  • the final product obtained above was quantified using the same Quant-iT PicoGreen dsDNA Assay Kits as above, followed by sequence analysis using Nanopore's NGS (MinION Mk1B).
  • the flow cell used for the NGS analysis was Nanopore's Flongle.
  • base calling was performed using MinKNOW, a software provided by Nanopore, Inc. (performed in SUP mode, which provides the highest base calling accuracy), and demultiplexing (the process of separating sequence information for each reaction using a barcode sequence as a marker) was performed.
  • the obtained sequence data was converted into base sequence information, and the number of gene sequences (hereinafter referred to as the number of reads) was counted for each target gene.
  • the number of reads per reaction was measured by using 5'LG oligos with different barcode sequences for each different reaction, but if 5'LG oligos with different barcode sequences are applied to different samples, it is also possible to perform sequence analysis of different samples at the same time.
  • the difference in reaction composition under each condition is the difference in the oligo DNA added, and the difference between conditions [1], [2] and conditions [3], [4] is the presence or absence of the addition of oligo for linkage, and the difference between conditions [1] and [2] and between conditions [3] and [4] is whether the forward side oligo DNA or the reverse side oligo DNA was used.
  • the reaction volume was 20 ⁇ l.
  • a linked primer (base length: 81 bases) was confirmed as a result of linking the 3' CC oligo and the 5' CC oligo, confirming the linking of oligos by click chemistry.
  • composition ratio of the target product (click chemistry reaction system)>
  • the composition ratio of the target product among the specific products in the present invention was confirmed using NGS manufactured by Nanopore.
  • the oligo DNA shown in Table 17 was used, and the click chemistry reaction and PCR reaction were carried out continuously without opening the reaction tube, using the reaction composition shown in Table 18 and the reaction conditions shown in Table 19.
  • the composition of the reaction solution is the same regardless of the conditions, and only the reaction conditions are different between the two conditions.
  • reaction condition A condition [1]
  • reaction condition B condition [2]
  • reaction condition A is a 3-step PCR like reaction condition B for only the first 2 cycles of PCR, and the subsequent cycles are 2-step PCR in which annealing and extension are performed in the same step (72°C).
  • reaction condition A the annealing temperature is raised during the process, and the temperature conditions are set so that unlinked 3'CC oligos with a low dissociation temperature do not function as primers, suppressing the amplification of non-target products, while the linked primers with a high dissociation temperature function preferentially, aiming to improve the composition ratio of the target product.
  • the target gene in this example is the 16S rRNA gene derived from eubacteria, as in Examples 1 and 3, and the template was also DNA-Mock (manufactured by NBRC), as in Examples 1 and 3. The subsequent steps are the same as in Example 3 above.
  • Table 20 shows the composition ratio of target products, non-target products, and non-specific products under each condition.
  • condition [1] The difference between conditions [1] and [2] is the PCR temperature conditions.
  • condition [1] the PCR process is performed in the first two cycles of 3-step PCR, followed by 2-step PCR (reaction condition A in Table 19).
  • condition [2] the PCR is completed without changing the temperature conditions under the same conditions as the 3-step PCR in condition [1] (reaction condition B in Table 19). Due to the difference in reaction temperature conditions, the composition ratio of the target product was significantly improved under condition [1] (reaction condition A) compared to condition [2] (reaction condition B). From these results, it was confirmed that the composition ratio of the target product can be improved by raising the annealing temperature during PCR (reaction condition A), as in Example 3.
  • Table 21 shows the theoretical composition ratios of the 16S rRNA genes derived from 15 types of bacteria contained in DNA-Mock and the actual composition ratios under each condition. Next, based on the numerical data in Table 21, a graph showing the relationship between the theoretical composition ratio and the actually measured composition ratio under each condition is shown in FIG.
  • Figure 19 shows the correlation between the actual composition ratio of target products and the actual composition ratio of non-target products.
  • the linear function is an approximation function, and the R2 value of the function is 0.97 for both conditions [1] and [2], and the slope of the function is 1.1 for condition [1] and 1.04 for condition [2], with almost no difference between the conditions.
  • This result suggests that there is a very high positive correlation between the measured composition ratios of the target product and the non-target product, and that this correlation is almost constant regardless of the conditions. From the above results, it was confirmed that, as in Example 3 above, the presence or absence of a barcode sequence has very little effect on the NGS analysis results.
  • the oligo DNA used in this example is shown in Table 22.
  • the target genes in this example were the same five human genes as in Example 4, and five pairs of 3'CC oligos were prepared corresponding to each target gene.
  • the reaction to prepare the target product was carried out five times, and sequence information was obtained for the target products obtained from the five reactions in a single NGS analysis. Therefore, in order to separate the gene sequence information for each reaction, five 5'CC oligos with different barcode sequences were prepared, and a different 5'CC oligo was used for each reaction. Note that, since the effect of the linking oligo could not be confirmed in Example 5 above, no linking oligo was used in this example, as in Example 6. For this reason, the same 5'CC oligo was added to the 5' end of the 3'CC oligo on both the forward and reverse sides, so only one type of 5'CC oligo was used per reaction.
  • the template added was commercially available human genomic DNA (Roche), as in Example 4 above, and 100 ng of this was added per reaction tube.
  • the preparation of the target product was carried out using the reaction solution composition shown in Table 23, based on the reaction temperature conditions shown as reaction condition A in Table 19. The subsequent steps were the same as those in Example 4 above.
  • the method of the present invention (a method for preparing a target product using click chemistry) can prepare target products for multiple genes by performing only one reaction, and that sequence information for multiple genes can be obtained all at once by analyzing the products by NGS.
  • the number of reads for each reaction was measured by using 5'CC oligos with different barcode sequences for each different reaction, but it is also possible to perform sequence analysis of different samples together by applying 5'CC oligos with different barcode sequences for different samples.
  • the target gene in this example was the 16S rRNA gene derived from eubacteria, as in Example 1, and the template used was DNA-Mock (NBRC), as in Example 1, and 10 copies of the 16S rRNA gene were added per reaction tube.
  • the oligo DNA used in this example was the oligo DNA shown in Table 2 of Example 1.
  • the DNA polymerases used in this example are listed in Table 25.
  • TITANIUM Taq DNA polymerase (Clontech) is not a high-speed PCR enzyme.
  • PrimeSTAR GXL DNA polymerase (Takara Bio) was added in double the normal amount, following the manufacturer's protocol, to enable a high-speed extension reaction.
  • the other three enzymes are all high-speed PCR enzymes.
  • Rotor-Gene Q (Qiagen) was used for gene amplification (PCR) reactions.
  • Amplification of the target product was carried out using the reaction solution composition shown in Table 26 and under the reaction temperature conditions shown in Table 27.
  • Amplification of the target product was confirmed by electrophoresis (apparatus used: Agilent 2100 Bioanalyzer Electrophoresis System [Agilent Technologies]), and the reaction time was measured from the start of the reaction to its end.
  • the table also lists the actual reaction time under each condition, as well as the reduction rate of reaction time based on the reaction time (124 minutes) in a three-step PCR (Test No. 1) using TITANIUM Taq DNA polymerase, a PCR enzyme with an average extension speed.
  • the results show that the reaction time can be reduced by performing three-step PCR for only the first two cycles and then changing to two-step PCR (Test Nos. 6-10). This is because in two-step PCR, annealing and extension were performed at a high and identical temperature, which reduced the time required for temperature transition. In addition, by setting the annealing and extension temperatures at higher temperatures (Test Nos. 11-15), it was possible to reduce the reaction time even further than Test Nos. 6-10, which used the lower temperature.
  • Test No. 15 The conditions under which amplification of the target product was confirmed and which showed the highest reaction time reduction rate compared to Test No. 1 were Test No. 15, which used repliQa HiFi ToughMix (Quantabio), with a reaction time of 35 minutes and a reaction time reduction rate of 72%. This was followed by Test No. 14, which used KOD ONE PCR Master Mix (Toyobo), with a reaction time of 36 minutes and a reaction time reduction rate of 71%.
  • reaction time of the method of the present invention can be significantly reduced by (1) shortening the reaction time by using a high-speed PCR enzyme, (2) changing from a three-step PCR to a two-step PCR during the gene amplification (PCR) reaction, and (3) increasing the annealing (/extension) temperature in the two-step PCR in (2) above.
  • repliQa HiFi ToughMix which enabled the reaction time to be reduced
  • KOD ONE PCR Master Mix Toyobo
  • they can be said to be DNA polymerases suitable for carrying out the method of the present invention.
  • the oligo DNA used in this example was the oligo DNA shown in Table 2 of Example 1.
  • the target gene in this example was the 16S rRNA gene derived from eubacteria, as in Example 1, and DNA-Mock (NBRC) was used as the template, as in Example 1, and this was added to the reaction tube at 105 copies in terms of 16S rRNA gene.
  • KOD ONE PCR Master Mix manufactured by Toyobo Co., Ltd.
  • EvaGreen manufactured by Biotium Co., Ltd.
  • the rest of the reaction solution composition was the reaction solution composition using KOD ONE PCR Master Mix, as described under condition [4] in Table 26.
  • the reaction temperature conditions except for the number of cycles, were based on the conditions of Test No. 4 in Table 27.
  • the number of cycles was set to 25, 27, 32, and 40 cycles so that the concentration of the target product would change, and one reaction was performed for each cycle number condition.
  • the target product obtained under the above conditions was purified using Agencourt AMPure XP (Beckman Coulter), and the purified product (double-stranded DNA) was quantified using Quant-iT PicoGreen dsDNA Assay Kits (Invitrogen) according to the kit's protocol. Fluorescence was measured using a fluorescence plate reader (Beckman Coulter DTX800).
  • the gene amplification reaction of the present invention was carried out using a real-time PCR device, Rotor-Gene Q 2plex (Qiagen), and fluorescence measurements were taken at 90°C and 98°C for each cycle.
  • the target product the Tm value of the target product in this example is 90°C
  • the fluorescence intensity at this temperature reflects the amount of the target product.
  • 98°C is a measurement at the dissociation step, and fluorescence measurement is performed in a state where double-stranded DNA is not present, so theoretically the fluorescence intensity will be constant regardless of the degree of gene amplification.
  • the fluorescence intensity values at 90°C and 98°C obtained in the above manner were used to calculate the final corrected fluorescence intensity value using the above-mentioned formula for the calculation of the final corrected fluorescence intensity value. More specifically, the fluorescence measurement value at 90°C in the final cycle was taken as the actual fluorescence intensity measurement value at a temperature where only the target product exists as dsDNA ( Ffc,TT in the calculation formula), and the fluorescence measurement value at 98°C in the final cycle was taken as the actual fluorescence intensity value at the dissociation step ( Ffc,DN in the calculation formula).
  • the cycle before the increase in fluorescence associated with gene amplification was confirmed was set to 10 cycles, and the fluorescence intensity value at 90°C in that cycle was taken as FCBFI,TT , and the fluorescence intensity value at 98°C was taken as FCBFI,DN .
  • FCBFI,TT the fluorescence intensity value at 90°C in that cycle was taken as FCBFI,TT .
  • FCBFI,DN the fluorescence intensity value at 98°C
  • Figure 21(a) shows the relationship between the quantitative value of purified double-stranded DNA and the uncorrected fluorescence intensity value of the final cycle in each reaction tube
  • Figure 21(b) shows the relationship between the quantitative value of purified double-stranded DNA and the final corrected fluorescence intensity value of the final cycle in each reaction tube.
  • the correlation coefficient is higher in FIG. 21(b), where fluorescence correction was performed, than in FIG. 21(a), where no correction was performed, indicating that the amount of double-stranded DNA can be quantified more accurately by performing fluorescence correction using the method described above.
  • the target product was obtained by the patent method using the DNA obtained above as a template.
  • the oligo DNA used in this example was the oligo DNA shown in Table 2 of Example 1 for the linking oligo and the 3'LG oligo containing a gene-specific sequence.
  • 5'LG oligos containing barcode sequences those listed in Table 29 were used so that 12 samples could be analyzed simultaneously in a single NGS analysis, and a pair of forward 5'LG oligos and reverse 5'LG oligos with the same barcode sequence were used so that the barcode sequence of the target product would be the same on the forward and reverse sides.
  • the amount of template added was 2 ⁇ l of the extracted DNA per reaction.
  • Other conditions were the same as those described in Example 9, except that the number of cycles was fixed at 40.
  • the target product was quantified by calculating the final corrected fluorescence intensity value in the final cycle (40th cycle) using the method described in Example 9, and the calculated value was calculated using the relationship in Figure 21 (b) as a calibration curve.
  • the unpurified reaction solutions were mixed in the test system so that the target products of each sample were equimolar, and this mixture was purified using Agencourt AMPure XP (manufactured by Beckman Coulter).
  • the double-stranded DNA concentration of the two-type mixture obtained above was measured using Quant-iT PicoGreen dsDNA Assay Kits (Invitrogen) according to the kit's protocol.
  • NGS analysis was performed on the mixture of two types of double-stranded DNA obtained above after quantification.
  • the conditions for NGS analysis were as described in Example 3, and Nanopore NGS was used.
  • NGS analysis was carried out on the mixture of two types of double-stranded DNA obtained above after quantification.
  • the conditions for NGS analysis were as described in Example 3, and Nanopore NGS was used.
  • NGS analysis was performed on a total of 24 samples (12 test samples and 12 control samples).
  • 5'LG oligos with different barcode sequences as described above, it is possible to simultaneously analyze up to 12 samples in one NGS analysis, so the number of NGS analyses performed in this example was two.
  • base calling is the process of converting the raw sequence data obtained by the flow cell into a gene sequence. This process was performed in SUP mode, which offers the highest accuracy), and demultiplexing (the process of separating sequence information for each sample using the barcode sequence as a marker) was performed. The resulting sequence data was then converted into base sequence information, and the number of reads for each sample was obtained using EPI2ME, a bioinformatics platform provided by Nanopore.
  • Results The results of this example are shown in Table 30.
  • the control system in which equal volumes of reaction solutions were mixed, there was a large variation in the number of reads per sample, with a standard deviation (%) of 26%.
  • the test system in which the dsDNA concentration in the reaction solution was measured using a low-inhibitory dsDNA dye, the variation in the number of reads per sample was kept low, with a standard deviation (%) of 13%, half that of the control system.
  • Example 9 show that by adding a low-inhibitory dsDNA dye with little inhibition of amplification to the amplification reaction solution before the reaction and performing fluorescence measurement without opening the reaction tube, it is possible to accurately quantify the target product amplified by the method described in this patent, and by mixing the reaction solution based on the quantitative value, it is possible to arbitrarily adjust the number of reads (number of gene sequences) per sample.
  • the amount of the target product can be quantified quickly and easily by adding a low-inhibitory dsDNA dye with little PCR inhibition to the reaction solution for preparing the target product and directly measuring the fluorescence of the reaction tube after the reaction is completed.
  • the target gene in this example was the 16S rRNA gene derived from eubacteria, as in Example 1, and the template used was DNA-Mock (NBRC), as in Example 1, and 10 copies of the 16S rRNA gene were added per reaction tube.
  • NBRC DNA-Mock
  • the oligo DNA used in this example was the oligo DNA shown in Table 2 of Example 1 for the linking oligo and the 3'LG oligo containing a gene-specific sequence.
  • the oligos listed in Table 31 were used. More specifically, in the test system using the A-attachment mix, 5'LG oligos with phosphorylated 5' ends (oligos listed in Nos. 1 and 2 in Table 31) were used, and in the control system where terminal modification was performed based on the manufacturer's recommended protocol, 5'LG oligos with non-phosphorylated 5' ends (oligos listed in Nos. 3 and 4 in Table 31) were used.
  • the amplification of the target product was performed using 40 cycles for both the test system and the control system, and other conditions were as described in Example 9.
  • 10x A-attachment mix was added to the reaction solution containing the target product obtained above, following the manufacturer's protocol, to give a final concentration of 1x, and the mixture was then incubated at 60°C for 10 minutes in a PCR device (iCycler (Bio-Rad)).
  • the above-mentioned terminally modified target product was purified using Agencourt AMPure XP (Beckman Coulter) according to the manufacturer's protocol to obtain a terminally modified purified target product.
  • the target product was first purified using Agencourt AMPure XP (Beckman Coulter) according to the manufacturer's protocol for the reagent.
  • the double-stranded DNA concentration of the purified product obtained above was measured using Quant-iT PicoGreen dsDNA Assay Kits (Invitrogen) according to the kit's protocol.
  • the purified target product obtained above was subjected to end processing using the NEB Next Ultra II End repair/dA-tailing Module reagents (manufactured by New England Biolabs) recommended by Nanopore, following the kit protocol (see Table 1).
  • Adapters were added to both ends of the purified, end-modified target products of the test system and the control system obtained above using Nanopore's Ligation Sequencing Kit [SQK-LSK114] according to the manufacturer's protocol.
  • the adapter-added product obtained above was quantified using the same Quant-iT PicoGreen dsDNA Assay Kits as above, using a fluorescent measurement method, and then the product was subjected to sequence analysis using a Nanopore NGS (MinION Mk1B).
  • the flow cell used for the NGS analysis was Nanopore's Flongle (version: R10.4.1). Since different barcode sequences were added to the final products of the test system and the control system, a solution containing equimolar amounts of each final product was prepared, and then NGS analysis was performed on this mixture, thereby obtaining gene sequence information for both products simultaneously in a single NGS analysis.
  • Base calling is the process of converting the raw sequence data obtained by the flow cell into a genetic sequence. This process was performed in SUP mode, which offers the highest accuracy.
  • demultiplexing the process of separating sequence information for each sample using the barcode sequence as a marker. The obtained sequence data was then converted into base sequence information, after which the bacteria were identified based on the genetic sequence information using EPI2ME, a bioinformatics platform provided by Nanopore.
  • Results The above results are shown in Tables 32 and 33.
  • the results in Table 32 show that no significant differences were found in the analysis results between the test and control systems in the items to be compared, such as the number of reads obtained, the barcode identification rate, which indicates the proportion of products to which a barcode is linked, and the phylogenetic classification name identification rate, which indicates the proportion of products derived from the gene to be analyzed (16S rRNA gene).
  • the results in Table 33 show that no significant differences were found in the microbial composition ratios.
  • the DNA concentration of the target product can be easily and quickly quantified by using the low-inhibitory dsDNA dye with little PCR inhibition described in Examples 9 and 10 and directly measuring the fluorescence in the reaction tube after amplification of the target product without going through a DNA purification process.
  • the method described in Examples 9 and 10 makes it possible to reduce the time by about 50 minutes (DNA quantification process: 20 minutes, purification process: 30 minutes) and significantly reduce the cost of purification (a 90% reduction assuming simultaneous processing of 10 samples).
  • Example 11 The application of the terminal modification method described in Example 11 is limited to the Nanopore sequencer, which requires terminal modification of the target product for analysis, and to analysis using Nanopore's Ligation Sequencing Kit, but by adopting this method, it was possible to reduce the time by approximately 50 minutes (reduction rate: 80%).
  • the reagent costs were reduced by using NEB Next Ultra II End repair/dA-tailing Module reagents (New England Biolabs) and A-attachment mix (Toyobo).
  • Examples 8 to 10 are applicable to the steps common to amplicon analysis using NGS (amplification, purification, and quantification of the target product), and are therefore recognized as effective methods when performing analysis using NGS other than that of Nanopore.
  • the present invention can be used as a novel method for preparing gene libraries.
  • ⁇ SEQ ID NOs: 1 to 72> Shows the base sequences of the oligo DNA used in the examples.

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Abstract

La présente invention concerne une technique permettant de préparer une banque contenant une proportion élevée d'un produit cible au moyen d'une opération économique et simple lors de la réalisation simultanée d'une analyse de séquence d'une pluralité d'échantillons par une utilisation unique d'un séquenceur de nouvelle génération.　Ce procédé de préparation d'un produit cible selon lequel une séquence d'acide nucléique artificiel est ajoutée à une séquence d'acide nucléique cible comprend les étapes (1a) et (2a) suivantes : (1a) faire réagir trois types de paires d'oligo ADN, c'est-à-dire les (ia)-(iiia) suivants en présence d'une ligase et à ligaturer ainsi l'oligo ADN (iia) et l'oligo ADN (iiia) suivants par une réaction de réparation de coupure à l'aide d'une ADN ligase pour synthétiser une paire d'amorces de ligature (une amorce de ligature (F) et une amorce de ligature (R) dans la FIG. 5), dans laquelle (ia) est une paire d'oligo (un oligo (F) pour la ligature et un oligo (R) pour la ligature dans la FIG. 5) pour la ligature qui est caractérisée par la présence d'une séquence complémentaire à une séquence (ci-après, une séquence de ligature) nécessaire pour ligaturer l'ADN oligo dans la réaction de réparation de coupure par l'utilisation de la ligase, (iia) est une paire d'oligo 3'LG (un oligo 3'LG (F) et un oligo 3'LG (R) dans la FIG. 5) présentant une partie de la séquence de l'extrémité 3' de la séquence de ligature à son extrémité 5', présentant une séquence spécifique d'un gène à analyser à son extrémité 3' et présentant une extrémité 5' phosphorylée, et (iiia) est une paire d'oligo 5'LG (oligo 5'LG (F) et oligo 5'LG (R) dans la FIG. 5) caractérisée par la présence d'une séquence d'acide nucléique artificiel, telle qu'une séquence de code-barres, à son extrémité 5' et par la présence d'une partie de la séquence de l'extrémité 5' de la séquence de ligature à son extrémité 3' ; (2a) réaliser une amplification génique par la paire d'amorces de ligature synthétisée à l'étape (1).
PCT/JP2024/009803 2023-03-15 2024-03-13 Nouveau procédé de préparation de banques à partir d'une réaction de ligature d'oligo adn et d'une réaction continue d'amplification de l'adn due à l'oligo adn ligaturé Pending WO2024190829A1 (fr)

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