JP2022170729A - Novel method for preparing gene libraries - Google Patents

Abstract

To provide a technique for economically, simply, and highly sensitively obtaining a final artificial sequence addition product when mainly targeting a plurality of genes using a next-generation sequencer.SOLUTION: Provided is a method for preparing a nucleic acid in which an artificial nucleic acid sequence is added to a target nucleic acid sequence (hereinafter referred to as an artificial sequence addition product). The method comprises the following steps (1) to (3): (1) a step of obtaining a 5P artificial AB adduct phosphorylated at one or both 5' ends of each primer pair specific to the target nucleic acid sequence (hereinafter referred to as a 5P artificial AB adduct); (2) a step of adding a complementary sequence of artificial nucleic acid sequence C or D to the 3' end of the 5P artificial AB addition product using a 3' artificial sequence introduction oligo pair (hereinafter referred to as a 3' artificial CD complementary addition product); and (3) a step of adding an artificial nucleic acid sequence C or D to the 5' end of the 3' artificial CD complementary addition product.SELECTED DRAWING: None

Description

The present invention relates primarily to assays for the detection or sequencing of specific populations of nucleic acid sequences. In certain embodiments, the present invention provides targeted nucleic acid sequences with one or more artificially designed nucleic acid sequences (hereinafter referred to as artificial nucleic acid sequences), including barcode sequences that are artificial nucleic acid sequences for identifying analytes. ) is added.

Detecting specific nucleic acid sequences is used in many fields, such as genetic diagnosis in medicine, sanitary inspection in food, and monitoring in the environment.

In addition, the emergence of next-generation sequencers in recent years has made nucleic acid sequencing much easier than conventional sequencing methods, and is spreading as a new analysis method.

In order to provide a mixture of amplification products derived from multiple specimens with a next-generation sequencer, a specific artificial nucleic acid sequence containing a barcode sequence for identifying specimens is added to both ends of the nucleic acid fragment to be sequenced. A nucleic acid amplification reaction step for that is required in addition to the step of amplifying the target nucleic acid sequence.

A nucleic acid fragment to be subjected to a next-generation sequencer can also be obtained by using a primer to which a specific artificial nucleic acid sequence containing a barcode sequence is added in advance to the 5' end of the primer specific to the target nucleic acid sequence. It is possible (prior art 1). However, this method requires preparation of primers with different barcode sequences for each type of nucleic acid sequence, which is extremely uneconomical (Fig. 1).

As a means for economically obtaining an amplification product to which an artificial nucleic acid sequence has been added, a method of performing two-step PCR (prior art 2) is generally used (non-patent document 1). In this technique, first, a primer obtained by adding a part of the 3' end side of the artificial nucleic acid sequence to the 5' end side of the specific sequence (hereinafter, artificially added specific primer) is used to perform the first PCR, and the target nucleic acid A derived product (hereinafter specific product) is obtained. Subsequently, after purifying the product, the product is used as a template for the second PCR using a primer having an artificial nucleic acid sequence such as a barcode sequence on the 3′ end side (hereinafter referred to as an artificial sequence primer). By the above two PCR amplifications, an amplification product (hereinafter referred to as final artificial sequence added product) in which artificial nucleic acid sequences are added to both ends of the target nucleic acid can be obtained (Fig. 2).

However, in Prior Art 2, the process is complicated because it is necessary to perform PCR twice, and it is necessary to purify the specific product obtained by the first PCR and adjust the concentration. is a time-consuming task.

As a means for obtaining the final artificial sequence addition product economically and simply, both the artificial addition specific primer pair and the artificial sequence primer pair are added to one PCR reaction solution at the same time, and then the amplification reaction is performed. A method (Prior art 3) capable of obtaining the final artificial sequence addition product in one round of PCR has been disclosed (Patent Documents 1 and 2).

According to Patent Documents 1 and 2, by lowering the concentration of the artificial addition specific primer to the concentration of the artificial sequence primer, the artificial sequence primer capable of binding to the artificial nucleic acid sequence commonly added to all target sequences is used in the amplification reaction. can be dominantly performed, the final artificial sequence addition product can be efficiently obtained, and when there are multiple target nucleic acids, variation in amplification efficiency between genes can be suppressed (Fig. 3) .

However, when preparing a nucleic acid sequence for use with a next-generation sequencer using prior art 3, the artificial sequence primer used at a high concentration becomes a long chain of about 60 to 70 bases, so primer dimers may be produced. sexuality increases. When there are multiple target nucleic acid sequences, the number of types of artificially added specific primers to be used increases, resulting in the frequent production of primer-dimers derived from the same primers. This raises the problem that the proportion of sequences other than the target sequence increases during next-generation sequencer analysis.

In addition, as the number of types of target nucleic acid sequences increases, the total amount of artificially added specific primers increases. There is a problem that the ratio of arrays becomes high.

A technique (prior art 4) capable of solving the above problem has been proposed (Patent Document 3). In prior art 4, in the reaction of adding an artificial nucleic acid sequence to a target gene sequence, in addition to the artificial addition specific primer, a long-chain artificial sequence primer (hereinafter referred to as a long-chain artificial sequence primer. "Primer pair Y" in FIG. ), by using two pairs of artificial sequence primers of short artificial sequence primers (hereinafter referred to as short artificial sequence primers, “primer pair Z” in FIG. 4), amplification of non-specific sequences derived from primers A technique is disclosed that can suppress the addition and efficiently amplify the desired final artificial sequence addition product. In addition, by lowering the concentration of the artificial sequence primer (primer pair Y) containing the artificial addition specific primer and the barcode sequence, etc. and increasing the concentration of the short-chain primer (primer pair Z), the final artificial sequence addition product can be produced more efficiently. It is described that it can be amplified well.

However, in prior art 4, at least three sets of primers, ie, an artificial addition specific primer pair, a long-chain artificial sequence primer pair, and a short-chain artificial sequence primer pair are used, and three types of PCR reactions proceed simultaneously. There is a need to rigorously optimize the reaction conditions so that the three sets of primers work properly simultaneously in one PCR reaction.

From the above-described principle characteristics, Prior Art 4 (1) requires strict optimization of reaction conditions for each target nucleic acid, requiring excessive time and labor for this work, (2) 1 primer pair It has the problems of low sensitivity and the need for a larger initial template (target gene) than conventional PCR that uses only pairs.

Applied and Environmental Microbiology, 2013 September 79(17):5112-5120.

WO2006/023919 Special table 2012-522517 Patent No. 5997407

Therefore, there is a need for a technology that can economically, easily, and highly sensitively obtain the final artificial sequence addition product when mainly targeting multiple genes using a next-generation sequencer.

In order to solve the above-mentioned problems, the present inventors have repeatedly studied an economical, simple, highly sensitive, and efficient method for obtaining the final artificial sequence adduct (see FIG. 5).

as a result,
(1) Using artificial addition-specific primers phosphorylated at one or both 5' ends (hereinafter referred to as 5P artificial addition-specific primers) of the artificial addition-specific primer pair as primers for nucleic acid amplification methods such as PCR. in which one or both 5′ ends are phosphorylated, having at least a partial sequence of the artificial nucleic acid sequence A on one 5′ end side, and at least a partial sequence of the artificial nucleic acid sequence B on the other 5′ end side. Step (2) of obtaining a specific product (hereinafter referred to as 5P artificial AB addition product) having a 'end' using a pair of oligo DNA (hereinafter referred to as 3' artificial sequence introduced oligo) characterized by the following three points, with DNA polymerase , a product obtained by extending the 3′ end of the 5P artificial AB addition product obtained in the step (1) and adding a sequence complementary to the artificial nucleic acid sequence C or D to the 3′ end of the 5P artificial AB addition product ( Hereafter, the process of adding 3' artificial CD complementary addition product) Feature 1: At least a part of the base sequence of the artificial nucleic acid sequence present in the 5P artificial addition specific primer pair has at the 3' end side.
Characteristic 2: It has an artificial nucleic acid sequence C or D added to the 5P artificial AB adduct on the 5′ end side.
Feature 3: The 3' end is labeled to prevent nucleic acid elongation by DNA polymerase.
(3) Performing one of the following two steps to add an artificial nucleic acid sequence to the 5' end of the 3' artificial CD complementary addition product Step (a): generated in the step (2) When either one or both of the 3' artificial CD complementary addition products are phosphorylated at the 5' ends, when the products form a double strand, among the single-stranded regions generated at both ends , At least one oligo DNA (hereinafter referred to as 5' The artificial sequence-introducing primer) and the 3' artificial CD complementary addition product obtained in the step (2) are ligated with DNA ligase to add the artificial nucleic acid sequence to the 5' end of the 3' artificial CD complementary addition product. Step of adding or step (b): when either one or both of the 3′ artificial CD complementary addition products generated in step (2) are phosphorylated at the 5′ ends, the products Of the single-stranded regions that occur at both ends when the double strand is formed in , at the end where the 5' end of the base pair at the site where the region is changed to double strands is phosphorylated, one of the ends At least one 5' artificial sequence-introducing primer complementary to a part of the main-strand region is filled in by an extension reaction using a DNA polymerase to fill the gap generated when the 3' artificial CD complementary addition product hybridizes. The step of adding an artificial nucleic acid sequence to the 5′ end of the 3′ artificial CD complementary addition product by ligating the extended 5′ artificial sequence introduction primer and the 3′ artificial CD complementary addition product with DNA ligase. It is possible to proceed simultaneously by a one-step reaction in which all the components (DNA polymerase, DNA ligase, various oligo DNAs, etc.) are added, and as a result, the desired final artificial sequence addition product can be produced economically, simply, and easily. It was possible to obtain with high sensitivity and high efficiency. The present invention has been completed based on this discovery.
In addition,
(4) Having an intramolecular secondary structure in the 5' end region and introducing a 3' artificial sequence by inserting a blocker into the 3' end of the sequence added for the purpose of forming the intramolecular secondary structure, or the sequence strand. By using an oligo (hereinafter referred to as 3' artificial sequence introduced SL oligo), the binding efficiency of the 5' artificial sequence introduction primer to the 3' artificial CD complementary addition product and the efficiency of introducing the 5' artificial sequence can be improved. As a result, it was possible to increase the yield of the final artificial sequence addition product.
(5) Primers that work in the reaction step (2) and beyond by increasing the annealing temperature after the third cycle of the reaction step (1) (PCR reaction) within a range that does not adversely affect the reaction step (1), or Oligo DNA can easily realize temperature conditions that do not function in the reaction step (1), and it is possible to reliably separate the reaction step (1) from the subsequent steps. The degree of freedom in designing the artificially added specific primer functioning in (1) has increased, leading to an expansion of genes to which the present invention can be applied (expansion of versatility).

Such techniques can be applied to analyzes for various purposes. For example, genes that can cause cancer can be comprehensively analyzed in order to identify the site of gene mutation. In addition, the present technology can be suitably used in various SNP analyses, gene mutation analyses, gene expression analyses, and microflora analyses, when using a next-generation sequencer. The scope of application is not limited as long as it is a method of analyzing a plurality of target genes by adding artificial nucleic acid sequences and identifying samples.

The gist of the present invention is as follows.
[1] A method for preparing a nucleic acid obtained by adding an artificial nucleic acid sequence to a target nucleic acid sequence (hereinafter referred to as an artificial sequence addition product), the method comprising the following steps (1) to (3).
(1) A primer pair in which at least a part of the sequence of the artificial nucleic acid sequence A or B is added to each primer pair specific to the target nucleic acid sequence, wherein , One or both 5' ends are phosphorylated by a nucleic acid amplification method using one or more pairs of artificial addition specific primers (hereinafter referred to as 5P artificial addition specific primer pairs) phosphorylated at one or both 5' ends obtaining a 5P artificial AB adduct;
(2) Using a pair of oligo DNA (hereinafter referred to as oligo pair with 3' artificial sequence introduction) characterized by the following three points, DNA polymerase is used to convert the 5P artificial AB addition product obtained in the step (1) above. A step of extending the 3' end and adding a complementary sequence of artificial nucleic acid sequence C or D (hereinafter referred to as 3' artificial CD complementary addition product) to the 3' end of the 5P artificial AB addition product, and feature 1: 5P artificial addition At the 3′-terminal side, it has the same sequence as at least part of the base sequence of the artificial nucleic acid sequence A or B present in the specific primer.
Characteristic 2: It has an artificial nucleic acid sequence C or D added to the 5P artificial AB adduct on the 5′ end side.
Feature 3: The 3' end is labeled to prevent nucleic acid elongation by DNA polymerase.
(3) Performing either of the following two steps (a) or (b) to add an artificial nucleic acid sequence C or D to the 5' end of the 3' artificial CD complementary addition product Step (a) : When either one or both of the 3′ artificial CD complementary addition products generated in the step (2) are phosphorylated, when the products form a double strand, both Of the single-stranded regions occurring at the ends, at least one complementary to the single-stranded region at the end where the 5' end of the base pair at the site where the region is converted to double strands is phosphorylated The 3' artificial CD complementary addition product is ligated with DNA ligase to the oligo DNA (hereinafter referred to as 5' artificial sequence-introduced oligo) and the 3' artificial CD complementary addition product obtained in the step (2) above. A step of adding an artificial nucleic acid sequence C or D to the 5′ end of or step (b): Either one or both of the 3′ artificial CD complementary addition products generated in the step (2) are In the case of phosphorylation, when the products form a double strand, of the single-stranded regions generated at both ends, the 5' of the base pair at the site where the single-stranded region changes to double strands At the phosphorylated end, at least one oligo DNA complementary to a part of the single-stranded region of the end (hereinafter referred to as a 5' artificial sequence introduction primer) is added as a 3' artificial CD complementary addition product After filling the gap generated when hybridizing with the DNA polymerase by extension reaction, the extended 5' artificial sequence introduction primer and the 3' artificial CD complementary addition product are ligated with DNA ligase to obtain the 3' artificial Step of adding an artificial nucleic acid sequence C or D to the 5' end of the CD complementary addition product [2] The 5P artificial addition specific primer pair has a sequence complementary to the target nucleic acid sequence on the 3' end side and 5' An artificial addition specific primer (a) having at least a partial sequence of the artificial nucleic acid sequence A on the terminal side, and a sequence complementary to the target nucleic acid sequence on the 3' terminal side and an artificial on the 5' terminal side An artificial addition-specific primer pair consisting of an artificial addition-specific primer (b) having at least a partial sequence of the nucleic acid sequence B, wherein at least one of the artificial addition-specific primer pairs is phosphorylated at the 5' end thereof. ,
The 3' artificial sequence-introducing oligo pair is labeled at the same end so that the 3' end is not extended by DNA polymerase, has at least a partial sequence of the artificial nucleic acid sequence A on the 3' end side, and has the 5' end A 3′ artificial sequence-introducing oligo (c) having an artificial nucleic acid sequence C on the side and a similarly labeled 3′ end, having at least a partial sequence of the artificial nucleic acid sequence B on the 3′ end side, 5 'Contains a 3' artificial sequence introduction oligo (d) having an artificial nucleic acid sequence D on the terminal side,
The 5' artificial sequence introduction oligo is both a 5' artificial sequence introduction oligo (e) having the entire sequence of artificial nucleic acid sequence C and a 5' artificial sequence introduction oligo (f) having the entire sequence of artificial nucleic acid sequence D. or including either
The 5' artificial sequence introduction primer is a 5' artificial sequence introduction primer (e) having a partial sequence of artificial nucleic acid sequence C and a 5' artificial sequence introduction primer (f) having a partial sequence of artificial nucleic acid sequence D. The method of [1] including both or one.
[3] The method according to [1] or [2], wherein the steps (1) to (3) are carried out simultaneously in one reaction.

[4] In addition to the features of the 3' artificial sequence-introducing oligo pair described in [1] to [3], by using the oligo pair having the following two features, a 3' artificial CD complementary addition product is obtained. When the products form a double strand, it is possible to improve the binding efficiency of the 5' artificial sequence-introducing primer pair to the single-stranded regions generated at both ends, and to increase the yield of the final artificial sequence-added product. The method according to any one of [1] to [4].
Feature 1: Introduction of a 3' artificial sequence in which an intramolecular secondary structure is artificially formed by introducing a sequence complementary to at least a partial sequence of the oligo to the 5' end of the 3' artificial sequence introduction oligo Oligo.
Feature 2: 3' artificial sequence introduced SL oligo [5] reaction in which a blocker is introduced into the 3' end of the sequence introduced at the 5' end of the 3' artificial sequence introduced oligo described in Feature 1 or into the sequence strand In step (1), a 5P artificial AB addition having at least a partial sequence of the artificial nucleic acid sequence A on one 5′ end side and having at least a partial sequence of the artificial nucleic acid sequence B on the other 5′ end side The 3' artificial sequence that works after the reaction step (2) by increasing the annealing temperature within a range that does not adversely affect the amplification reaction of the reaction in the cycle following the cycle in which the product is first generated. Any of [1] to [4], wherein the reaction step (1) is performed under temperature conditions at which the introduced oligo pair and/or the 5' artificial sequence-introduced primer pair do not function in the reaction step (1). The method described in Crab.
[6] The method of any one of [1] to [5], wherein the artificial nucleic acid sequence is added to one or more target nucleic acid sequences in one reaction.
[7] A kit for producing an artificial sequence addition product, comprising the following (i) to (iii).
(i) an artificial addition specific primer (a) having a sequence complementary to the target nucleic acid sequence on the 3' end side and having at least a partial sequence of the artificial nucleic acid sequence A on the 5' end side; An artificial addition specific primer pair consisting of an artificial addition specific primer (b) having a sequence complementary to the target nucleic acid sequence on the 'end side and having at least a partial sequence of the artificial nucleic acid sequence B on the 5' end side and a 5P artificial addition-specific primer pair group characterized by comprising at least one pair of artificial addition-specific primer pairs in which at least one of the 5′ ends is phosphorylated,
(ii) The 3' end is labeled so that it is not extended by DNA polymerase, has at least a partial sequence of the artificial nucleic acid sequence A on the 3' end side, and the artificial nucleic acid sequence C on the 5' end side. The 3' artificial sequence introduction oligo (c) having the a 3' artificial sequence-introducing oligo pair comprising a 3' artificial sequence-introducing oligo (d) having sequence D; and
(iii) (a) and/or (b) below
(a) including both or one of a 5' artificial sequence-introducing oligo (e) having the entire sequence of artificial nucleic acid sequence C and a 5' artificial sequence-introducing oligo (f) having the entire sequence of artificial nucleic acid sequence D; 5' artificial sequence introduction oligo characterized by
(b) both or one of a 5' artificial sequence introduction primer (e) having a partial sequence of artificial nucleic acid sequence C and a 5' artificial sequence introduction primer (f) having a partial sequence of artificial nucleic acid sequence D a 5' artificial sequence-introducing primer [8], which further comprises a DNA polymerase [7].

The present invention
(1) Artificial addition specific primer (a) having a sequence complementary to the target nucleic acid on the 3' end side and having at least a partial sequence of the artificial nucleic acid sequence A on the 5' end side, and the 3' end side an artificial addition specific primer pair consisting of an artificial addition specific primer (b) having a sequence complementary to the target nucleic acid and having at least a partial sequence of the artificial nucleic acid sequence B on the 5' end side, at least one of which 5P artificial addition specific primer pair group characterized by containing at least one pair of artificial addition specific primer pairs phosphorylated at the 5' end of
(2) The 3' end is labeled so that it is not extended by DNA polymerase, has at least a partial sequence of the artificial nucleic acid sequence A on the 3' end side, and the artificial nucleic acid sequence C on the 5' end side. The 3' artificial sequence introduction oligo (c) having the A 3' artificial sequence-introducing oligo pair characterized by comprising a 3' artificial sequence-introducing oligo (d) having a sequence D
(3) 5' artificial sequence introduction primer (e) having at least a partial sequence of artificial nucleic acid sequence C, and 5' artificial sequence introduction primer (f) having at least a partial sequence of artificial nucleic acid sequence D ), a primer/oligo DNA set composed of a 5′ artificial sequence-introducing primer characterized by including both or one of ), a reaction reagent such as a thermostable DNA polymerase for carrying out an amplification reaction, a thermostable DNA ligase reaction Provided is a method for obtaining a final artificial sequence addition product derived from one or more target nucleic acids using a reaction reagent or the like for carrying out the above.

The present invention provides, for example, a method of amplifying one type of nucleic acid sequence in a sample and adding an artificial nucleic acid sequence containing a barcode sequence to both ends of the amplified product. A specific embodiment will be given and its contents will be described below (see FIG. 5).

The reaction solution used in the embodiment described in FIG. The final artificial sequence addition product of interest is obtained by carrying out the following reaction steps (1) to (3) using these reagents, including a DNA ligase.
Reaction step (1) Amplification reaction using a 5P artificial addition specific primer pair results in at least a partial sequence of the artificial nucleic acid sequence A at one end of the target nucleic acid and at least a portion of the artificial nucleic acid sequence B at the other end. and the 5′ end of the artificial nucleic acid sequence A is phosphorylated to obtain a target nucleic acid-derived 5P artificial AB addition product. By adding a complementary sequence of the artificial nucleic acid sequence C to one 3′ end of the 5P artificial AB addition product obtained in the step (1), and complementing the artificial nucleic acid sequence D to the other 3′ end Reaction Step for Obtaining 3' Artificial CD Complementary Product by Addition of Target Sequence Reaction Step (3) Adjust the temperature of the reaction mixture to a temperature at which the 5' artificial sequence-introducing primer can hybridize, and transfer the oligo to the above ( Hybridize to the single-stranded site of the 3′ artificial CD complementary addition product obtained in step 2), and fill the gaps generated in the single-stranded site with a thermostable DNA polymerase for amplification, and , by repairing the nick generated between the 3' end of the 5' artificial sequence introduction primer after extension and the 5' end of the phosphorylated artificial nucleic acid sequence A with a thermostable DNA ligase. Reaction step to obtain the final artificial sequence adduct

Embodiments of the present invention have various variations as described below.
(1) Two or more pairs of 5P artificially added specific primer pairs are used together with a 3' artificial sequence-introducing oligo pair and a 5' artificial sequence-introducing primer or oligo to carry out the reaction steps (1) to (3) above. Thus, a plurality of final artificial sequence addition products of two or more types derived from the target nucleic acid are obtained.
(2) The reaction steps (1) to (3) can be carried out separately, and the reaction steps can be arbitrarily separated and utilized depending on the purpose. However, an embodiment in which all the necessary materials are added to the same reaction solution before starting the reaction and the reaction steps are continuously carried out in one reaction is advantageous in terms of economy and simplicity.
(3) In the method of the present invention, the reaction conditions of the reaction steps (1) and (2) and the reaction step (3) are separated by shortening the 5' artificial sequence-introducing primer or oligo, etc. It is possible to allow reactions to proceed in isolation. In that case, since the final artificial sequence addition product does not occur in the reaction steps (1) and (2), but only in the reaction step (3), the concentration of the product that allows introduction of the 5' artificial sequence is generated depending only on the concentration of the 5' artificial sequence-introducing primer or oligo. Therefore, by adjusting the concentration of the 5' artificial sequence-introducing primer or oligo, it is possible to adjust the final artificial sequence addition product to the target concentration.

When the reaction step (3) proceeds simultaneously with the reaction steps (1) and (2), the final artificial sequence addition product is generated during the amplification reaction in the reaction step (1), and the product is used as a template. As a result, it becomes difficult to adjust the concentration of the final artificial sequence addition product by adjusting the concentration of the 5' artificial sequence-introducing primer or oligo. In order to prevent the reaction step (3) from proceeding in the reaction steps (1) and (2), the 5' artificial sequence introduction primer or oligo is shortened (hereinafter referred to as a short 5' artificial sequence introduction primer or oligo ), and in reaction steps (1) and (2), the temperature condition of the reaction step is adjusted to the Tm of the short-chain 5' artificial sequence-introducing primer or oligo so that the oligo does not react with the 3' artificial CD complementary addition product. This can be achieved by setting the temperature higher than the value.

On the other hand, when the reaction steps (1) and (2) proceed simultaneously in the reaction step (3), the 3′ artificial CD complementation which is the substrate of the reaction and the product of the reaction step (2) in the reaction step (3) Since the addition product increases, it becomes difficult to adjust the concentration of the final artificial sequence addition product by adjusting the addition concentration of the 5' artificial sequence-introducing primer or oligo. In order to prevent the reaction steps (1) and (2) from proceeding simultaneously in the reaction step (3), it can be achieved by setting the conditions in the reaction step (3) so that no amplification reaction occurs. For example, with regard to the reaction step (3), the temperature conditions are set such that the products produced in the reaction steps (1) to (3) do not dissociate, or the various products do not repeatedly dissociate and melt. can be achieved.

From the above, in adjusting the concentration of the 5' artificial sequence introduction primer or oligo and preparing the final artificial sequence addition product to the target concentration, using a short 5' artificial sequence introduction primer or oligo, and the reaction step ( It is desirable to set various conditions, including the temperature condition in 3), so that amplification does not occur.

(4) In the reaction step (3), using a 5' artificial sequence-introducing oligo that does not cause a gap in the single-stranded site of the 3' artificial CD complementary addition product, between the oligo and the 3' artificial CD complementary addition product It is also possible to obtain the final artificial sequence addition product by a method of repairing the nicks generated in the DNA ligase.

However, in that case, the following problems arise.
1. Since the 5' artificial sequence-introduced oligo is lengthened, at the reaction temperature of the reaction steps (1) and (2), the oligos, the oligo and the artificial addition specific primer, the 3' artificial sequence-introduced oligo, 5P Artificial AB adducts, 3' artificial CD complementary adducts, etc. are likely to interact, resulting in increased possibility of generating unwanted non-specific products.
2. Since the 5′ artificial sequence-introducing oligo is long, the reaction step (3) proceeds simultaneously under the temperature conditions of the reaction steps (1) and (2), and during the amplification reaction of the reaction step (1) A final artificial sequence addition product is generated, and an amplification reaction using this product as a template proceeds with a primer pair consisting of a 5′ artificial sequence-introducing oligo and a 5P artificial addition specific primer. As a result, it becomes difficult to adjust the concentration of the final artificial sequence addition product by adjusting the concentration of the 5′ artificial sequence-introducing oligo.
3. If the artificial nucleic acid sequences C and D or the artificial nucleic acid sequences A and B that are not included in the artificial additional specific primer have an artificial nucleic acid sequence (barcode sequence) for identifying the sample, the 5' artificial sequence introduction oligo also Since it has the above-mentioned barcode sequence, it is necessary to prepare the same number of oligos as the number of types of barcodes, so the economic efficiency is low.

From the above, unless there is a particular reason, the 5' artificial sequence-introducing oligo does not contain a barcode sequence, and is as short as possible so as not to interact with the various nucleic acids described above in the reaction steps (1) and (2). It is preferable that the reaction step (3) is carried out separately from the reaction steps (1) and (2).

(5) If the annealing temperature in reaction step (1) can be set to a high temperature, the difference between the annealing temperature of reaction step (1) and reaction step (2), and the difference between reaction step (2) and reaction step (3) It is possible to sufficiently secure the difference from the annealing temperature of . As a result, the 3' artificial sequence-introducing oligo pair that functions in the reaction step (2) does not function in the reaction step (1), and the 5' artificial sequence-introducing primer that functions in the reaction step (3) does not function in the reaction step (1). ) and (2) do not function, and by greatly changing the annealing temperature conditions for each reaction step, reaction steps (1) to (3) can proceed in order. Become.

As a result, in each reaction step, it is possible to reduce the possibility that oligo-DNAs and primers that function in other reaction steps will function, thus simplifying the reaction scheme in each reaction step. In addition to facilitating condition optimization and troubleshooting, the reactions other than the reaction step (1) are common regardless of the target nucleic acid. 1) only, and there is an advantage that the reaction conditions in the reaction steps (2) and (3) do not need to be optimized for each target nucleic acid.

In order to realize the above, the following condition setting is required.
Condition 1. The difference in annealing temperature between the reaction steps (1) and (2) is ensured by providing a sufficient difference between the Tm value of the artificially added specific primer and the Tm value of the 3' artificial sequence-introducing oligo pair.
Condition 2. Ensuring a difference in annealing temperature between reaction steps (2) and (3) by providing a sufficient difference between the Tm values of the 3' artificial sequence-introducing oligo pair and the 5' artificial sequence-introducing primer. .
Condition 3. The annealing temperature in reaction step (3) is set to a temperature at which the reaction proceeds sufficiently.

However, since the annealing temperature in the reaction step (1) is set to a temperature at which only the sequence complementary to the target nucleic acid among the artificial addition specific primers can bind alone, depending on the target nucleic acid, the reaction steps (1) to ( There may be cases where it is difficult to secure a sufficient difference in annealing temperature when proceeding with 3) in order. For example, the annealing temperature in reaction step (3) is set to 40° C., which is assumed to be the lower limit for ensuring normal reaction in the step, and the difference in annealing temperature between reaction steps (1) and (2), And when the difference in annealing temperature between reaction steps (2) and (3) is secured by 10 ° C., the annealing temperature in reaction step (1) is 60 to 72 ° C., which is within the general annealing temperature range of PCR. (50-72° C.) (FIG. 6a). As described above, the method described above imposes greater restrictions on the design of artificially added specific primers, which narrows the applicable range of target nucleic acids in the present invention. Therefore, some improvement is desirable.

The improved method is shown in FIG. The annealing temperature of the reaction step (1) in this improved method is 5P artificial AB having the same sequence as one artificial addition specific primer at the 5′ end and a complementary sequence of the other artificial addition specific primer at the 3′ end. At least until the PCR cycle in which the addition product is generated, the sequence complementary to the target nucleic acid contained in the 5P artificial addition specific primer pair is at a temperature at which it can bind alone, ensuring amplification using the initially added nucleic acid as a template. . Since the 5P artificial AB addition product can bind to the entire region of the artificial addition specific primer, in any cycle after the cycle in which the 5P artificial AB addition product is amplified for the first time, the reaction step (1) Amplification of the product is thought to be possible even if the annealing temperature of is increased. Specifically, since the 5P artificial AB addition product is amplified for the first time in the second cycle (see FIG. 7), the annealing temperature in the reaction step (1) can be adjusted from any number of cycles after the third cycle. Amplification of 5P-artificial AB adducts is thought to be possible even if the temperature is raised based on the Tm value of the entire region of the artificially added specific primers. Thus, by increasing the annealing temperature in the middle of the reaction step (1) and performing PCR using only the 5P artificial AB addition product as a template, the design limitations of the artificial addition specific primer (specifically, the reaction The annealing temperature in step (1) is the temperature set from the Tm value of the sequence complementary to the target nucleic acid among the artificial addition specific primers, and the difference between this annealing temperature and the annealing temperature in reaction step (2) is kept constant. Since the limitation of ensuring the above is eliminated, it becomes possible to expand the range of applicable target nucleic acids (see FIG. 6b).

(6) Phosphorylate the 5′ ends of all the artificial addition-specific primers used in the reaction step (1) to obtain 5P artificial AB addition products in which both 5′ ends are phosphorylated, Two 5' artificial sequences that bind to single-stranded sites existing at both ends of the 3' artificial CD complementary addition product in the reaction step (3) after being converted into the 3' artificial CD complementary addition product in the reaction step (2) It is also possible to obtain the desired final artificial sequence addition product by double-stranding the single-stranded sites present at both ends using the introduced primers. Both DNA strands are effective when artificial nucleic acid sequences are added to both ends. For example, for the purpose of simplifying the purification process of the final artificial sequence addition product, enzymes using ExoSAP-IT Express PCR Cleanup Reagents (manufactured by Thermo Fisher Scientific), etc., which can be performed only by adding reagents If a single-stranded site is present in the final artificial sequence addition product, the site will be degraded when the purification method is used. There is a need to obtain a final artificial sequence addition product with double-stranded ends.

However, the 5' artificial sequence-introducing primer is not modified at the 3' end of the oligo and functions as a primer. If there are many types of primers to be added, the possibility of generating non-specific amplification products such as primer dimers is likely to increase. If the purpose can be achieved by adding an artificial nucleic acid sequence to both ends of one of the DNA strands, it is basically desirable to use one type of 5' artificial sequence-introducing primer.

(7) The binding of the 3′ artificial sequence-introducing oligo and the 5P artificial AB adduct in the reaction step (2) includes the rebinding of the 5P artificial AB adducts and the binding of the 5P artificial addition specific primer to the 5P artificial AB adduct. Inhibited by binding, the tendency becomes stronger as the concentration of 5P artificial AB adduct and 5P artificial adduct specific primer increases. As a result, the binding between the 3' artificial sequence-introducing oligo and the 5P artificial AB adduct may be inhibited, and the production of the 3' artificial CD complementary adduct may be suppressed.

For the purpose of solving the above problem, among the sequence regions of the 3' artificial sequence-introducing oligo, the sequence region that binds to the 5P artificial AB adduct or the 5P artificial AB adduct is modified with the function of increasing the melting temperature of the oligo. By using the 3' artificial sequence-introducing oligo, the concentration of the 3' artificial CD complementary addition product in the reaction step (2) can be increased in some cases. Examples of such modifications include artificial nucleic acids (LNA, BNA, etc.).

(8) The binding of the 5′ artificial sequence-introducing primer and the 3′ artificial CD complementary addition product in the reaction step (3) is inhibited by the recombination of the 3′ artificial CD complementary addition product and the 3′ artificial sequence-introducing oligo, and the 5′ Introduction of artificial sequences may be suppressed.

For the purpose of solving the above, by using a modified 5' artificial sequence introduction primer that has a function of increasing the melting temperature of the oligo in the sequence region of the 5' artificial sequence introduction primer, in the reaction step (3) It may be possible to increase the efficiency of introduction of 5' artificial sequences. Examples of such modifications include artificial nucleic acids (LNA, BNA, etc.).

Moreover, it can be solved by using a 3' artificial sequence-introduced SL oligo having an intramolecular secondary structure in the 5' terminal region. The principle is shown as FIG.

The 3' artificial sequence-introduced SL oligo described in FIG. It has an intramolecular secondary structure (Stem-loop structure in FIG. 8). In addition, a blocker is introduced between the sequence introduced for the purpose of forming an intramolecular secondary structure and the artificial nucleic acid sequence C or D.

When using a 3' artificial sequence-introduced SL oligo having such a structure, it is possible to achieve the following two points.
1. Since the 3' artificial sequence-introduced SL oligo forms an intramolecular secondary structure, the oligo cannot bind to the sequence complementary to the artificial nucleic acid sequence C or D of the 3' artificial CD complementary addition product. . Therefore, the 5' artificial sequence-introducing primer can easily bind to the complementary sequence of the artificial nucleic acid sequence C or D in the 3' artificial CD complementary addition product.
2. When a blocker is not introduced into the 3' artificial sequence-introduced SL oligo (Fig. 9), the 3' artificial sequence-introduced SL oligo is extended including the intramolecular secondary structure in the reaction step (2), so the product ( 3' artificial CD complementary addition product) forms an intramolecular secondary structure at the complementary sequence portion of the artificial nucleic acid sequence C or D, which inhibits the binding of the 5' artificial sequence-introducing primer.

On the other hand, when a blocker is introduced into the 3' artificial sequence-introduced SL oligo (Fig. 10), DNA elongation is blocked immediately before the extension of the sequence necessary for the formation of the intramolecular secondary structure. CD complementary addition product) does not form an intramolecular secondary structure, and the 5′ artificial sequence-introducing primer binds to the complementary sequence of the artificial nucleic acid sequence C or D in the 3′ artificial CD complementary addition product. do not interfere. As a result, it leads to an improvement in the efficiency of introducing the 5' artificial sequence into the 3' artificial CD complementary addition product.

According to the present invention, the following effects can be expected.
(1) A final artificial sequence addition product that can be analyzed by a next-generation sequencer can be obtained in a one-step work process.
(2) The primers used in the exponential amplification reaction in the technique of the present invention are only artificial addition-specific primers that amplify the target nucleic acid, and amplification using the target nucleic acid as a template as in the techniques of prior arts 3 and 4. Since there is no need to use a primer for amplification and a primer for amplification using the amplification product derived from the target nucleic acid as a template in the same reaction solution, the generation of non-specific amplification products such as primer dimers can be suppressed. Therefore, the minimum required amount of the initial template can be kept lower than in the above-described prior art, and sensitivity similar to general PCR using only target nucleic acid-specific primers can be ensured.
(3) As in prior arts 3 and 4, in the case of a method in which amplification using a target nucleic acid as a template and amplification using an amplification product derived from the target nucleic acid as a template are simultaneously performed in the same reaction solution, There is a need to rigorously optimize the reaction conditions so that two or more different amplification reactions proceed properly and simultaneously.
On the other hand, in the case of the present invention, by changing the reaction conditions such as temperature conditions, it is possible to separately perform the reaction of adding the artificial nucleic acid sequence and the amplification reaction within one step of the reaction. It is possible to carry out each reaction under optimum conditions. In addition, since the reaction to add the artificial nucleic acid sequence can be performed 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 amplification using an amplification product derived from the target nucleic acid as a template. From this, a reaction system can be constructed simply and quickly like general PCR.
(4) In the case of a method using a primer to which a specific artificial nucleic acid sequence containing a barcode sequence is added in advance to the 5' end of the primer specific to the target nucleic acid sequence (prior art 1), it is easy to A final artificial sequence addition product that can be subjected to next-generation sequencer analysis can be obtained in a single-step work process. However, it is necessary to prepare primers with different barcode sequences for each target nucleic acid, which is extremely uneconomical. For example, 100 specimens are simultaneously analyzed in one analysis, and 100 types of target nucleic acids are analyzed per specimen by a method of identifying specimens by combining bar code sequences added to both ends and the sequences. In this case, 10 types of forward primers and 10 types of reverse primers are required per target nucleic acid, for a total of 20 types of primers. have a nature.
On the other hand, in the technique based on the present invention, the amplification of the target nucleic acid is carried out by the artificially added specific primer, and the addition of the artificial nucleic acid sequence including the barcode sequence is performed by the 3′ artificial sequence introduction oligo and the 5′ artificial sequence introduction primer or the 5′ artificial sequence introduction primer. Since the sequence-introducing oligo is responsible, the types of primers or oligos required respectively depend on the type of target nucleic acid for the former and the number of specimens to be analyzed for the latter. For example, if the same premises as above (target nucleic acid: 100 types, number of specimens: 100 specimens) are used, there are 200 types of artificially added specific primers in total for forward / reverse primers, 20 types of 3' artificial sequence introduction oligos, 5' Artificial sequence-introducing primers or 5' artificial sequence-introducing oligos are one type when the oligo does not contain a barcode sequence, and 10 types when the oligo contains a barcode sequence, for a total of 221 or 230 types. In addition, the 3' artificial sequence-introducing oligo and the 5' artificial sequence-introducing primer or 5' artificial sequence-introducing oligo can be used in common even if the target nucleic acid is changed.
(5) Since the concentration of the final artificial sequence addition product to be subjected to the next-generation sequencer needs to be constant, it is required to adjust the concentration after the final artificial sequence addition product is prepared.
In the case of the method of obtaining the final artificial sequence addition product based on the amplification reaction as in Prior Art 1 to 4, the final concentration of the product depends on the amount of non-specific amplification product, the amount of initial template, the concentration of amplification inhibitors, etc. Therefore, the above concentration adjustment is often necessary because it changes for each amplification reaction.
On the other hand, in the method of the present invention, the concentration of the 5' artificial sequence-introducing primer or 5' artificial sequence-introducing oligo is adjusted within the range of the concentration of the target product into which the 5' artificial sequence can be introduced as the upper limit. Sequence adducts can be prepared at target concentrations. Therefore, the density adjustment described above is not necessarily required.

As described above, the method based on the present invention has the effect of being able to economically, easily, and highly sensitively obtain the final artificial sequence addition product that can be suitably used for analysis using a next-generation sequencer. Therefore, it can be said to be efficient in many respects compared to the prior art.

1 is a principle diagram of prior art 1. FIG. Using a primer to which a specific artificial nucleic acid sequence including a barcode sequence has been added to the 5' end of the primer specific to the target nucleic acid sequence, a final artificial sequence addition product that can be subjected to a next-generation sequencer is obtained. method. In this method, it is necessary to prepare primers with different barcode sequences for each type of nucleic acid sequence as many as the number of specimens. FIG. 2 is a principle diagram of prior art 2; In this method, the first amplification is performed using an artificially added specific primer in which a part of the 3' end side of the artificial nucleic acid sequence is added to the 5' end side of the specific sequence to obtain a specific product derived from the target nucleic acid. . Subsequently, after purifying the product, the product is used as a template for the second amplification using an artificial sequence primer having an artificial nucleic acid sequence such as a barcode sequence on the 3′ end side. A final artificial sequence addition product in which the artificial nucleic acid sequences are added to both ends of the target nucleic acid can be obtained by the above two amplifications. FIG. 2 is a principle diagram of prior art 3; In this method, the concentration of the artificial addition specific primer is set lower than the concentration of the artificial sequence primer, and both primers are added to the same reaction solution and amplified, so that the final artificial sequence addition product can be obtained. FIG. 4 is a principle diagram of prior art 4; In this method, two pairs of artificial sequence primers, a long chain artificial sequence primer and a short chain artificial sequence primer, are used in addition to the artificial addition specific primer in the reaction to add the artificial nucleic acid sequence to the target nucleic acid sequence. By setting the primer concentration higher than that of the artificially added specific primer and the concentration of the long-chain artificial sequence primer, the final artificial sequence added product can be amplified more efficiently. The figure which showed an example of embodiment regarding this invention. Reaction steps (1) and (2) The figure which showed an example of embodiment regarding this invention. Reaction step (3) (There is a gap between the 5' artificial sequence-introducing oligo (e) or (f) and the 3' artificial CD complementary addition product, and only the 5' artificial sequence-introducing primer (e) or (f) is used. if you did this) The figure which showed an example of embodiment regarding this invention. Reaction step (3) (there is a gap between the 5' artificial sequence-introducing oligo (e) or (f) and the 3' artificial CD complementary addition product, and both the 5' artificial sequence-introducing primers (e) and (f) used) The figure which showed an example of embodiment regarding this invention. Reaction step (3) (no gap between 5' artificial sequence introduced oligo (e) or (f) and 3' artificial CD complementary addition product, using only 5' artificial sequence introduced oligo (e) or (f)) if you did this) The figure which showed an example of embodiment regarding this invention. Reaction step (3) (no gap between 5' artificial sequence-introduced oligo (e) or (f) and 3' artificial CD complementary addition product, and both 5' artificial sequence-introduced oligos (e) and (f) used) Comparison with and without changing the annealing temperature in the reaction step (1) Reaction scheme diagram in the initial cycle (1-3 cycles) of reaction step (1) Improvement of artificial sequence introduction efficiency by 3' artificial sequence introduction oligo (hereinafter referred to as 3' artificial sequence introduction SL oligo) having intramolecular secondary structure Reaction scheme diagram when using 3' artificial sequence-introduced SL oligo without blocker Reaction scheme when using 3' artificial sequence-introduced SL oligo with blocker Intramolecular secondary structure of 3′ artificial sequence-introduced SL oligo with blocker Chemical structural formulas of various spacer groups with blocker functions. Chemistry of dT reagents capable of forming 3'-3', 5'-5' bonds and acting as blockers (left) and dT reagents forming the normal 5'→3' backbone (right). Structural formula. FIG. 4 is a diagram showing the relationship between the concentration of the 5′ artificial sequence-introducing primer and the amount of the resulting product. The figure which showed the read number in each target gene for every embodiment.

Embodiments of the present invention will be described in detail.

The present invention provides a method for preparing a final artificial sequence addition product by adding an artificial nucleic acid sequence to a target nucleic acid sequence, the method comprising the following steps (1) to (3).
(1) A primer pair in which at least a part of the sequence of the artificial nucleic acid sequence A or B is added to each primer pair specific to the target nucleic acid sequence, wherein , One or both 5' ends are phosphorylated by a nucleic acid amplification method using one or more pairs of artificial addition specific primers (hereinafter referred to as 5P artificial addition specific primer pairs) phosphorylated at one or both 5' ends obtaining a 5P artificial AB adduct;
(2) Using a pair of oligo DNA (hereinafter referred to as oligo pair with 3' artificial sequence introduction) characterized by the following three points, DNA polymerase is used to convert the 5P artificial AB addition product obtained in the step (1) above. A step of extending the 3' end and adding a complementary sequence of artificial nucleic acid sequence C or D (hereinafter referred to as 3' artificial CD complementary addition product) to the 3' end of the 5P artificial AB addition product, and feature 1: 5P artificial addition At the 3′-terminal side, it has the same sequence as at least part of the base sequence of the artificial nucleic acid sequence A or B present in the specific primer.
Characteristic 2: It has an artificial nucleic acid sequence C or D added to the 5P artificial AB adduct on the 5′ end side.
Feature 3: The 3' end is labeled to prevent nucleic acid elongation by DNA polymerase.
(3) Performing either of the following two steps (a) or (b) to add an artificial nucleic acid sequence C or D to the 5' end of the 3' artificial CD complementary addition product Step (a) : When either one or both of the 3′ artificial CD complementary addition products generated in the step (2) are phosphorylated, when the products form a double strand, both Of the single-stranded regions occurring at the ends, at least one complementary to the single-stranded region at the end where the 5' end of the base pair at the site where the region is converted to double strands is phosphorylated The 3' artificial CD complementary addition product is ligated with DNA ligase to the oligo DNA (hereinafter referred to as 5' artificial sequence-introduced oligo) and the 3' artificial CD complementary addition product obtained in the step (2) above. A step of adding an artificial nucleic acid sequence C or D to the 5′ end of or step (b): Either one or both of the 3′ artificial CD complementary addition products generated in the step (2) are In the case of phosphorylation, when the products form a double strand, of the single-stranded regions generated at both ends, the 5' of the base pair at the site where the single-stranded region changes to double strands At the phosphorylated end, at least one oligo DNA complementary to a part of the single-stranded region of the end (hereinafter referred to as a 5' artificial sequence introduction primer) is added as a 3' artificial CD complementary addition product After filling the gap generated when hybridizing with the DNA polymerase by extension reaction, the extended 5' artificial sequence introduction primer and the 3' artificial CD complementary addition product are ligated with DNA ligase to obtain the 3' artificial Step of adding an artificial nucleic acid sequence C or D to the 5' end of the CD complementary addition product

In the method of the present invention, the 5P artificial addition specific primer pair has a sequence complementary to the target nucleic acid sequence on the 3' end side, and at least a partial sequence of the artificial nucleic acid sequence A on the 5' end side. and an artificial addition specific primer (a) having a sequence complementary to the target nucleic acid sequence on the 3' end side and having at least a partial sequence of the artificial nucleic acid sequence B on the 5' end side An artificial addition-specific primer pair consisting of primer (b), at least one of which is phosphorylated at the 5' end thereof, and a 3' artificial sequence-introducing oligo pair, the 3' end of which is a DNA polymerase 3' artificial sequence-introducing oligo ( c), similarly labeled at the 3' end, has at least a partial sequence of the artificial nucleic acid sequence B on the 3' end side, and introduces a 3' artificial sequence having an artificial nucleic acid sequence D on the 5' end side Oligo (d), wherein the 5' artificial sequence introduction oligo is a 5' artificial sequence introduction oligo (e) having the entire sequence of artificial nucleic acid sequence C and a 5' artificial sequence having the entire sequence of artificial nucleic acid sequence D containing both or one of the introduction oligos (f),
The 5' artificial sequence introduction primer is a 5' artificial sequence introduction primer (e) having a partial sequence of artificial nucleic acid sequence C and a 5' artificial sequence introduction primer (f) having a partial sequence of artificial nucleic acid sequence D. Both or one may be included.

In the present invention, an amplification product derived from a target nucleic acid is obtained by amplification using a group of 5P artificial addition specific primer pairs, one end of the product has at least a partial sequence of the artificial nucleic acid sequence A, and the other One or more 5P artificial AB addition products having at least a partial sequence of the artificial nucleic acid sequence B at the end of are amplified from one or more target nucleic acids, and for the resulting product group, the 3' artificial sequence introduction oligo, and artificial nucleic acid sequence C is introduced into the end having artificial nucleic acid sequence A of the product, and artificial nucleic acid sequence D is introduced into the end having artificial nucleic acid sequence B by a reaction that does not rely on an amplification reaction using a 5′ artificial sequence-introducing primer. Obtain the final artificial sequence addition product.

The 5P artificial addition specific primer pair has a sequence complementary to the target nucleic acid on the 3' end side, and at least a partial sequence (3' end side sequence) of the artificial nucleic acid sequence A on the 5' end side. Artificial addition specific primer (a), having a sequence complementary to the target nucleic acid on the 3' end side, and at least a partial sequence (3' end side sequence) of the artificial nucleic acid sequence B on the 5' end side It consists of an artificially added specific primer (b) with In the present invention, at least one pair of 5P artificial addition-specific primer pairs is used. Generally, it is said that uniform PCR amplification becomes more difficult as the number of types of primers increases, but amplification is easily possible with about 5 pairs, and even if there are 20 pairs or more, the combination of sequences should be optimized. can be amplified by

In addition, one or both of the two artificially added specific primers that constitute the primer pair are phosphorylated at the 5' ends.

In the 5P artificial addition specific primer pair groups (a) and (b), the sequence complementary to the target nucleic acid is preferably 10 to 40 bases long, preferably 15 to 30 bases long, more preferably It is 15-25 bases long. The sequence of the artificial nucleic acid sequence A or B to be introduced into the artificial addition specific primer pair group may be a sequence obtained by removing any number of nucleotides from the 5' end side of the artificial nucleic acid sequence A or B, and at least the artificial nucleic acid sequence It can be designed to include the 3'-end sequence of A or B. The artificial additional specific primer preferably contains 0 to 40 bases, preferably 0 to 30 bases, and more preferably 0 to 20 bases of the 3′ terminal side sequence of the artificial nucleic acid sequence A or B.

Also, one or both of the artificially added specific primers are phosphorylated at the 5' end.

The 3' artificial sequence-introducing oligo pair has at least a partial sequence on the 5'-terminal side of the artificial nucleic acid sequence A on the 3'-terminal side, and a 3' artificial sequence-introducing artificial nucleic acid sequence C on the 5'-terminal side. Oligo (c) and a 3' artificial sequence introduction oligo (d ), and the 3′ end is labeled with phosphoric acid or the like so as not to be extended by DNA polymerase.

The 3' artificial sequence-introducing oligo preferably contains 10 to 60 bases, preferably 10 to 40 bases, and more preferably 15 to 25 bases of the 5' terminal sequence of the artificial nucleic acid sequence A or B.

In addition, the 3' artificial sequence introduction oligo shares at least a partial sequence of the artificial nucleic acid sequence A or B at the 5' end of the 5P artificial addition specific primer pair and the 3' end of the 3' artificial sequence introduction oligo. It should be designed as follows. The sequence shared by the primer and oligo may be 0-40 bases, preferably 0-30 bases, more preferably 0-20 bases.

When the reaction is performed using such primers, in the reaction step (2), the complementary strands of the artificial nucleic acid sequences A and B added in the reaction step (1) are added to the 3′ artificial sequence-introducing oligo. Complementary strands are formed with the artificial nucleic acid sequences A and B, and as a result, the 3′ ends of the complementary strands of the artificial nucleic acid sequences A and B are extended to form the full-length complementary strands of the artificial nucleic acid sequences A and B. becomes (Fig. 5-1). Subsequently, by performing the reaction step (3), an elongation reaction occurs using the full-length complementary strands of the artificial nucleic acid sequences A and B generated in the reaction step (2) as templates. will be formed (Figs. 5-2, 5-3, 5-4 and 5-5).

In the examples described later, the 3′ artificial sequence-introducing oligos (c) and (d) have all the sequences of the artificial sequences A and B, but the 5P artificial addition specific primers (a) and (b) have artificial sequences. It has only part of the 3' terminal side of A and B. By using a 5P artificial addition specific primer having only a part of the artificial nucleic acid sequences A and B sequences, the common sequence part of the 3' artificial sequence introduction oligo and the 5P artificial addition specific primer is shortened. By adjusting the length of the sequence portion, the binding temperature between the 3′ artificial sequence-introducing oligo and the 5P artificial AB adduct produced in the reaction step (1) can be adjusted to the binding temperature between the 5P artificial addition specific primer and the target gene. can be lower than By doing so, it is possible to carry out the reaction of only the reaction step (1) at a temperature at which the 3′ artificial sequence-introducing oligo does not bind to the 5P artificial AB adduct, and the reaction step (1) and the reaction step (2) are performed. ) will no longer run at the same time. As a result, the reaction can be carried out under the optimum conditions for each reaction step, and as a result, it is thought that the reaction specificity is improved. In other words, by adjusting the length of the common sequence between the 5P artificial addition specific primer pair and the 3′ artificial sequence-introducing oligo, the reaction conditions (mainly temperature) in the reaction step (2) can be arbitrarily adjusted. .

In addition, the 3' artificial sequence introduction oligo has artificial nucleic acid sequences C and D, artificial nucleic acid sequences A and B not included in the artificial addition specific primer, or an artificial nucleic acid sequence (barcode sequence) for identifying the sample. You may The barcode sequence may be 3-20 bases long, preferably 5-15 bases long, more preferably 5-10 bases long.

In addition, the 3' ends of the 3' artificial sequence-introducing oligos (c) and (d) are labeled so as not to be extended by DNA polymerase. The labeling is generally modified with phosphorylation, fluorescent dyes, amino linkers, biotin, etc., but any modification method that can prevent elongation of the 3′ end by DNA polymerase can be applied depending on the labeling method. The scope is not limited.

In the reaction step (3), if the purpose is to eliminate the suppression of introduction of the 5' artificial sequence due to the recombination of the 3' artificial CD complementary addition product and the 3' artificial sequence-introducing oligo, 5 of the 3' artificial sequence-introducing oligo By arranging a sequence complementary to at least a partial sequence of the oligo at the 'end, an intramolecular secondary structure is introduced on the 5' end side, and the 3' of the sequence introduced for the purpose of forming the intramolecular secondary structure This inhibition can be overcome by using 3' artificial sequence introduced SL oligos with blockers introduced at the end or in the strand of the sequence.

In order to promote the binding of the 5' artificial sequence-introduced primer and the 3' artificial CD complementary addition product, the entire region of the 3' artificial sequence-introduced SL oligo excluding the sequence introduced for the formation of the stem-loop structure and the 3' artificial CD When the temperature reaches the temperature at which the complementary addition product can bind, the oligo has already formed a stem-loop structure, and the oligo cannot bind to the 3′ artificial CD complementary addition product in the entire region. For that reason, the dissociation temperature (Tm value) of the Stem-loop structure of the oligo is lower than the dissociation temperature (Tm value) of the entire region of the oligo excluding the sequence introduced for the formation of the Stem-loop structure. high temperature is desirable. The difference in Tm values is preferably 1 to 30°C, preferably 3 to 20°C, more preferably 5 to 15°C. The Tm value of the Stem-loop structure can be predicted by the web application mfold (http://www.unafold.org/mfold/applications/dna-folding-form.php) etc., and the predicted value is By adjusting the length of the Stem while confirming, it is possible to adjust the Tm value with high accuracy. It is 5 to 25 bases long, more preferably 10 to 20 bases long.

On the other hand, the loop portion is desirably a sequence that does not form a secondary structure other than the designed stem-loop structure. In addition, when the sequence length of the loop portion is short, the distance between DNAs forming the Stem structure decreases and the microscopic concentration increases, so the Tm value of the structure increases. Since the distance between the DNAs forming the structure increases and the microscopic concentration decreases, the Tm value of the structure decreases. Therefore, it is possible to adjust the Tm value of the Stem-loop structure by adjusting the sequence length of the loop portion, but the length of the loop portion is usually 0 to 30 nucleotides, preferably is 8 to 24 bases long, more preferably 10 to 20 bases long.

The blocker used in the artificially added specific SL primer is assumed to be HEG, but like HEG, any substance that has the function of stopping DNA elongation may be used, and the blocker does not limit its application range. . Substances having functions similar to HEG are shown in FIGS. The substance shown in FIG. 12 is a structure having no base, and is a substance that can be introduced into the phosphodiester bond skeleton of a DNA chain, and is an alkyl linker (Spacer C2, C3, C4, C6, C9, C12 ), a polyethylene glycol linker (Spacer 9, 18), and a baseless deoxyribose ring (dSpacer, ethynyl dSpacer) are used as spacers and have similar functions to HEG.
The substances shown on the left side of FIG. 13 form 3′-3′, 5′-5′ bonds by introducing the substances into the strand during oligo-DNA synthesis, and insert nucleosides in the opposite direction. and a dT reagent (dT-5'-CE Phosphoramidite (Glenn Research)) that functions as a blocker at the introduction site. In normal oligo DNA synthesis, the monomer reagent of the type on the right side of Fig. 13 is placed at the 5' position of DMT, and the phosphodiester bond on the 3' side repeats polymerization, from the 3' side to the 5' direction. Although the sequence is synthesized, the reagent (left side of FIG. 13) has a portion that forms a phosphodiester bond at the 5′ position, so when the substance is introduced at an arbitrary portion, it proceeds from the 3′ side. The 5' position of the sequence and the 5' position of the substance of interest are combined to form a 5'-5' bond, followed by polymerization of a conventional monomeric reagent to form a 3'-3' bond, followed by In this case, normal polymerization proceeds.

On the other hand, DNA elongation by DNA polymerase proceeds from the 5' side to the 3' side, and a sequence complementary to the template is synthesized sequentially from the 3' side of the template. (because it changes from 3' to 5' to 5' to 3'), synthesis stops at the insertion point. Therefore, DNA elongation can be blocked by introducing the substance into oligo DNA.

The blocker can be inserted at two or more sites in one oligo DNA, and as a result, it becomes possible to more reliably block DNA elongation by DNA polymerase. In this case, the blockers can be inserted in various forms, such as inserting two or more blockers in succession, inserting the blockers in such a manner that bases are usually arranged between the blockers, By inserting more than one type of different blockers into one oligo DNA, or by inserting any combination of the above three types of insertion forms, it is possible to achieve the above-mentioned purpose (blocking DNA elongation more reliably) is possible).

The 5' artificial sequence-introducing primer pair consists of a 5' artificial sequence-introducing primer (e) having at least a partial sequence of the artificial nucleic acid sequence C and a 5' having at least a partial sequence of the artificial nucleic acid sequence D. Although it consists of an artificial sequence-introducing primer (f), it is not always necessary to use a pair of 5' artificial sequence-introducing primers, and the present invention can be practiced by using either one of the 5' artificial sequence-introducing primers. .

The base length of the 5' artificial sequence-introducing primer and 5' artificial sequence-introducing oligo is desirably designed in the range of 5 to 40 bases, preferably 5 to 30 bases, more preferably 7 to 15 bases.

The 5' artificial sequence introduction oligo pair consists of a 5' artificial sequence introduction oligo (e') having the entire sequence of the artificial nucleic acid sequence C and a 5' artificial sequence introduction having the entire sequence of the artificial nucleic acid sequence D Although it consists of oligo (f'), it is not always necessary to use a pair of 5' artificial sequence-introducing oligos, and the present invention can be practiced by using either one of the 5' artificial sequence-introducing oligos. When the 5P artificial addition specific primer (a) or (b) does not have the full length of the artificial nucleic acid sequence A or B, the 5' artificial sequence introduction oligo (e) or (f) is the artificial nucleic acid sequence A or Contains a partial sequence of the 5' end of B.

Further, in Examples described later, the 5' artificial sequence-introducing primers (e) and (f) have only a part of the artificial sequence C or D on the 5' terminal side. Thus, there are two major advantages of shortening the 5' artificial sequence-introducing primer. One is to shorten the 5′ artificial sequence-introducing primer, so that the reaction step (3) can be performed separately from the reaction steps (1) and (2). can be carried out under optimum conditions. The other is that the 5' artificial sequence-introducing primer is not phosphorylated at the 3' end, so it functions as a PCR primer, but when this primer functions in the reaction step (1), it induces amplification of non-specific products. there's a possibility that. However, shortening the 5' artificial sequence-introducing primer makes it possible to prevent the primer from functioning in the reaction step (1), making it possible to suppress amplification of non-specific products. Therefore, by shortening the 5' artificial sequence introduction primer,
(1) Since reaction step (3) can be separated from reaction steps (1) and (2), reaction step (3) can be carried out under optimum conditions.
(2) In the reaction step (1), the 5' artificial sequence-introducing primer does not function as a PCR primer, so formation of non-specific amplification products can be prevented.
There will be an advantage.

again,
As artificial nucleic acid sequences A, B, C, D and barcode sequences, sequences used for next-generation sequencer analysis can be suitably used. For example, all or part of the sequence described as Primer in the Illumina Adapter Sequence Document (http://support.illumina.com/downloads/illumina-customer-sequence-letter.html) disclosed by Illumina is artificial As nucleic acid sequences A, B, C, D, the sequences described as Index Adapters are suitable for use as barcode sequences.

In the present invention, the final artificial sequence addition product is generated by first combining the 5P artificial addition specific primer pair group, the 3′ artificial sequence introduction oligo pair, and the 5′ artificial sequence introduction primer or 5′ artificial sequence introduction oligo. A series of reactions can be completed without mixing, purification or dilution in the middle.

The present invention suppresses the amplification of non-specific sequences, and thus enables more efficient amplification by combining with various methods and reagents that are said to be able to suppress the activity particularly in the early stages of the amplification cycle. Specifically, techniques and reagents that gradually increase amplification reaction efficiency, such as touchdown PCR and the use of chemically modified hot-start DNA polymerase, are preferred.

Chemically modified hot-start DNA polymerases are DNA polymerases into which thermolabile blocking groups have been introduced that inactivate the enzyme at room temperature, and these blocking groups reach high temperatures prior to amplification. It is then removed and the enzyme becomes activated. Such modification can be accomplished by, for example, binding citraconic anhydride, cis-aconitic anhydride, etc. to lysine residues of proteins (Patent No. 3026554). Compared to another hot-start modification method, monoclonal antibody-based method (U.S. Pat. No. 5,338,671), activation time is longer and activity increases step by step during the amplification cycle. , is characterized by high specificity in the early stages of amplification.

For the present invention it is preferred to use a DNA polymerase that does not have 5' to 3' exonuclease activity. This is because when a DNA polymerase having 5'→3' exonuclease activity is used, in the reaction step (3), the 5' artificial sequence-introducing primer is extended while excluding base pairs existing in the extension direction. This is because the complementary strand of the artificial nucleic acid sequence C or D added to the 3′ end of the 3′ artificial CD complementary addition product, which is a degradation product, may be removed.

As for the DNA polymerase used in the present invention, those without 5′→3′ exonuclease activity and strand displacement activity can be preferably used, and Hot-Start Gene Taq (Nippon Gene), Tks Gflex DNA Polymerase ( Takara Bio Co., Ltd.), KOD plus (Toyobo Co., Ltd.), and the like.

Table 1 Presence or absence of 5'-3' exonuclease activity and strand displacement activity of various DNA polymerases

The DNA ligase used in the present invention is preferably a thermostable DNA ligase because it must maintain its activity after amplification when all steps are performed in one step. Specific examples include Taq DNA Ligase (New England Biolabs), Ampligase Thermostable DNA Ligase (Air Braun), Pfu DNA Ligase (Agilent Technologies) and the like.

In the present invention, the reaction step (1) for amplifying the 5P artificial AB adduct using a group of 5P artificial addition specific primer pairs, the artificial nucleic acid sequence at the 3' end of the 5P artificial AB adduct using a 3' artificial sequence-introducing oligo pair Reaction step (2) for obtaining a 3′ artificial CD complementary addition product to which complementary strands of C and D are added, and 5′ of the 3′ artificial CD complementary addition product using a 5′ artificial sequence-introducing primer or a 5′ artificial sequence-introducing oligo For the reaction step (3) to obtain the final artificial sequence addition product with the artificial nucleic acid sequence (C or / and D) added to the 'end, setting three different temperature conditions for each is more efficient. Allows amplification and addition of artificial nucleic acid sequences.

Since the reaction step (1) is an amplification reaction, the amplification cycle consists of three steps: dissociation step, annealing step, and elongation step, or annealing and elongation are performed in one step, and two steps, the same step and the dissociation step, are performed. Desirably, 10 to 70 cycles of amplification are performed, preferably 30 to 60 cycles, more preferably 35 to 50 cycles.

When reacting in the order of (1) to (3) without the reaction steps (1) to (3) proceeding simultaneously, "(1) Among the sequences of the artificial addition specific primers that function in the reaction step (1) Tm value of the sequence complementary to the target nucleic acid", "(2) the Tm value of the sequence complementary to the 5P artificial AB adduct among the sequences of the 3' artificial sequence-introducing oligo that functions in the reaction step (2)", and In "(3) the Tm value of the sequence complementary to the 3' artificial CD complementary addition product among the 5' artificial sequence-introducing primers functioning in the reaction step (3)", (1) > (2) > (3) It is necessary to set the annealing temperature of each reaction step to be reaction step (1)>reaction step (2)>reaction step (3), and to provide a certain difference in each annealing temperature.

In the above-mentioned reaction system characterized in that the annealing temperature decreases as the reaction process shifts, the primer or oligo DNA that functions in the reaction process with a high annealing temperature is Theoretically it should work. In that case, at the stage of the reaction step where the annealing temperature is high, the primer or oligo DNA that functions in the reaction step is allowed to sufficiently proceed until reaching a saturation state (plateau), thereby lowering the annealing temperature. Functioning in the reaction step can be minimized. As a result, it is possible to create a reaction environment in which only the primers or oligo-DNAs that should function in each reaction step function properly, making it possible to clearly separate and carry out each reaction step.

Of the three reaction steps (1) to (3), when reaction steps (1) and (2) are reacted simultaneously and only reaction step (3) is separated and reacted from the above reaction steps, the reaction step The Tm value of the sequence complementary to the 5P artificial AB adduct among the sequences of the 3' artificial sequence-introducing oligo that functions in (2), and the 3' artificial sequence of the 5' artificial sequence-introducing primer that functions in the reaction step (3) Provide a certain or more difference between the Tm value of the CD complementary addition product and the complementary sequence, and the annealing temperature difference such that the annealing temperature in the reaction steps (1) and (2) > the annealing temperature in the reaction step (3) can be achieved by ensuring a certain level of

Further, of the three reaction steps (1) to (3), when the reaction steps (2) and (3) are reacted simultaneously, and only the reaction step (1) is separated and reacted from the reaction step, The Tm value of the sequence complementary to the target nucleic acid among the sequences of the artificial addition specific primer functioning in the reaction step (1), and the 5P artificial AB addition among the sequences of the 3′ artificial sequence introduction oligo functioning in the reaction step (2) Provide a certain or more difference between the Tm value of the product and the complementary sequence, and the annealing temperature in the reaction step (1) > the annealing temperature in the reaction steps (2) and (3). It can be achieved by ensuring

When the reaction steps are carried out separately as described above, the difference in Tm value may be 5 to 30°C, preferably 7 to 25°C, and more preferably 10 to 20°C. Also, the difference in annealing temperature between reaction steps may be 5 to 30°C, preferably 7 to 25°C, and more preferably 10 to 20°C (see Table 1).

The annealing temperature in the reaction step (3) needs to be set to a temperature at which the reaction proceeds sufficiently, and the temperature may be from 30° C. to the elongation temperature, preferably 40 to 65° C., more preferably. is preferably 50 to 60°C. Since the elongation temperature varies depending on the DNA synthetase used, it is recommended to adopt the recommended elongation temperature described in the instruction manual of each manufacturer.

However, depending on the target nucleic acid, it may be difficult to design an artificially added specific primer that satisfies the above conditions. It is possible to expand the range of applicable target nucleic acids by being freed from restrictions on addition-specific primer design.

The number of cycles for increasing the annealing temperature in the reaction step (1) may be 3 to 10 cycles, preferably 3 to 5 cycles, more preferably the second cycle in which the 5P artificial AB adduct is first generated. The next cycle, 3 cycles, is good.

In addition, between the Tm value of the sequence complementary to the 5P artificial AB adduct in the sequence of the 3′ artificial sequence-introducing oligo and the Tm value of the sequence complementary to the target nucleic acid in the sequence of the artificial addition specific primer, Under conditions where the temperature difference required to separate reaction step (1) and reaction step (2) is not guaranteed, the annealing temperature is increased even after the third cycle when the 5P artificial AB adduct functions as a template. If not, it is assumed that the reaction step (2) proceeds simultaneously with the reaction step (1), since the 3' artificial sequence-introducing oligo binds to the 5P artificial AB adduct. If there is a need to avoid the above situation, it is theoretically considered most favorable to increase the annealing temperature in reaction step (1) from the third cycle onwards.

The annealing temperature in the reaction step (1) before the change may be ±10°C, more preferably ±5°C, of the Tm value of the sequence complementary to the target nucleic acid of the artificial addition specific primer. good. On the other hand, the annealing temperature in the reaction step (1) after the change may be ±10° C., more preferably ±5° C., of the Tm value of the entire region of the artificial addition specific primer, with the elongation temperature being the upper limit. (Reason: If the annealing temperature is set to a temperature exceeding the elongation temperature, there is a concern that amplification failure may occur due to a decrease in the activity of DNA synthetase).

In the reaction step (2), after the 5P artificial AB adduct is dissociated, the temperature is changed to a temperature at which the 3′ artificial sequence-introduced oligo can bind to the 5P artificial AB adduct, and the temperature is maintained for a certain period of time to complete the reaction. Because it progresses, it is not always necessary to cycle the temperature multiple times as in PCR. However, the binding of the 3′ artificial sequence-introduced oligo to the 5P artificial AB adduct is inhibited by the binding between the 5P artificial AB adducts. By performing multiple temperature cycles configured at a temperature at which can bind to the 5P artificial AB adduct, the chances of binding between the 3′ artificial sequence-introducing oligo and the 5P artificial AB adduct are increased, and as a result, the 3′ In some cases, the amount of artificial CD complementary adduct can be increased. The temperature cycle is desirably 1 to 20 cycles, preferably 1 to 10 cycles, more preferably 1 to 5 cycles.

In the reaction step (3), the reaction proceeds only by setting the temperature at which the 5' artificial sequence-introducing primer or 5' artificial sequence-introducing oligo can bind to the 3' artificial CD complementary addition product, and maintaining the temperature for a certain period of time. Therefore, it is not always necessary to repeat the temperature cycle multiple times. However, even after carrying out the reaction step (2), if a certain amount of the 5P artificial AB addition product to which the complementary strands of the artificial nucleic acid sequences C and D are not added at the 3' end remains, in the reaction step (3) , an artificial nucleic acid sequence at the 5' end of not only the 5' end of the 3' artificial CD complementary addition product, but also the 5' end of the product in which the single strand of the 5P artificial AB addition product and the single strand of the 3' artificial CD complementary addition product are bound. In some cases, C and D are added, and the target amount of the final artificial sequence addition product cannot be obtained. In such a case, the temperature at which the 3' artificial sequence introduction oligo and the complementary strand of the product dissociate from the 3' artificial CD complementary addition product and the 5' artificial sequence introduction primer or the 5' artificial sequence introduction oligo 5′ artificial sequence-introducing primer or 5′ artificial sequence-introducing oligo and complementary artificial nucleic acid sequences C and D at the 3′ end by performing a temperature cycle consisting of a temperature capable of binding to the CD complementary addition product multiple times. It may be possible to increase the chances of binding of the 3' artificial CD complementary adduct to which the strand is added, thus increasing the final artificial sequence adduct. The temperature cycle is desirably 1 to 20 cycles, preferably 1 to 10 cycles, more preferably 1 to 5 cycles.

The first heat denaturation step before entering the temperature cycle of this method is different depending on the DNA polymerase used, but when using a chemically modified hot-start DNA polymerase, it is recommended to carry out at 94-98°C for 1-15 minutes. desirable. More preferably 1 to 10 minutes, still more preferably 2 to 8 minutes.

Regarding the heat denaturation step in the reaction step (1), although it varies depending on the DNA polymerase used, it is generally desirable to carry out at 90 to 98°C for 5 to 90 seconds, more preferably 5 to 60 seconds, and still more preferably 10 to 30 seconds. seconds.

The annealing step in the reaction step (1) is preferably carried out for 30 to 120 seconds because the artificial nucleic acid sequence A or B is commonly added to each 5P artificial addition specific primer. More preferably 60 to 120 seconds, still more preferably 60 to 90 seconds. The temperature is desirably −10° C. to +10° C., preferably −5° C., relative to the Tm of the portion complementary to the target nucleic acid of the 5P artificial addition specific primer (usually 55° C. to 65° C., preferably around 60° C.). ~+5°C.

The elongation step in the reaction step (1) depends on the length of the amplified region and the elongation speed of the DNA polymerase used, but it is usually desirably performed for 30 to 120 seconds, preferably 30 to 90 seconds, and more preferably. is 45-90 seconds. The temperature of the elongation step is usually 60-80°C, preferably 60-75°C, more preferably 65-75°C.

The heat denaturation step in the reaction step (2) is desirably carried out at 90 to 98° C. for 5 to 90 seconds, more preferably 5 to 60 seconds, still more preferably 10 to 30 seconds.

The annealing step in the reaction step (2) is preferably carried out for a relatively short time of 5 to 30 seconds, more preferably 5 to 15 seconds, in order to suppress hybridization of the 5P artificially added specific primer pair group. The temperature is desirably −10° C. to +10° C., preferably −5° C. to +5° C. relative to the Tm of the 5′ artificial sequence-introducing primer or 5′ artificial sequence-introducing oligo (usually 50 to 65° C.).

The elongation step in the reaction step (2) is desirably performed for 0 to 20 seconds, preferably 0 to 10 seconds, more preferably 0 to 5 seconds, since the elongation region is short. The temperature of the elongation step depends on the properties of the DNA polymerase used, but is usually 60-80°C, preferably 60-75°C, more preferably 65-75°C.

It is desirable that the Tm value of each primer pair in the 5P artificially added specific primer pair group has as little variation as possible. Specifically, it is desirable to be in the range of ±10°C, preferably ±5°C, more preferably ±3°C, relative to the average Tm (usually 45 to 75°C) of all primers.

The concentration of each primer pair in the 5P artificially added specific primer pair group is desirably in the range of 1-2000 nM, preferably 10-1000 nM, more preferably 40-500 nM. By adjusting the concentration of each primer pair in the 5P artificially added specific primer pair group in the sample solution, the amplification efficiency of each target gene can be made nearly uniform.

The concentration of the 3' artificial sequence-introducing oligo is preferably as high as possible so that the oligo can efficiently bind to the 5P artificial AB adduct. Specifically, it is desirably in the range of 2 to 4000 nM, preferably 20 to 2000 nM, more preferably 50 to 1000 nM.

The concentration of the 5' artificial sequence-introducing primer and 5' artificial sequence-introducing oligo is desirably in the range of 2-4000 nM, preferably 20-2000 nM, more preferably 50-1000 nM.

Amplification can be performed by the PCR method, the LAMP method, the NASBA method, the ICAN method, the LCR method, the Rolling Cycle method, the SMAP method, the PALSAR method, and the like.

Nucleic acids used for primers/oligos may be natural nucleic acids such as DNA and RNA, or 2',4'-BNA ^coc , 3'-Amino-2',4'-BNA, 2',4' - Artificial nucleic acids such as BNA ^NC (BNA is an abbreviation for Bridged Nucleic Acid), PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid), TNA (Throse nucleic acid), GNA (Glycol nucleic acid), etc. .

The above steps (1) to (3) can be carried out simultaneously in one reaction.

Also, according to the present invention, an artificial nucleic acid sequence can be added to one or more target nucleic acid sequences in a single reaction.

The present invention also provides kits for producing artificial sequence addition products, including the following (i) to (iii).
(i) an artificial addition specific primer (a) having a sequence complementary to the target nucleic acid sequence on the 3' end side and having at least a partial sequence of the artificial nucleic acid sequence A on the 5' end side; An artificial addition specific primer pair consisting of an artificial addition specific primer (b) having a sequence complementary to the target nucleic acid sequence on the 'end side and having at least a partial sequence of the artificial nucleic acid sequence B on the 5' end side and a 5P artificial addition-specific primer pair group characterized by comprising at least one pair of artificial addition-specific primer pairs in which at least one of the 5′ ends is phosphorylated,
(ii) The 3' end is labeled so that it is not extended by DNA polymerase, has at least a partial sequence of the artificial nucleic acid sequence A on the 3' end side, and the artificial nucleic acid sequence C on the 5' end side. The 3' artificial sequence introduction oligo (c) having the a 3' artificial sequence-introducing oligo pair comprising a 3' artificial sequence-introducing oligo (d) having sequence D; and
(iii) (a) and/or (b) below
(a) including both or one of a 5' artificial sequence-introducing oligo (e) having the entire sequence of artificial nucleic acid sequence C and a 5' artificial sequence-introducing oligo (f) having the entire sequence of artificial nucleic acid sequence D; 5' artificial sequence introduction oligo characterized by
(b) both or one of a 5' artificial sequence introduction primer (e) having a partial sequence of artificial nucleic acid sequence C and a 5' artificial sequence introduction primer (f) having a partial sequence of artificial nucleic acid sequence D 5' artificial sequence introduction primer characterized by comprising

The kit of the invention may further contain a DNA polymerase.

Elements such as primers, oligo DNA, and DNA polymerase that constitute the kit of the present invention are as described above.

The kit of the present invention may additionally contain reaction containers, reaction buffers, instructions for use, target nucleic acids for positive control tests, primers, oligo DNAs and the like.

The present invention will now be described more specifically with reference to Examples, but these Examples are merely illustrative of the present invention and are not intended to limit the present invention.

<Confirmation of detection sensitivity>
The final artificial sequence addition product was prepared in Prior Art 1, Prior Art 4, and the present invention, and the amount of the resulting product was quantified to compare the detection sensitivity of each method.
Genomic DNA derived from Escherichia coli K-12 strain was used as a template, the 16S rRNA gene was used as the target gene, and two target gene concentrations of 10 ³ copies/μl and 10 ¹ copies/μl were used. 10 μl of this was added per reaction. Table 2 shows the composition of the reaction solution.

Table 2 Reaction liquid composition

Table 3 shows the base sequences of the primers/oligo DNAs used.

Table 3 Nucleotide sequences of primers/oligo DNA used

The above reaction solution was subjected to reaction under the temperature conditions shown in the table below using a PCR amplification device (Life touch (manufactured by Nippon Genetics)).

Table 4 Temperature conditions of Example 1

The resulting amplified product was purified using AmpureXP (manufactured by Beckman Coulter, Inc.) and diluted 10,000-fold.

Next, using the above amplified product as a sample, real-time PCR using the NEBNext Library Quant Kit for Illumina (manufactured by New England Biolabs) was carried out according to the manual attached to the kit to obtain an amplified product with an adapter sequence (hereinafter referred to as Quantitation of adapter sequence-retained products) was performed. The quantitative value of the measurement is obtained as the copy number of the gene, and this quantitative value was converted to the weight concentration as the double-stranded final artificial sequence addition product. Rotor-Gene Q (manufactured by Qiagen) was used as a real-time PCR device.

Furthermore, real-time PCR using QProbe was performed using the same amplified product as the sample for quantification of the amplified product having the adapter sequence. In this quantification, since QProbe that is complementary to the amplification site of the target gene is used, only specific amplification products that have adapter sequences can be quantified. It is possible to The quantitative value of the measurement is obtained as the copy number of the gene, and this quantitative value was converted to the weight concentration as the double-stranded final artificial sequence addition product. Tables 5 and 6 show the composition of the reaction mixture (including the primers used and the sequences of QProbe) and the temperature conditions. Rotor-Gene Q (manufactured by Qiagen) was used as a real-time PCR device.

Table 5 Composition of the reaction solution for quantification of the final artificial sequence addition product (including primers used and QProbe sequences)

Table 6 Temperature conditions for quantification of the final artificial sequence adduct

The results are shown as Table 7.

Table 7 Amount of final artificial sequence adduct produced under each condition

From Table 7, it can be seen that prior art 1 has the largest amount of final artificial sequence adduct when the initial template concentration is 10 ⁴ copies/reaction. However, when the initial added template concentration was 10 ² copies/reaction, which was two orders of magnitude lower, the amount of the final artificial sequence addition product decreased significantly, showing a value lower than that of the present invention. Products with adapter sequences showed high values regardless of the initial template concentration added. This is because the primer used in prior art 1 is very long, around 70 bases, and when the initial template concentration is low, non-specific amplification products such as primer dimers are predominantly amplified, and this is the adapter sequence It was speculated that it was detected as a retained product.

It was shown that the amount of the final artificial sequence addition product of prior art 4 was the smallest regardless of the initial template concentration added. Although Prior Art 4 has the advantage of being able to prepare primers at low cost, the efficiency of conversion to the final artificial sequence addition product is extremely low, and this tendency is particularly pronounced when the initial template concentration is low. was shown.

On the other hand, in the present invention, the amount of the final artificial sequence adduct showed a high value regardless of the initial template concentration added. In particular, even when the initial template concentration was low, almost the same final artificial sequence addition product was obtained as when the template was added at a high concentration, and the quantification value was higher than that of the conventional technique.

From the above, it was shown that the method according to the present invention has a higher target gene detection sensitivity than the conventional technique.

In addition, the final artificial sequence addition product obtained in this study was selected one each from Prior Art 1, Prior Art 4, and the present invention, and the product was decoded with a next-generation sequencer (experimental method, etc. is Example 3 ). As a result, the sequences of all products agreed with the E. coli 16S rRNA gene sequence.

<Concentration adjustment of final artificial sequence adduct>
In the present invention, the concentration of the 5' artificial sequence-introducing primer is adjusted by varying the concentration of the 5' artificial sequence-introducing primer, preparing the final artificial sequence-added product, and quantifying the amount of the resulting product. We examined whether it is possible to control the amount of the final artificial sequence addition product.

Genomic DNA derived from E. coli K-12 strain was used as a template in the same manner as in Example ¹ , and the 16S rRNA gene was used as the target gene. 10 μl was added. The composition of the reaction solution was the same as in Table 2, except for the concentration of the 5' artificial sequence-introducing primer. The concentrations of the 5' artificial sequence-introducing primer were 25, 50, 75, 100, 150, 200 and 300 nM. The base sequences of the primers/oligo-DNAs used and the reaction temperature conditions were also the same as in Example 1.

The resulting amplified product was purified using AmpureXP (manufactured by Beckman Coulter, Inc.) in the same manner as in Example 1, and diluted 10,000-fold.

Using this diluted solution as a target, the total amount of specific product and the final artificial sequence addition product were quantified. The amount of the final artificial sequence addition product was quantified by the same method as in Example 1, and the total amount of the specific product was quantified by the same method as in Example 1 except for the primers used.

Table 8 shows the primers used in the quantification of total specific product.

Table 8 Primers for specific product total amount quantification

The sequence of the primer for quantification of the total amount of specific products is a sequence specific to the target gene. It is possible to quantify the total amount of specific products including those not introduced. Quantitative values for both the final amount of the artificial sequence addition product and the total amount of the specific product can be obtained in terms of gene copy number, and this quantitative value was converted to the weight concentration as the double-stranded final artificial sequence addition product.
The results are shown in FIG.

The total amount of specific products was almost constant regardless of the concentration of the 5' artificial sequence-introducing primer.

On the other hand, the amount of the final artificial sequence-added product increased in proportion to the added amount of the 5' artificial sequence-introducing primer up to a concentration of 100 nM. At 150 nM or more, the increase in the amount of the final artificial sequence addition product peaked out, but this is because the molar concentration calculated from the total amount of specific product was about 100 nM. was added, there was no target product into which the 5' artificial sequence was introduced, and the amount of the final artificial sequence-added product did not increase.

From the above results, the final artificial sequence addition product can be adjusted to the target concentration by adjusting the concentration of the 5' artificial sequence introduction primer within the range where the concentration of the target product into which the 5' artificial sequence can be introduced is the upper limit. was shown to be possible.

<Simultaneous amplification of dozens of items and dozens of specimens>
Using the human genomes of 48 people as a template, according to prior art 4 and the present invention, the amount of the final artificial sequence addition product is simultaneously amplified and prepared from 26 human genes in one reaction, and the product is used as the target for the next generation. Sequence analysis (hereinafter, NGS analysis) was performed.

As a template, 48 specimens of DNA extracted from swabs collected from human oral mucosa were used. DNA concentrations ranged from 1 to 4 ng/μl.

Table 9 shows the composition of the reaction solution of the present invention, excluding the final concentration of the artificially added specific primer. The composition of the reaction solution in Prior Art 4 is the same as in Example 1, except for the final concentration of the artificially added specific primer. The final concentrations of artificially added specific primers are listed in Table 10.

Table 9 Reaction solution composition of the present invention [Example 3]

In addition, Table 10 shows the artificially added specific primers used in the Examples, and Table 11 shows other primers or oligo DNAs.

Table 10 List of artificial addition specific primers and final addition concentrations used in Example 3

Table 11 List of other primers and oligo DNAs used in Example 3

Using a PCR amplifier (Life touch (manufactured by Nippon Genetics)), the final artificial sequence addition product was amplified and prepared. The temperature conditions are given in the table below (Table 12) for the present invention. The temperature conditions of Prior Art 4 are the same as those described in Example 1.

Table 12 Temperature conditions of Example 3

Subsequently, 2 μl of each amplified product was mixed in equal amounts, purified using AmpureXP (manufactured by Beckman Coulter), and subjected to NGS analysis (equipment used: Miseq; manufactured by Illumina).

The obtained base sequences were separated according to the barcode sequence, and the number of reads for each target gene in the sample was counted.

The results are shown in FIG. From this figure, it can be seen that reads were obtained for all 26 types of target genes in any embodiment.

However, in prior art 4, there were a plurality of specimens with 0 reads. Specifically, in the ABO261 gene, ABO703/796 gene, AGXT ProLeu gene, GPX1 gene, GYPA gene, and ITPA gene, the number of reads was 1, 1, 7, 2, 1, and 2, respectively. There were 0 specimens. On the other hand, in the present invention, the overall number of reads was large and the variation in the number of reads between samples was small.

Table 13 shows the average number of reads for each gene and its standard deviation.

The average number of reads was higher for the present invention than for prior art 4. This is presumed to be the result of reflecting the difference in artificial sequence introduction efficiency among the techniques.

On the other hand, the standard deviation (%) of the present invention showed a smaller value than Prior Art 4. This result shows that the present invention has less variation in the number of reads between genes than prior art 4.

Table 13 About the average number of reads and its standard deviation

<Effect of increasing the amount of final artificial sequence addition product produced by SL oligo with 3′ artificial sequence introduction>
Reaction steps (1) to (3) were carried out using a 3' artificial sequence-introducing oligo (3' artificial sequence-introducing SL oligo) having an intramolecular secondary structure at the 5' end and a blocker in the chain. A system (test system) and a system (control system) in which reaction steps (1) to (3) were performed using a normal 3' artificial sequence introduction oligo (control system), the final artificial sequence addition product was obtained. By quantifying the product, the effect of improving the production amount of the final artificial sequence-added product by the 3′ artificial sequence-introduced SL oligo was verified.

As a template, genomic DNA derived from Escherichia coli K-12 strain (genomic DNA extracted from Competent E. coli JM109 (Nippon Gene)) was used, and the 16S rRNA gene was used as the target gene. was used and 10 μl of this was added per reaction.

For the composition of the reaction solution, the 3′ artificial sequence-introducing oligo used was different between the test system and the control system, and the addition concentration of the 5′ artificial sequence-introducing primer was 150 nM. As specified in the column marked with . The 3' artificial sequence-introduced oligo (3' artificial sequence-introduced SL oligo) used in the test system is shown in the table below.

Table 14 SL oligo with 3′ artificial sequence introduced in the test system

In addition, the base sequences of the primers/oligo DNAs used are as specified in the column labeled "Invention" in Table 3 above, except that SL oligos with 3' artificial sequences introduced were used in the test system. Using a PCR amplification device (Life touch (manufactured by Nippon Genetics Co., Ltd.)), the above-described reaction solution was performed under the conditions indicated in the column "Present Invention" in Table 4 above.

The resulting amplified product was purified using AmpureXP (manufactured by Beckman Coulter) and diluted 10,000-fold.

Subsequently, using the diluted amplification product as a sample, the final artificial sequence addition product having adapter sequences (P1, P2) at both ends was quantified. The quantitative value of the measurement is obtained as the copy number of the gene, and this quantitative value was converted to the weight concentration as the double-stranded final artificial sequence addition product.

The primers used for this quantification, other reaction mixture compositions (including the QProbe sequence), and temperature conditions are as shown in Tables 5 and 6 above, respectively. Rotor-Gene Q (manufactured by Qiagen) was used as a real-time PCR device.

In addition, the amount of 5P artificial AB adducts was quantified in the same manner as in Example 2 using the same diluted solution as described above.
The results are shown as Table 15.

Table 15 Amount of 5P artificial AB adduct and quantitative results of final artificial sequence adduct

From Table 15 above, the amount of the final artificial sequence addition product in the test system using the 3' artificial sequence-introduced SL oligo having a secondary structure was obtained using a normal 3' artificial sequence-introduced oligo having no secondary structure. It was shown to be about 1.8 times more than the control system. From this result, it was confirmed that the 3' artificial sequence-introduced SL oligo having a secondary structure was effective in improving the yield of the final artificial sequence-added product.

<Reaction step (1) Confirmation of amplification under conditions where the annealing temperature is raised in the middle of the cycle>
Reaction step (1) The presence or absence of amplification of 5P artificial AB adducts under reaction conditions in which the annealing temperature was increased in the middle of the cycle was examined. In addition, the presence or absence of the formation of 3′ artificial CD complementary addition products in the reaction step (2) using the 5P artificial AB addition product as a template was examined.

Table 16 shows the reaction conditions for each test system (6 series).

The 5P artificially added specific primer pair and the 3' artificial sequence-introducing oligo were those described in the column labeled "Present invention" in Table 3. In this Example, no 5' artificial sequence-introducing primer was added. The composition of the reaction solution is as specified in the column labeled "Invention" in Table 2 above.

As a template, genomic DNA derived from Escherichia coli K-12 strain (genomic DNA extracted from Competent E. coli JM109 (Nippon Gene)) was used, and the 16S rRNA gene was used as the target gene. was used and 10 μl of this was added per reaction.

The reaction in the test system was carried out under the temperature conditions shown in Table 19 using a PCR amplifier (Life touch (manufactured by Nippon Genetics)).

The resulting amplified product was purified using AmpureXP (manufactured by Beckman Coulter) and diluted 10,000-fold.

Subsequently, using the diluted amplified product as a sample, the 5P artificial AB addition product was quantified by the same method as in Example 2, and the 3' artificial CD complementary addition product was quantified by the same method as in Example 4. did. The quantitative value of the measurement is obtained as the copy number of the gene, and this quantitative value was converted to the weight concentration as the double-stranded final artificial sequence addition product.

Other reaction mixture compositions (including the QProbe sequence) and temperature conditions used for this quantification are as shown in Tables 5 and 6 above, respectively. Rotor-Gene Q (manufactured by Qiagen) was used as a real-time PCR device.

Table 16 Oligos/primers used in Example 7 and reaction conditions for each test system

The results are shown as Table 17.

Table 17 Effect on the amount of various products when the annealing temperature in the reaction step (1) is changed in the middle of the cycle

In test system 1 in which the annealing temperature was constant at 63° C., which is the proper annealing temperature for the primer pair specific to the 5P artificial addition used in this example, the amount of 5P artificial AB addition products was confirmed as expected. Also, in test system 2 in which the annealing temperature was constant at 72°C, which is 5°C higher than the proper annealing temperature for the 5P artificial addition specific primer pair, no 5P artificial AB addition product was generated. On the other hand, after performing 2-cycle reaction at the proper annealing temperature (63°C) of the 5P artificial addition specific primer pair, 50-cycle reaction was performed at the annealing temperature (72°C) where amplification was not confirmed in test system 2. In test system 3, almost the same amount of 5P artificial AB adducts as in test system 1 was confirmed.

From this result, even if the annealing temperature is raised to a temperature (72° C.) at which normal PCR cannot be amplified in the next cycle (3rd cycle and later) after the 2nd cycle in which the 5P artificial AB addition product is first generated, It was possible to confirm that amplification occurred. This is because the 5P artificial AB addition product has a sequence complementary to the entire region of one 5P artificial addition specific primer pair at the 3′ end of the product, so that the 5P artificial Since the entire region of the addition-specific primer pair binds to the 5P artificial AB addition product, it was determined that PCR amplification was possible even if the temperature was raised to the annealing temperature at which amplification could not be confirmed in normal PCR cycles. .

Table 18 shows the estimated Tm value of the sequence region complementary to the target nucleic acid of the 5P artificial addition specific primer pair used in this example and the estimated Tm value of the entire region of the same primers.

Table 18 Sequences complementary to target nucleic acids of 5P artificial addition specific primer pairs, estimated Tm values for the entire region

From this table, the difference between the Tm value of the sequence complementary to the target nucleic acid and the annealing temperature after change (72 ° C.) is about 14 ° C. Therefore, the annealing temperature was set to 72 ° C. from the beginning of the reaction step (1). , it is assumed that amplification does not occur, as in test system 2 of this example. On the other hand, the estimated Tm value of the entire region of the 5P artificial addition specific primer pair is almost the same as the annealing temperature after the change. It was assumed that amplification would be confirmed even if the annealing temperature was increased to 72° C. in the presence of a 5P artificial AB adduct with the desired sequence. Thus, it was possible to explain the results in this Example also from the estimated Tm value of the artificially added specific primer.

Test systems 1 and 4, and test systems 3 and 6 have the same reaction step (1) conditions. About the same degree of 3′ artificial CD complementation product was detected in test systems 4 and 6, which added the introduction oligo. From this result, the 5P artificial AB adduct amplified under the conditions (test system 6) in which the annealing temperature is raised from the middle of the cycle in the reaction step (1) serves as a template for the reaction step (2), and the 3' artificial CD It was shown to be convertible into complementary adducts.

From the above results, the annealing temperature was increased within the temperature range where the entire region of the 5P artificial addition-specific primer pair can bind to the 5P artificial AB addition product from any cycle after the 3rd cycle of the reaction step (1). Even if it is, it became clear that the present invention can be implemented, and as a result, the above-mentioned merit (5P artificial addition specific primer pair design limitation [specifically, the annealing temperature in the reaction step (1) The temperature is set according to the Tm value of the sequence complementary to the target nucleic acid in the 5P artificially added specific primer pair, and the difference between this annealing temperature and the annealing temperature in reaction step (2) is set to a certain level or more. It was shown that it is possible to enjoy the advantage of being able to expand the range of applicable target nucleic acids because it is released.

INDUSTRIAL APPLICABILITY The present invention can be used as a novel method for preparing gene libraries.

<SEQ ID NOs: 1 to 128> These show the base sequences of the primers/oligo DNAs used in the Examples.

Claims (9)

A method for preparing a nucleic acid in which an artificial nucleic acid sequence is added to a target nucleic acid sequence (hereinafter referred to as an artificial sequence addition product), the method comprising the following steps (1) to (3).
(1) A primer pair in which at least a part of the sequence of the artificial nucleic acid sequence A or B is added to each primer pair specific to the target nucleic acid sequence, wherein , One or both 5' ends are phosphorylated by nucleic acid amplification using one or more pairs of artificial addition specific primer pairs (hereinafter referred to as 5P artificial addition specific primer pairs) phosphorylated at one or both 5' ends obtaining a specific product (hereinafter referred to as 5P artificial AB adduct);
(2) Using a pair of oligo DNA (hereinafter referred to as oligo pair with 3' artificial sequence introduction) characterized by the following three points, DNA polymerase is used to convert the 5P artificial AB addition product obtained in the step (1) above. A step of extending the 3' end and adding a complementary sequence of artificial nucleic acid sequence C or D (hereinafter referred to as 3' artificial CD complementary addition product) to the 3' end of the 5P artificial AB addition product, and feature 1: 5P artificial addition It has the same sequence as at least part of the base sequence of the artificial nucleic acid sequence A or B present in the specific primer pair on the 3′ end side.
Characteristic 2: It has an artificial nucleic acid sequence C or D added to the 5P artificial AB adduct on the 5′ end side.
Feature 3: The 3' end is labeled to prevent nucleic acid elongation by DNA polymerase.
(3) Performing either of the following two steps (a) or (b) to add an artificial nucleic acid sequence C or D to the 5' end of the 3' artificial CD complementary addition product Step (a) : When either one or both of the 3′ artificial CD complementary addition products generated in the step (2) are phosphorylated, when the products form a double strand, both Of the single-stranded regions occurring at the ends, at least one complementary to the single-stranded region at the end where the 5' end of the base pair at the site where the region is converted to double strands is phosphorylated The 3' artificial CD complementary addition product is ligated with DNA ligase to the oligo DNA (hereinafter referred to as 5' artificial sequence-introduced oligo) and the 3' artificial CD complementary addition product obtained in the step (2) above. A step of adding an artificial nucleic acid sequence C or D to the 5′ end of or step (b): Either one or both of the 3′ artificial CD complementary addition products generated in the step (2) are In the case of phosphorylation, when the products form a double strand, of the single-stranded regions generated at both ends, the 5' end of the base pair at the site where the region changes into a double strand is phosphorylated. At the oxidized end, at least one oligo DNA complementary to a part of the single-stranded region of the end (hereinafter referred to as a 5' artificial sequence-introducing primer) hybridizes with the 3' artificial CD complementary addition product. 3' artificial CD complementary addition is performed by filling the gaps generated during the above by an extension reaction with DNA polymerase, and then ligating the extended 5' artificial sequence-introducing primer and the 3' artificial CD complementary addition product with DNA ligase. Adding an artificial nucleic acid sequence C or D to the 5' end of the product The 5P artificial addition specific primer pair has a sequence complementary to the target nucleic acid sequence on the 3' end side and an artificial addition specific primer ( a) and an artificial addition specific primer (b) having a sequence complementary to the target nucleic acid sequence on the 3' end side and at least a partial sequence of the artificial nucleic acid sequence B on the 5' end side an artificial addition-specific primer pair, wherein at least one of the artificial addition-specific primer pairs is phosphorylated at the 5' end thereof;
The 3' artificial sequence-introducing oligo pair is labeled at the same end so that the 3' end is not extended by DNA polymerase, has at least a partial sequence of the artificial nucleic acid sequence A on the 3' end side, and has the 5' end A 3′ artificial sequence-introducing oligo (c) having an artificial nucleic acid sequence C on the side and a similarly labeled 3′ end, having at least a partial sequence of the artificial nucleic acid sequence B on the 3′ end side, 5 'Contains a 3' artificial sequence introduction oligo (d) having an artificial nucleic acid sequence D on the terminal side,
The 5' artificial sequence introduction oligo is both a 5' artificial sequence introduction oligo (e) having the entire sequence of artificial nucleic acid sequence C and a 5' artificial sequence introduction oligo (f) having the entire sequence of artificial nucleic acid sequence D. or including either
The 5' artificial sequence introduction primer is a 5' artificial sequence introduction primer (e) having a partial sequence of artificial nucleic acid sequence C and a 5' artificial sequence introduction primer (f) having a partial sequence of artificial nucleic acid sequence D. 2. The method of claim 1, including both or one. In addition to the features related to the 3' artificial sequence-introducing oligo pair described in claims 1 and 3, by using the oligo pair containing at least one 3' artificial sequence-introducing oligo having the following two features, the 3' When the artificial CD complementary addition product forms a double strand between the products, the binding efficiency of the 5' artificial sequence introduction primer pair to the single-stranded regions generated at both ends is improved, and the amount of the final artificial sequence addition product produced 3. A method according to either claim 1 or claim 2, wherein the is capable of increasing the .
Feature 1: A 3' artificial sequence-introduced oligo in which a sequence intended to artificially form an intramolecular secondary structure is introduced at the 5' end.
Feature 2: A 3' artificial sequence-introducing oligo in which a blocker is introduced into the 5' end of the sequence introduced at the 5' end of the 3' artificial sequence-introducing oligo described in Feature 1 above, or into the sequence strand. In the reaction step (1), the annealing temperature is raised within a range that does not adversely affect the amplification reaction of the reaction in the cycle following the cycle in which the 5P artificial AB adduct is first generated. The reaction step (1) is characterized in that the 3' artificial sequence-introducing oligo pair and/or the 5' artificial sequence-introducing primer that work in step (2) and thereafter do not function in the reaction step (1). The method according to any one of claims 1 to 3. The method according to any one of claims 1 to 4, wherein steps (1) to (3) are carried out simultaneously in one reaction. The method according to any one of claims 1 to 5, wherein the artificial nucleic acid sequence is added to one or more target nucleic acid sequences in one reaction. The method according to any one of claims 1 to 6, wherein the artificial nucleic acid sequence is added to one or more target nucleic acid sequences in one reaction. A kit for producing an artificial sequence addition product, comprising the following (i) to (iii).
(i) an artificial addition specific primer (a) having a sequence complementary to the target nucleic acid sequence on the 3' end side and having at least a partial sequence of the artificial nucleic acid sequence A on the 5' end side; An artificial addition specific primer pair consisting of an artificial addition specific primer (b) having a sequence complementary to the target nucleic acid sequence on the 'end side and having at least a partial sequence of the artificial nucleic acid sequence B on the 5' end side and a 5P artificial addition-specific primer pair group characterized by comprising at least one pair of 5P artificial addition-specific primer pairs in which at least one of the 5′ ends is phosphorylated,
(ii) The 3' end is labeled so that it is not extended by DNA polymerase, has at least a partial sequence of the artificial nucleic acid sequence A on the 3' end side, and the artificial nucleic acid sequence C on the 5' end side. The 3' artificial sequence introduction oligo (c) having the a 3' artificial sequence-introducing oligo pair comprising a 3' artificial sequence-introducing oligo (d) having sequence D; and
(iii) (a) and/or (b) below
(a) including both or one of a 5' artificial sequence-introducing oligo (e) having the entire sequence of artificial nucleic acid sequence C and a 5' artificial sequence-introducing oligo (f) having the entire sequence of artificial nucleic acid sequence D; 5' artificial sequence introduction oligo characterized by
(b) both or one of a 5' artificial sequence introduction primer (e) having a partial sequence of artificial nucleic acid sequence C and a 5' artificial sequence introduction primer (f) having a partial sequence of artificial nucleic acid sequence D 5' artificial sequence introduction primer characterized by comprising 9. The kit of Claim 8, further comprising a DNA polymerase.

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