JP5300335B2 - Highly efficient method for preparing mismatched base pair-containing nucleic acid fragments - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently selecting and preparing only a mismatched base pair-containing heteroduplex nucleic acid fragment from a mixture of homo- and hetero-double-stranded nucleic acid fragments. <P>SOLUTION: A nucleic acid amplification reaction is carried out by using two pairs of primers which are hybridized with two kinds of template nucleic acids, respectively, and in which in one primer pair, one of F/R primers carries a selective molecule and in the other primer pair, a primer in the opposite direction to the primer carries a selective molecule. After the degeneration, only a single-stranded amplified nucleic acid fragment carrying the selective molecule is selected and after annealing, a mismatched base pair-containing hetero-double-stranded nucleic acid fragment is prepared by using a detection reagent specifically associated with a mismatched base pair. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a method for efficiently preparing a heteroduplex nucleic acid fragment containing a mismatched base pair from a mixture of a plurality of types of nucleic acid fragments, and a method for efficiently detecting a single base substitution in a nucleobase sequence.

  There are various mutations in the genome sequence between individuals within a species. Most of them are mutations in which only one base is substituted (single base substitution). Among such single nucleotide substitutions, mutations present at a frequency of 1% or more in the species population are particularly referred to as single nucleotide polymorphisms (SNPs), and about one in every 1000 bases in the human genome. It is known to exist in proportions. Since single nucleotide polymorphisms are useful in obtaining information on disease susceptibility and drug responsiveness of individuals and in the development of tailor-made medicine, analysis thereof has been urgent in recent years.

  However, single nucleotide polymorphisms are present relatively randomly on the genome base sequence, and therefore it is very difficult to search for them. Conventionally, the restriction fragment length polymorphism (RFLP) method has been used to search for single nucleotide polymorphisms (Non-patent Document 1). Although this method can analyze the sunshade region, it has a problem that a single nucleotide polymorphism not associated with a restriction enzyme site cannot be detected. On the other hand, in order to exhaustively search for single nucleotide polymorphisms existing in a specific region, a method of determining all the nucleotide sequences in the region after positional cloning and identifying the mutated portion is used. (Non-Patent Document 2). However, this method has a problem that it takes a lot of time, labor, and cost to obtain a result because the area that can be analyzed at one time is narrow.

  In order to solve the above problem, recently, a mismatched base pair present in a chimeric double-stranded DNA in which heterologous complementary single-stranded DNAs are associated with each other, that is, a hetero-double-stranded DNA, is detected to detect a single base multiple. A new method of exhaustively searching for types has been developed (Patent Document 1, Non-Patent Document 3). This is a technique for detecting a single base substitution existing between individuals as a mismatched base pair by mixing the same genes of two individuals of the same species and annealing them after denaturation. However, in this method, in addition to the intended hetero double-stranded DNA in the annealing step, its own complementary strands are re-annealed. As a result, there was a problem that unnecessary homo double-stranded DNA was always mixed in the product. The amount of such homoduplex DNA contamination is theoretically half of the product after annealing. Therefore, contamination with homodouble-stranded DNA not only lowers the detection sensitivity of single base substitution, but also has a risk of causing erroneous identification of single base substitution. However, in order to selectively separate only the desired heteroduplex DNA from such a homo- and heteroduplex DNA mixture, it is necessary to go through extremely complicated steps, resulting in this new comprehensive one. The base polymorphism method was also in the current state of being not efficient.

Japanese Patent No. 3705975 Wyman AR, White R., 1980, Proc Natl Acad Sci U S A. 77 (11): 6754-8 Clifford R, Edmonson M, Hu Y, Nguyen C, Scherpbier T, Buetow KH., 2000, Genome Res. 10 (8): 1259-65 Sando S, et al., 2000, Nucleic Acid Symp Ser, 44: 119-120

  In view of the above problems, an object of the present invention is to provide a method for efficiently preparing only a heteroduplex nucleic acid fragment containing a mismatched base pair from a mixture of homo and heteroduplex nucleic acid fragments.

  Another object of the present invention is to provide a method for efficiently and / or exhaustively detecting the position and base of single base substitution existing between two kinds of nucleic acid base sequences.

  Furthermore, an object of the present invention is to provide a kit for detecting a single base substitution for efficiently and / or exhaustively detecting a single base substitution existing between heteroduplexes.

In order to achieve the above-described object, the present application provides the following inventions.
In one embodiment, the present invention provides (1) a nucleic acid amplification step in which a nucleic acid amplification reaction is performed using a template nucleic acid and a primer pair that hybridizes to the template nucleic acid in each of the two reaction systems, The template nucleic acid in the reaction system is derived from two different nucleic acid molecules of the same origin including the base sequences of the regions corresponding to each other, and in one reaction system, either the forward / reverse primer carries the selection molecule, In the other reaction system, the nucleic acid amplification step in which the primer in the opposite direction carries the selected molecule, (2) a single strand preparation step in which the nucleic acid fragment amplified in each reaction system is denatured into a single strand, (3) a selection step of selecting a single-stranded amplified nucleic acid fragment carrying the selected molecule using the characteristics of the selected molecule, and (4) mixing the single-stranded amplified nucleic acid fragments obtained in the respective reaction systems. You A mixing step, (5) a double-stranded preparation step in which single-stranded amplified nucleic acid fragments derived from the different sources are annealed to form double-stranded nucleic acid fragments, and (6) detection specifically associated with mismatched base pairs Provided is a method for preparing a heteroduplex nucleic acid fragment containing mismatched base pairs, which comprises a separation step of separating a heteroduplex nucleic acid fragment associated with the detection reagent using a reagent.

  In another embodiment, the present invention provides (1) two template nucleic acids derived from different sources and containing the corresponding region base sequences, and each of the template nucleic acids hybridizing, In either primer pair, either forward / reverse primer carries a selection molecule, and in the other primer pair, a primer in the opposite direction carries a nucleic acid amplification reaction using two pairs of primers carrying a selection molecule. A nucleic acid amplification step to be performed; (2) a single-stranded preparation step in which the amplified nucleic acid fragment is denatured into a single strand; and (3) a single-stranded amplified nucleic acid fragment carrying the selected molecule by utilizing the characteristics of the selected molecule. (4) a double-stranded preparation step in which a single-stranded amplified nucleic acid fragment derived from the different source is annealed to form a double-stranded nucleic acid fragment, and (5) a mismatch To provide a process for the preparation of heteroduplex nucleic acid fragments comprising mismatched base pairs, including a separation step of separating the heteroduplex nucleic acid fragments associated with detection reagents using a detection reagent that specifically relevant base pairs.

  Furthermore, the present invention determines the base sequence of each strand of the heteroduplex nucleic acid fragment obtained by the method for preparing a heteroduplex nucleic acid fragment containing the mismatched base pair, and a single base existing between the nucleic acid fragments. A single base substitution detection method for identifying a substitution is provided.

  The present invention also provides a kit for detecting a single base substitution using the method for preparing a heteroduplex nucleic acid fragment containing the mismatched base pair.

  According to the method for preparing a heteroduplex nucleic acid fragment containing a mismatched base pair of the present invention, only a heteroduplex nucleic acid fragment containing a mismatched base pair can be prepared with high efficiency.

  Moreover, according to the single base substitution detection method of the present invention, single base substitution existing between heteroduplexes can be detected efficiently and / or exhaustively.

  Furthermore, according to the kit for detecting a single base substitution of the present invention, a kit for efficiently and / or exhaustively detecting a single base substitution existing between heteroduplexes can be provided.

<Definition>
The definitions of the following terms used in this specification will be explained.

  The “template nucleic acid” is a nucleic acid molecule that serves as a template in this step. The nucleic acid molecule is usually DNA (including a single strand). For example, the DNA includes genomic DNA, cDNA, plasmid DNA, mitochondrial DNA or chloroplast DNA, or a fragment thereof. More specific examples of the template nucleic acid include a genomic library and a cDNA library. In addition, the template nucleic acid can also be RNA containing mRNA, hnRNA, tRNA, rRNA, snRNA, or a fragment thereof. However, when the template nucleic acid is RNA, the template nucleic acid once converted to cDNA by the reverse transcription reaction is used as the final template nucleic acid. The template nucleic acid is a nucleic acid molecule having a naturally derived sequence (including a chimeric sequence derived from two or more different kinds of organisms), and all or a part of the nucleotides of the naturally derived sequence is modified (for example, introduced by mutation by gene manipulation, etc.) Or a nucleic acid molecule to which a functional group is added), a nucleic acid molecule containing a base sequence (for example, a multicloning site) artificially synthesized on all or part of the naturally-occurring sequence, or a combination thereof. The length of the base sequence of the template nucleic acid is not particularly limited, but may be in the range of 60base to 5Mb (Mega base).

  “Nucleic acid molecules of the same origin” as used herein refers to nucleic acid molecules having a common source. “Common source” means, for example, but not limited to, the same source category such as genus, species, breed (subspecies), ethnicity, age, gender, individual, disease, and gene. Say. Therefore, specific examples of “two different nucleic acid molecules of the same origin” include the genomes of two different members (eg, individuals) belonging to the same species, the genomes of two different members belonging to the same ethnic group, and the same species Examples include the same type of genes of different members, or different genes that are homologous to each other within the same individual (ie, paralog).

  The “corresponding region” is a target region to be amplified in the present invention, and refers to a region homologous to each other between template nucleic acids derived from two different nucleic acid molecules of the same origin. For example, the same region between the genomes of two different individuals belonging to the same species, the entire region or a common partial region between the genes of the same species of two different individuals belonging to the same species, or between two different paralogs within the same individual This corresponds to the whole area or a part of the common area. Although the base length of the base sequence of the corresponding region is not particularly limited, it may be in the range of 30base to 5Mb. The corresponding region may be inserted into a vector such as a plasmid or phage. In addition, a tag sequence may be added to the 5 'end and / or the 3' end of the corresponding region. The base sequence of the corresponding region can contain the desired single base substitution.

  When used in the present invention, the sense strand / antisense strand refers to a strand complementary to a sense strand when one strand of a double-stranded nucleic acid is used as a sense strand. What is necessary is just to process as a sense strand the strand which has a 5 'terminal in the side which an implementer judges as 5' side (upstream side) in the said area | region.

  “Single base substitution” is a mutation in which one nucleotide is substituted with another nucleotide in a certain base sequence. For example, single nucleotide polymorphism (SNP) is typical of single nucleotide substitution.

  “Primer” is synonymous with a term used in the art. The primer of the present invention is composed of an oligonucleotide composed of DNA or RNA, or a combination thereof. These may contain artificial nucleic acid molecules such as LNA (Locked Nucleic Acid) and phosphorothioate type oligonucleotides as necessary. In addition, among the bases constituting the primer, any base that does not inhibit primer extension in the nucleic acid amplification reaction can be modified with an appropriate functional group (for example, a thiol group). Such a primer can be prepared by chemical synthesis by a technique known in the art. It is also possible to entrust a primer having a desired base sequence or modifying group to a nucleic acid synthesis contractor.

  The base sequence of each primer is not particularly limited as long as it hybridizes to a position where a desired nucleic acid region on the template nucleic acid can be amplified. In any primer, it is preferable to select a base sequence to constitute so that the melting temperature (Tm) is higher than the annealing temperature in the nucleic acid amplification reaction. The base length of the primer may be at least 10 to 50 bases, preferably 15 to 40 bases, more preferably 20 to 35 bases, in the region hybridizing to the template nucleic acid. Moreover, each primer may have an additional sequence of an appropriate length (5 to 50 bases) on the 5 'side as necessary. Such an additional sequence can introduce a restriction enzyme site, and can also function as a spacer sequence for binding a selective molecule or support described later to the 5 'end side. The length of the base sequence amplified between each primer pair may be appropriately determined according to the type and length of the template nucleic acid to be verified, the type of nucleic acid polymerase to be used, and the like. Preferably, it is in the range of 100 base to 3 kb (kiro base), more preferably 150 base to 2 kb, still more preferably 200 base to 1.5 kb.

  In addition, as the primer, for example, a multi-step primer such as a Nested primer can be used as necessary in order to increase the amplification rate or specificity. The use of such a clever primer may be appropriately determined by those skilled in the art within the technical scope known to those skilled in the art without departing from the configuration of the present invention described later.

  The forward / reverse notation in the primer of the present invention is based on the directionality of the base sequence of the corresponding region. That is, forward means the same direction as the sense strand of the corresponding region, and reverse means the same direction as the antisense strand of the corresponding region. As described above, the sense strand / antisense strand orientation of the corresponding region is the same between two template nucleic acids derived from different sources. Therefore, the directionality of the primer between both template nucleic acids is also the same. Here, the primer of the present invention is characterized in that either one of the forward / reverse primers carries a selective molecule.

  “Selected molecule” refers to a molecule that can be specifically selected using the properties of the molecule. As used herein, “molecule” includes a carrier, a compound, or a combination thereof. “Carrier” includes, but is not limited to, magnetic beads, polymeric polysaccharide supports (eg, Sepharose or Sephadex), silica, glass, metals (eg, gold, platinum, silver), plastics, ceramics, latex, etc. . The shape of the carrier may be any shape that does not inhibit the nucleic acid amplification reaction. Spherical particles such as beads are particularly preferable as the shape of the carrier because they have a large binding surface area and high operability. The “compound” includes a fluorescent dye (for example, cy3, cy5, FAM, HEX, VIC, TAMRA), a luminescent substance, or biotin or (strept) avidin. The “characteristic” here is a property peculiar to the selected molecule and is not limited, but includes, for example, magnetic force, specific gravity, fluorescence, luminescence, affinity, and the like. The selection molecule used in the present invention can be directly or indirectly bound to a nucleic acid, and its characteristics are inactivated, denatured and cleaved depending on the denaturation temperature of the nucleic acid amplification reaction and / or the denaturation conditions in the single-stranded preparation step. If it does not decompose, there is no particular limitation. A magnetic bead is preferable as a selection molecule of the present invention because it hardly reduces the magnetic force due to the above-described denaturation temperature and denaturation conditions, and also facilitates selective operability in the selection step described later.

  As a means for loading a selected molecule on a primer, that is, immobilization, it may be either direct or indirect. The support in the present invention is usually preferably an irreversible bond, that is, a bond that is once formed and does not dissociate by a reverse reaction, or a bond that can be disregarded. Examples of such irreversible bonds include covalent bonds by chemical reactions such as nucleophilic addition reactions between functional groups, nucleophilic substitution reactions or electrophilic substitution reactions, or bonds between biotin and avidin or streptavidin. Non-covalent bonds with high affinity can be mentioned. For example, in the case of a direct bond, a covalent bond by a chemical reaction can be achieved by modifying a primer and a selection molecule with a functional group, and forming a covalent bond by a chemical reaction between both functional groups. In this case, both functional groups should be a combination that can form a covalent bond. Examples of such combinations include an amino group and an aldehyde group, a thiol group and a maleimide group, an azide group and an acetylene group, an azide group and an amino group, a hydrazine group and a ketone group, and a hydrazine group and an aldehyde group. A method of forming a covalent bond by such a chemical reaction is a technique well known to those skilled in the art. For example, see the PIERCE manual. When a functional group is introduced into a primer, the position may be any position as long as it does not inhibit primer hybridization or extension. For example, a 5 'terminal and / or an internal base can be mentioned. Such functional groups can be introduced into the primer during primer synthesis. On the other hand, when it is desired to dissociate the selected molecule and the nucleic acid moiety as necessary during the preparation process or after the preparation according to the present invention, the support in the present invention can be made reversible. In this case, the selected molecule and the primer are strongly bound in a normal state (for example, under each step of the present invention), but dissociate only when treated under specific conditions (for example, treatment with an acid). You can do it. Alternatively, a restriction enzyme (for example, 8-base recognition restriction enzyme: AscI, FseI, NotI, SgfI, SrfI, SmiI, PmeI, PacI, etc.) with a low frequency of appearance is introduced into the spacer sequence of the primer, and prepared. Thereafter, the selected molecule and the amplified nucleic acid product may be separated by treatment with the restriction enzyme. The selective molecule can be carried at any site on the primer as long as it does not inhibit primer hybridization or extension. The 5 'end is preferred.

<Embodiment 1>
The first embodiment of the present invention relates to a method for preparing a heteroduplex nucleic acid fragment containing mismatched base pairs. In this embodiment, two template nucleic acids containing base sequences of corresponding regions derived from different sources are divided into (1) each template nucleic acid to perform a nucleic acid amplification reaction, or two template nucleic acids are mixed. After carrying out the nucleic acid amplification reaction, it is divided into two template nucleic acids. (2) Each is denatured into a single strand, the sense strand of one template nucleic acid is separated, and the sense strand in the other template nucleic acid is further separated. (3) Annealing the sense strand and the antisense strand to prepare a duplex, and using a detection reagent specifically associated with the mismatched base pair, A method for preparing a heteroduplex nucleic acid fragment containing mismatched base pairs, comprising separating a heteroduplex nucleic acid fragment associated with the detection reagent.

  One embodiment of the present invention is illustrated in FIG. As shown in this figure, the method for preparing a heteroduplex nucleic acid fragment containing mismatched base pairs of this embodiment includes a nucleic acid amplification step 11, a single strand preparation step 12, a selection step 13, a mixing step 14, and a double strand preparation. Step 15 and separation step 16 are included. In FIG. 1, the S chain represents the sense strand, the A chain represents the antisense strand, and the two template nucleic acids 1 are indicated by a thin line and a thick line, respectively. In this figure, the position of the other template nucleic acid corresponding to the CG base pair of one template nucleic acid is mutated to a GC pair by single base substitution. Hereinafter, this embodiment will be described in detail with reference to the drawings.

The nucleic acid amplification step “nucleic acid amplification step 11” is a step of performing a nucleic acid amplification reaction using the template nucleic acid 1 and the primer pair (31/41) that hybridizes to the template nucleic acid in each of the two reaction systems. . In each reaction system, the template nucleic acid is derived from two different nucleic acid molecules of the same origin including the base sequences of regions corresponding to each other.

  “Nucleic acid amplification reaction” refers to a reaction in which a specific nucleic acid region is amplified by a nucleic acid polymerase. The nucleic acid amplification method used in this reaction uses forward / reverse primers, and any method known in the art can be used as long as both sense / antisense strands of the template are amplified by these primers. Also good. For example, a PCR (polymerase chain reaction) method, an ICAN (isothermal gene amplification) method, or an application method thereof (for example, a real-time PCR method) can be mentioned. The PCR method is preferred. This is the most widely used method in the field of molecular biology all over the world. Reagents, kits, reaction devices, etc. are extensive, and various applied technologies are known. Because.

  The nucleic acid polymerase used in the reaction is appropriately determined depending on the nucleic acid amplification method to be used, and is usually a DNA polymerase, particularly a heat resistant DNA polymerase. Various types of such nucleic acid polymerases are commercially available, and they can also be used. However, in the present invention, it is preferable to use a nucleic acid polymerase having high fidelity. This is because a new mutation is not introduced in the nucleic acid amplification step. For example, in the case of a DNA polymerase, it is preferable to use a DNA polymerase having 3 'to 5' exonuclease activity. In general, as the nucleic acid region to be amplified becomes longer, the incidence of unintended mutations introduced during the nucleic acid amplification process increases. Therefore, when amplifying 500 bases or more, it is desirable to use pfu DNA polymerase or a DNA polymerase having high fidelity equivalent to or higher than that.

  The reaction conditions for the nucleic acid amplification reaction are based on the known reaction conditions for the nucleic acid amplification method used, the length of the base sequence to be amplified, the amount of nucleic acid for template, the Tm value of the primer, and the optimum reaction temperature for the nucleic acid polymerase to be used. And it may be determined in consideration of requirements such as optimum pH. For example, in the case of PCR, the temperature and reaction time of each step are as follows: the heat denaturation step at 90 ° C. to 98 ° C. for 30 seconds to 1 minute, and the annealing step at 52 ° C. to 60 ° C. for 30 seconds to 1 minute. May be performed at 70 ° C. to 75 ° C. for about 40 seconds to 3 minutes. The number of cycles greatly depends on the above-mentioned requirements, but usually 25 cycles to 45 cycles are sufficient. It is preferably 28 cycles to 42 cycles, particularly preferably 30 cycles to 40 cycles. These are carried out in the reaction system.

  In this embodiment, the conditions for the nucleic acid amplification reaction in the two reaction systems are not necessarily the same. Appropriate reaction conditions can be set for each reaction system within the technical scope of those skilled in the art according to the conditions (for example, concentration and length of base sequence) of each template nucleic acid.

  In this step, with respect to the primer pair (31/41) in each reaction system, one of the forward / reverse primers carries the selection molecule 2 in one reaction system, and the reverse primer in the other reaction system Is characterized by carrying a selective molecule. Therefore, in one reaction system (the first reaction system in FIG. 1), the antisense strand side occurs as a single-stranded nucleic acid fragment carrying the selection molecule 2, and from the other reaction system (the second reaction system in FIG. 1) The sense strand side is generated as a single-stranded nucleic acid fragment carrying the selection molecule 2.

  The template nucleic acid in each reaction system of this embodiment can have a common base sequence in a region other than the corresponding region. For example, the case where the corresponding region is inserted into a common vector can be mentioned. In addition, each base sequence of the primer pair can be the same between the two reaction systems. That is, at least two types of primers, that is, a forward primer and a reverse primer may be used. In this case, as described above, the forward / reverse primer carrying the selection molecule is reversed between the two reaction systems. Furthermore, it should be noted that if the primer hybridizes outside the corresponding region in the template nucleic acid, the direction of the base sequence of the corresponding region in the template nucleic acid is determined. That is, it is necessary that the base sequences of the corresponding regions are in the same direction in the two template nucleic acids. This is because when the base sequence of the corresponding region is inserted in the opposite direction in each template nucleic acid, a chain carrying a selective molecule in the same direction is generated from both reaction systems. Orientation of the corresponding region in the template nucleic acid can be easily achieved, for example, by inserting the corresponding region between two types of restriction enzyme sites having different cleavage sites on the vector. .

  You may refine | purify the obtained amplified nucleic acid fragment as needed after completion | finish of this process to a single strand preparation process. Unreacted deoxynucleotides and primers, or nucleic acid polymerase can be removed by purification. The purification method may be any method known in the art. For example, a purification method using a spin-type gel filtration column is preferable because nucleic acid can be purified quickly and easily. Such columns are commercially available from companies in the bio-related business, and they can also be used.

The single-stranded preparation step “single-stranded preparation step 12” is a step of denaturing the amplified nucleic acid fragment into a single strand 6. It is desirable to completely denature the double-stranded amplified nucleic acid fragment in the reaction system in this step.

  As the denaturing method, any method known in the art may be used. Conventionally known modification methods include a heat modification method or a modification (alkali modification) method by treatment with an alkaline solution. Among them, the heat denaturation method is preferable because the amplified nucleic acid fragment is less likely to be scratched like Nick and the neutralization treatment is unnecessary and simple.

  Thermal denaturation is only required to heat-treat nucleic acid fragments in a double-stranded state. The denaturation temperature depends on the GC content and / or base length of the target double-stranded amplified nucleic acid fragment, and may be adjusted as appropriate according to these conditions. Usually, if it is set at 96 ° C. or higher, it can be completely denatured regardless of the above conditions. Similarly, since the heat denaturation time depends on the GC content and / or base length of the double-stranded amplified nucleic acid fragment and the denaturation temperature, it is appropriately adjusted according to these conditions. Usually, the above purpose can be achieved by heating at 95 ° C. for 10 to 20 minutes and at 98 ° C. for 5 to 10 minutes.

  The single-stranded amplified nucleic acid fragment prepared in this step must be kept in a single-stranded state until at least the single-stranded amplified nucleic acid fragment carrying the selected molecule is selected in the following selection step. Therefore, a treatment for maintaining a single-stranded state is appropriately performed according to the denaturing method. For example, in the case of heat denaturation, a method of performing a selection step immediately after heating and before annealing due to a temperature decrease, or a method of quenching by immediately transferring the reaction system to ice, etc. Further, in the case of alkali modification, a method of adding a denaturing agent such as urea before, during or after modification. The method of performing the selection step immediately after heat denaturation is particularly preferable because it is simple and rapid.

The selection step “selection step 13” is a step of selecting a single-stranded amplified nucleic acid fragment carrying the selection molecule using the characteristics of the selection molecule.

The “characteristics of the selected molecule” are as described in the “Definition” above. “Selecting a single-stranded amplified nucleic acid fragment carrying the selection molecule using characteristics” means, for example, using a magnet 7 as shown in FIG. 1 if the characteristics of the selection molecule are magnetic. The specific gravity is selected by centrifugation (including density gradient centrifugation), the fluorescence is selected by a fluorescence detector, or the affinity is selected by binding based on the affinity. Each specific selection method may be appropriately determined based on the characteristics of the selected molecule using techniques known to those skilled in the art, and is not particularly limited. For example, if the selection molecule is a magnetic bead, after the single-strand preparation step, a single-strand amplified nucleic acid fragment and a primer carrying the selection molecule are brought into contact with the wall of the reaction tank in which each reaction system is carried. Examples include a method in which a liquid phase containing a single-stranded amplified nucleic acid fragment that does not carry a selective molecule and a template nucleic acid is attracted to, removed by decantation, or removed from the wall while being attracted to the wall surface. If the selected molecule is sepharose beads, after the single strand preparation step, the reaction system is centrifuged at an appropriate centrifugal acceleration to precipitate the single-stranded amplified nucleic acid fragment and primer carrying the selected molecule, and the liquid phase is A method of removing by suction is mentioned. Furthermore, if the selected molecule is biotin, the reaction solution after the single-strand preparation step is transferred to a reaction system in which avidin or streptavidin is immobilized, and after binding both molecules, the liquid phase is aspirated, decanted, or A method of washing and removing by distribution is mentioned. In this case, it is preferable to use a biotin- (strept) avidin bond capable of reversible binding. This is because the target single-stranded amplified nucleic acid molecule immobilized on the wall of the reaction vessel is recovered after the selection step. A commercially available biotin or (strept) avidin reagent capable of such reversible binding can be used. Examples thereof include DSB-X ^™ biotin (Invitrogen).

The mixing step “mixing step 14” is a step of mixing single-stranded amplified nucleic acid fragments obtained from each reaction system in one reaction system. By this step, amplified nucleic acid fragments obtained in each reaction system can be encountered. In FIG. 1, although the mixing process 14 is performed after the selection process 13, it is not restricted to this. That is, after the nucleic acid amplification step 11 and before the double strand preparation step 15, it may be performed after any step. Preferably after the selection step 13. It is preferable to quantitate the single-stranded amplified nucleic acid fragments obtained in each reaction system before mixing, and adjust so that equal amounts of the single-stranded amplified nucleic acid fragments in both reaction systems are mixed. By this operation, the single-stranded amplified nucleic acid fragment derived from each reaction system can be efficiently annealed in the double-stranded preparation step 15.

The double-stranded preparation step “double-stranded preparation step 15” is a step of forming a hetero-double-stranded nucleic acid fragment by annealing single-stranded amplified nucleic acid fragments derived from two different nucleic acid molecules of the same origin. By this step, a hetero double-stranded nucleic acid fragment is formed.

  In this step, it is preferable to anneal the base sequence of the corresponding region of the single-stranded amplified nucleic acid fragment as accurately and completely as possible between the hetero-complementary strands. Such annealing can be achieved, for example, by gradually lowering the temperature in the solution after the selection step. If the concentration of the desired single-stranded amplified nucleic acid fragment present in the solution is sufficiently high, the annealing can be achieved even if the temperature is lowered to room temperature and then kept at that room temperature. When a large number of unintended single-stranded amplified nucleic acid fragments are mixed in the solution, or when the concentration of either one of the hetero-complementary strands is extremely low, the annealing is performed at 65 to 70 ° C, preferably 68 ° C. It is preferable to achieve this by holding at temperature for a long time.

The separation step “separation step 16” is a step of separating a hetero double-stranded nucleic acid fragment associated with the detection reagent using a detection reagent specifically associated with the mismatched base pair. By this step, only those containing mismatched base pairs can be selected from the heteroduplex nucleic acid fragments obtained in the double-stranded preparation step.

  A “detection reagent specifically associated with a mismatched base pair” refers to a substance that specifically recognizes (for example, by steric distortion) a mismatched base pair present in a double-stranded nucleic acid and associates with it. Here, “relation” means to be involved, for example, binding or adhesion. The detection reagent is not particularly limited as long as it is a substance that can specifically recognize and relate to mismatched base pairs. Examples of such detection reagents include mismatch repair enzymes (eg, MutY, MutM, MutH, MutL, MutS, MutD, MutT, etc.) (Modrich P., 1989, J Biol Chem. Apr 25; 264 (12): 6597-600), and mismatch-binding ligand NC-link (Mismatch-binding Ligand NC-link: MBL) (see Patent Document 1, JP-A 2006-104159). Of these, MBL is particularly preferred. Multiple types of MBL are known, and depending on the type, there are mismatched base pairs that are specifically related, such as G (guanine) -G, GA (adenine), C (cytosine) -T (thymine), CA, or TT. Different. Which MBL should be used may be appropriately determined according to the purpose and application. Of course, a plurality of MBLs having different mismatched base pairs to be recognized can be mixed and used.

  The detection reagent may be used alone or may be immobilized on a carrier. The solid phase referred to here is not particularly limited as long as the detection reagent is fixed to a substance serving as a carrier. For example, a state in which the detection reagent and the carrier are bonded by chemical bonding (for example, covalent bonding) can be mentioned. The carrier is not particularly limited, and examples thereof include resins (for example, plastics such as polystyrene, polyacrysamide, etc.), polymeric polysaccharides (for example, agarose, sepharose, or sephadex), silica, glass, and ceramics. Further, the shape of the carrier is not particularly limited. For example, a bead shape or a porous particle shape is preferable because it has a large surface area and can be easily packed in a column.

  The method for separating nucleic acid fragments with a detection reagent is not particularly limited, and may be carried out in accordance with a known method in consideration of the conditions of the present invention. For example, there is an affinity separation method in which the sample after the double-strand preparation step is applied to a column packed with beads in which a detection reagent is immobilized (Goto Y, et al., 2007, Anal Bioanal Chem., 388). (5-6): 1165-1173; see Peng T, et al., 2004, Nucleic Acid Symp Ser (Oxf), 48: 123-124). Alternatively, a fluorescent substance is supported on the detection reagent, the sample after the double-strand preparation process treated with the detection reagent is washed, and then a double-stranded nucleic acid fragment having a fluorescent substance (a particulate molecule such as a sepharose bead on the selected molecule). A carrier carrying a carrier is preferable) may be separated using a cell sorter. As an example, a specific method for separating heteroduplex nucleic acid fragments associated with MBL using MBL is described in Example 3.

  According to the present embodiment, a heteroduplex nucleic acid fragment containing a mismatched base pair can be prepared very efficiently even after going through a nucleic acid amplification step. That is, according to the conventional method, the homoduplex nucleic acid fragment not containing about half of the mismatched base pairs was included in the final product through the nucleic acid amplification step (see the result section of Example 3). However, according to this embodiment, it is possible to remove 100% of such homoduplex nucleic acid fragments, and theoretically, a nucleic acid fragment consisting only of a heteroduplex nucleic acid fragment containing mismatched base pairs is prepared. be able to. As a result, the screening efficiency and analysis efficiency of single base substitution can be improved, and a substantially comprehensive analysis becomes possible. Moreover, according to this aspect, a nucleic acid amplification reaction and a double strand preparation can be performed efficiently. In addition, it is sufficient that at least two kinds of primers are necessary for the nucleic acid amplification reaction, and base sequences of corresponding regions derived from two different nucleic acid molecules of the same origin may be inserted at the same position in the same vector. It is also convenient for using existing genomic libraries and cDNA libraries.

<Embodiment 2>
An example of another embodiment of the present invention is shown in FIG. As shown in this figure, the basic principle of the second embodiment is not different from that of the first embodiment. That is, the second embodiment also includes a nucleic acid amplification step 21, a single strand preparation step 22, a selection step 23, a double strand preparation step 24, and a separation step 25, as in the first embodiment. However, the second embodiment is different from the first embodiment in that a nucleic acid amplification reaction is performed by mixing two template nucleic acids derived from different sources in the nucleic acid amplification step 21, and therefore the mixing step 14 is not required. . Therefore, the description of the above-described steps and the like that are common to the first embodiment will be omitted, and here, the nucleic acid amplification step 21 that is mainly characteristic of the second embodiment will be described below with reference to the drawings. The embodiment will be described in detail. In FIG. 2, as in FIG. 1, the S strand represents the sense strand, the A strand represents the antisense strand, and the two template nucleic acids 1 are indicated by thin lines and bold lines, respectively. Further, the position of the other template nucleic acid corresponding to the CG base pair of one template nucleic acid is mutated to a GC pair by single base substitution.

The nucleic acid amplification step “nucleic acid amplification step 21” is a step of performing a nucleic acid amplification reaction using a template nucleic acid and a primer pair in each of the two reaction systems. That is, the nucleic acid amplification step of the above aspect is characterized in that the nucleic acid amplification reaction is performed separately for each template nucleic acid, whereas the nucleic acid amplification step of the present aspect is performed by mixing two template nucleic acids and amplifying the nucleic acid. It differs in that it reacts.

  In this embodiment, two independent nucleic acid amplification reactions are performed in one reaction system. As described above, the corresponding region has the same or similar base sequence in two template nucleic acids derived from different sources. Therefore, two pairs of primers are required to independently carry out the nucleic acid amplification reaction for each template nucleic acid, and each template nucleic acid has a nucleotide sequence region in which one pair of primers can specifically hybridize. It must be in the desired position. This can be done, for example, by changing the type of vector (for example, plasmid) into which the corresponding region is inserted between the template nucleic acids, or by changing the tag sequence added to the end of the corresponding region between the template nucleic acids. Can be easily achieved.

  The “two pairs of primers” can amplify a desired nucleic acid region independently using each of two template nucleic acids derived from different sources as templates. Each primer has a different base sequence. Therefore, at least four types of primers in this embodiment are necessary. In addition, it is preferable to design the primers in advance so that the lengths of the base sequences amplified by each of the two primer pairs are the same or substantially the same in both. This is to make the yields of both nucleic acid amplification products as equal as possible in a single reaction system.

  A characteristic point of Embodiment 2 is that, in the two pairs of primers, one of the forward / reverse primers of one primer pair carries a selection molecule, and the primer in the opposite direction is supported in the other primer pair. It is carrying a selective molecule. That is, of two template nucleic acids derived from different sources, when a forward primer carries a selection molecule in a primer pair that hybridizes to one template nucleic acid, a primer pair that hybridizes to the other template nucleic acid Then, the reverse primer carries the selected molecule. Also in the nucleic acid amplification step, as in the first embodiment, a sense strand carrying a selected molecule is obtained from one template nucleic acid, and an antisense strand carrying a selected molecule is obtained from the other template nucleic acid.

The nucleic acid amplification reaction conditions and the like may be performed according to the nucleic acid amplification step of the first embodiment.
According to the present embodiment, it is possible to reduce work time and cost. Furthermore, in this embodiment, since all reactions can be performed in one reaction system in principle, the operation is simple and economical.

<Single base substitution detection method>
In the detection method of the present invention, the base sequence of each strand of the fragment is determined using the heteroduplex nucleic acid fragment obtained by the preparation method of Embodiment 1, and the result is analyzed to be present between the nucleic acid fragments. This is accomplished by identifying a single base substitution.

  Prior to determining the base sequence of each strand of the obtained hetero double-stranded nucleic acid fragment, a linker sequence that hybridizes a sequence primer to both ends of the fragment may be added by ligation reaction, if necessary. As a method of adding a linker sequence to the terminal, a technique known in the art can be used.

  If necessary, the obtained hetero double-stranded nucleic acid fragment can be subcloned prior to base sequence determination. For subcloning, any method known in the art may be used. For example, the hetero double-stranded nucleic acid fragment obtained in Embodiment 1 or 2 is inserted into a vector such as a TA cloning vector and then introduced into Escherichia coli, and a single transformant clone is isolated and cultured, and then the target The method of preparing a vector is mentioned. In this method, two types of vectors having insert fragments derived from respective strands of mismatched base pairs are obtained in a single clone. Therefore, when the automatic sequencer is used, the target mismatch site is detected as two overlapping different signal peaks. In the present invention, since the nucleic acid polymerase having high fidelity is used, the end of the hetero double-stranded nucleic acid fragment obtained after the nucleic acid amplification step may not have an A (adenine) overhang. This can be easily solved by, for example, reacting the fragment with dNTP or dATP, Taq polymerase and the like at about 72 ° C. for several minutes in the absence of a primer, and adding A to the 3 ′ end.

  The base sequence may be determined using a technique well known in the art. Usually, a base sequencing method based on the dideoxy method (Sanger method) is used. Reagents used for sequencers and nucleotide sequences use commercially available equipment (eg 3130 Genetic Analyzer (ABI), CEQ8000 (BECKMAN COULTER), etc.) and reagents (eg BigDye (registered trademark) Terminator (ABI)) can do. About the method of base sequence determination, what is necessary is just to follow the manual attached to the sequencer and reagent to be used.

  According to the detection method of the present invention, single-base substitutions present in genomes and genes can be detected. In particular, this detection method can comprehensively search and identify unknown single nucleotide polymorphisms present in genomes and genes, point mutations in causative genes such as genetic diseases or pest resistance. Thereby, not only can information on disease susceptibility and drug responsiveness be provided more efficiently than in the prior art, but it can also contribute to the development of genetic diagnosis and plant variety improvement.

<Single base substitution detection kit>
The single-base substitution detection kit of the present invention includes the primer and / or detection reagent described in Embodiment 1 or 2, and detects the single-base substitution using the preparation method described in Embodiment 1 or 2. Can do. In addition, although not limited, reagents necessary for nucleic acid amplification reaction (for example, nucleic acid polymerase, dNTP, buffer), materials used for selection of selected molecules (for example, magnets if selected molecules are magnetic beads), templates Vectors used for the preparation of nucleic acids for nucleic acids, nucleic acid purification instruments (for example, gel filtration columns), containers such as various tubes used as reaction systems, and / or instruction manuals for kits can also be included.

  The purpose of this kit is to provide materials, reagents, and / or instruments necessary for preparing a heteroduplex nucleic acid fragment containing a mismatched base pair as a final product. Therefore, a commercially available base sequencing reagent or a kit thereof may be used as a reagent or the like for identifying the single base substitution by sequencing the obtained hetero double-stranded nucleic acid fragment. For example, BigDye (registered trademark) Terminator (ABI) can be used. However, if necessary, this kit may include a nucleotide sequencing reagent.

  What is necessary is just to define suitably about the usage frequency (minute) amount of a primer, a detection reagent, etc. which are included in a kit.

  ADVANTAGE OF THE INVENTION According to this invention, the hetero double stranded nucleic acid fragment containing the mismatched base pair used for single base substitution detection can be obtained simply and easily.

  EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are only specific illustrations of the present invention, and do not limit the technical scope of the present invention.

<Preparation of template nucleic acid>
(the purpose)
As a template nucleic acid, a single base substitution is introduced at a specific site of a plasmid.

(Materials and methods)
Single-base substitution was introduced into plasmid pBR322 using Quik Change (registered trademark) Site-Directed Mutagenesis Kit (Stratgene). PCR was performed using 50 ng of pBR322 in 5 μl of 10 × PCR buffer, 5 μl of 2 mM dNTP mixture, 2.5 μl of each 5 pmol / μl primer (SEQ ID NO: 1 and 2), 1 μl of 2.5 U / μl PfuTurbo® DNA polymerase And a total volume of 50 μl were mixed and run using a thermal cycler (DNAengine: BIORAD). The reaction conditions were denaturation at 95 ° C. for 30 seconds, 18 cycles of 95 ° C. for 30 seconds, 55 ° C. for 1 minute, 68 ° C. for 5 minutes 30 seconds, and then placed at 4 ° C. The synthesis of each primer used was outsourced to Operon.

  After completion of the PCR, 1 μl of methylated DNA-specific restriction enzyme 10 U / μl DpnI attached to the kit was added, and the reaction was performed at 37 ° C. for 1 hour. Using 1 μl of the obtained DpnI-treated sample, E. coli competent cells attached to the kit were transformed according to the instructions attached to the kit. After culturing the transformant, a plasmid was extracted from Escherichia coli using QIAprep Spin Miniprep Kit (manufactured by QIAGEN) according to the instructions attached to the kit.

  The base sequence of the extracted plasmid was determined, and introduction of a single base substitution was confirmed. The base sequence was determined using the DTCS Quick Start Master Mix (BECKMAN COULTER) and the sequencer CEQ8000 (BECKMAN COULTER) in accordance with the company's instruction manual.

(result)
From the base sequence, the 796th base of the target pBR322 (Genbank (ACCESSION N0 .: J01749)
It was confirmed that a single base substitution product obtained by substituting C with G for the first base of the registered sequence (the first base), that is, a single base substitution product was obtained. The plasmid into which this single base substitution was introduced was named pBR322M, and was used as a template nucleic acid together with pBR322 in the following examples.

<Preparation of primer>
(the purpose)
A magnetic bead-carrying primer to which magnetic beads as selective molecules are added and a normal primer that does not carry magnetic beads are prepared.

(Materials and methods)
In order to activate magnetic beads M-PVA N11 (Chemagen), the following operation was performed. First, it transferred the beads 100μl a (3.75μmol NH ₂ / 5mg) in 1.5ml tubes, to remove suspended extracellular fluid for storage using a magnet. Next, the beads remaining in the tube were suspended in 100 μl of PBS-EDTA (20 mM Na—PO 4, 150 mM NaCl, EDTA 1 mM pH 7.2) solution, and then the solution was removed again using a magnet. After this operation was repeated twice, the beads were suspended in 100 μl of 50 mM sulfo-LC-SPDP (Pierce) solution, which is a hetero-2 crosslinking reagent. Subsequently, the tube was incubated at room temperature for 60 minutes, after which the solution was removed using a magnet and resuspended in 200 μl of PBS-EDTA. This operation was repeated 5 times, and finally suspended in 375 μl of PBS-EDTA solution to obtain 10 nmol / μl of sulfo-LC-SPDP-bead suspension.

  Hybridization was carried out at a specific site of pBR322 or pBR322M serving as a template, and forward / reverse primers composed of the nucleotide sequences shown in SEQ ID NOs: 3 and 4, respectively, were synthesized (referred to as “PBRF” and “PBRR”, respectively). At this time, for each of the above primers, a primer having a thiol group introduced at the 5 ′ end for the preparation of a magnetic bead-carrying primer and a primer having no thiol group at the 5 ′ end were prepared for the preparation of a normal primer. The synthesis was outsourced to Operon.

  The thiol group-introduced oligonucleotide was subjected to a deprotection group treatment before adding the magnetic beads. A specific method of deprotecting group treatment is as follows. First, the dried thiol group-introduced oligonucleotide was dissolved in 500 μl of 100 mM 1,4-dithio-DL-threitol and allowed to stand at room temperature for 30 minutes, after which 500 μl of MQ water was added. A Sep Pak Plus C18 column (manufactured by Waters) was previously washed with 10 ml of acetonitrile, and equilibrated by flowing 10 ml of 2M TEAA. The entire amount of the thiol group-introduced oligonucleotide was applied to this column. The Pass fraction was reapplied and then washed with 10 ml of 5% acetonitrile / 0.1M TEAA solution. Subsequently, it was washed with 10 ml of MQ water. Finally, the column was eluted with 3 ml 30% acetonitrinyl. The resulting eluate was lyophilized and redissolved with MQ water. The concentration of the resulting thiol group-introduced oligonucleotide solution was measured and adjusted to 0.25 nmol / μl.

  The magnetic bead-carrying primer was prepared by reacting the thiol group-introduced oligonucleotide with the activated magnetic beads according to the following procedure. First, 40 μl of the thiol group-introduced oligonucleotide solution (0.25 nmol / μl) and 10 μl of the sulfo-LC-SPDP-bead suspension (10 nmol / 1 μl) were mixed in a tube and then incubated at room temperature for 18 hours. Next, the solution in the tube was removed using a magnet, and the remaining beads were suspended in 100 μl of PBS-EDTA (composition as described above) solution. This operation was repeated 5 times. Finally, the suspension was resuspended in 100 μl of PBS-EDTA to obtain a 100 pmol / ml magnetic bead primer. The magnetic beaded forward / reverse primers are called “Magnet-PBRF” and “Magnet-PBRR”, respectively.

  The normal primers were prepared so that the synthesized lyophilized oligonucleotide was 10 pmol / μl with MQ water for each of the forward / reverse primers.

<Preparation of heteroduplex nucleic acid fragment containing mismatched base pairs>
(the purpose)
It is verified that a heteroduplex nucleic acid fragment containing the target mismatch base pair can be prepared using the method for preparing a heteroduplex nucleic acid fragment containing a mismatch base pair of the present invention.

(Materials and methods)
1. Nucleic acid amplification step PCR was carried out in two tubes using each primer prepared in Example 2 using pBR322M and pBR322 prepared in Example 1 as template nucleic acids. In each tube, 1 μl of template nucleic acid is converted into 5 μl of 10 × PCR buffer (attached to AmpliTaq Gold Polymerase below), 4 μl of 2.5 mM dNTP mixture, 3 μl of 25 mM MgCl ₂ , 5 μl of 100 pmol / μl magnetic beads. A total volume of 50 μl was mixed with the primer, 2.5 μl of 10 pmol / μl normal primer, and 0.25 μl of 5 U / μl DNA polymerase. Here, in the first tube, pBR322 was used as a template nucleic acid, and PBRF / Magnet-PBRR was used as a normal primer / magnetic bead primer combination. On the other hand, in the second tube, pBR322M was used as the template nucleic acid, and Magnet-PBRF / PBRR was used as the magnetic bead primer / normal primer combination. As the DNA polymerase, AmpliTaq Gold Polymerase (Applied Biosystems) was used for both tubes. The reaction conditions are as follows: heat treatment at 95 ° C. for 10 minutes, 30 cycles of 95 ° C. for 30 seconds, 60 ° C. for 30 seconds and 72 ° C. for 2 minutes, followed by treatment at 72 ° C. for 8 minutes. , Placed at 4 ° C. These reactions were performed using DNAengine (BIORAD).

2. Single-stranded preparation step to double-stranded preparation step After the nucleic acid amplification step, the sample in each tube was incubated at 96 ° C. for 5 minutes to denature the amplified double-stranded nucleic acid fragment into a single strand (single-stranded preparation). Process). Then, immediately press the magnet against the tube wall, adsorb the nucleic acid amplification fragment or primer carrying the magnetic beads in the reaction solution, or only the magnetic beads on the wall, and remove all the solution in the tube by aspiration (selection) Process). Subsequently, the samples remaining in the first and second tubes were mixed (mixing step). Here, in order to confirm the success or failure of amplification by PCR, the obtained mixture was electrophoresed on an agarose gel. As a result, a band of the desired size was confirmed (data not shown). Next, the single-stranded amplified nucleic acid fragment mixed in the mixing step was annealed to reconstruct the double-stranded nucleic acid fragment (double-stranded preparation step). For annealing, the mixed sample was incubated at 96 ° C. for 10 minutes, and then the temperature was gradually lowered to 25 ° C. at a rate of 0.2 ° C./min. Thereafter, the solution was subjected to ethanol precipitation, and the resulting precipitate was dissolved in 20 μl of MQ water. This was used as a double-stranded nucleic acid fragment sample.

3. Separation process In order to separate heteroduplex nucleic acid fragments containing mismatched base pairs from the double-stranded nucleic acid fragments obtained after the double-stranded preparation process, affinity chromatography using a solid-phase detection reagent is performed on the double-stranded nucleic acid fragments. did. As a detection reagent, Mismatch-binding Ligand NC-link (MBL) (see Patent Document 1) was used. This MBL has the ability to bind to GG mismatch. The detection reagent was synthesized according to the method described in Patent Document 1. MBL was immobilized by the following procedure. First, a column packed with a solid support HiTrap NHS-activated HP (GE Healthcare) was washed with 6 ml of cooled 1 mM hydrochloric acid. Next, 1.0 ml of 1.78 mg / ml MBL dissolved in a solution of 0.5 M NaCl / 100 mM NaHCO _{3 was} immediately injected onto the column. The outlet screw was closed and left at room temperature for 30 minutes. Then, the column was washed with 6 ml of 0.5 M NaCl / 100 mM NaHCO ₃ , and then washed with 6 ml of 0.5 M NaCl / 20 mM Na—PO ₄ (pH 7.0). Washing with these solutions was repeated twice, followed by washing with 6 ml of 0.5 M NaCl / 50 mM NaOH solution, followed by washing with 0.5 M NaCl / 20 mM Na—PO 4 (pH 7.0). This was used as an MBL solid phase column.

90 μl of the double-stranded nucleic acid fragment sample was diluted 100 to 100000 times with 2 ng / μl of ColE1 DNA solution and applied to the column. After the application, the column was washed with 6 ml of 0.5 M NaCl / 20 mM Na—PO ₄ (pH 7.0) /0.01% NaN ₃ and eluted with 6 ml of 0.5 M NaCl / 50 mM NaOH. The eluate (6 ml) was neutralized by adding 12 μl of acetic acid. The column was washed with 6 ml of 0.5 M NaCl / 20 mM Na—PO 4 (pH 7.0) /0.01% NaN ₃ .

The pre-application sample (diluted solution) and the eluted sample (neutralized eluate) were each quantified by real-time PCR. PRISM7700 (ABI) was used as a measuring instrument. As a kit, SYBR Premix Ex Taq ^™ (Perfect Real Time) (TAKARA) was used. Plasmid pBR322 was used as a standard for the template sample, and PBRF and PBRR were used as primers. For PCR, 5 μl of 10 × PCR buffer of pBR322, 5 μl of 2 mM dNTP mixture, 2.5 μl of each 5 pmol / μl primer (SEQ ID NOs: 3 and 4), 1 μl of 2.5 U / μl PfuTurbo® DNA polymerase 50 μl Was mixed up. A thermal cycler (DNAengine: BIORAD) was used. The reaction conditions were 50 ° C for 2 minutes (Ramp time: automatic), 95 ° C for 10 minutes (Ramp time: automatic), 95 ° C for 5 seconds (Ramp time: automatic), and 60 ° C for 30 seconds (Ramp time: automatic). (time: automatic) 40 cycles of 1 cycle, then 95 ° C for 15 seconds (Ramp time: automatic), 60 ° C for 20 seconds (Ramp time: automatic), 95 ° C for 15 seconds (Ramp time: 20 seconds) After that, it was finally treated at 20 ° C. for 1 minute (Ramp time: automatic).

4). Comparative control experiment by the conventional method PCR was performed using pBR322 and pBR322M described in Example 1 as templates, and PBRF and PBRR as primers. The reaction conditions and the like were in accordance with the method described in Example 3. Amplification products obtained from each template were quantified, equal amounts were mixed and denatured at 98 ° C., and then slowly cooled to reconstitute the duplex. In the same manner as in “3. Separation step”, this was diluted with a ColE1 DNA solution and applied to the MBL-immobilized column. The column was washed and then eluted with alkali. Thereafter, the pre-application sample and the eluted sample were each quantified by real-time PCR.

(result)
FIG. 3 shows the results of the method for preparing heteroduplex nucleic acid fragments containing mismatched base pairs of the present invention, and FIG. 4 shows the results of the conventional method.

  In FIG. 3, it can be seen that almost equal amounts of the pre-application sample and the eluted sample are recovered at any dilution ratio. On the other hand, in FIG. 4, about 20% of the sample before application was recovered as an elution sample at each dilution rate.

  Here, when an equal amount of a wild-type DNA fragment and the DNA fragment having a single base substitution are mixed and annealed after heat denaturation, the mutant sense, which is usually a heteroduplex nucleic acid fragment, is probabilistic. Of wild-type and wild-type antisense strands, wild-type sense strands and mutant-type antisense strands, and homo- and double-stranded nucleic acid fragments of mutant sense strands and mutant-type antisense strands and wild-type sense strands and wild-type antisense strands Four types will be obtained equally. As described above, MBL binds to GG mismatch base pairs but does not bind to CC mismatch base pairs. Therefore, in the conventional method, theoretically, 1/4 of the whole, that is, 25% binds to MBL (the hetero-duplex nucleic acid fragment containing mismatched base pairs itself is 1/2 of the whole). . In fact, the result of FIG. 4 showed a value close to this theoretical value (about 20%).

  In contrast, in the method for preparing a heteroduplex nucleic acid fragment containing a mismatched base pair according to the present invention, the heteroduplex nucleic acid fragment is selected from the four types of double strand nucleic acid fragments in the selection step prior to the separation step by MBL. In other words, the sense / antisense strand constituting either the mutant sense strand and the wild type antisense strand or the wild type sense strand and the mutant antisense strand is specifically selected. When only two types of templates are used as in this example, 100% of the nucleic acid fragments contained in the pre-application sample theoretically bind to MBL at the time after the double-stranded preparation step that has passed through the selection step. This is a heteroduplex nucleic acid fragment containing mismatched base pairs. In fact, FIG. 3 showed a result that almost matched this theoretical value.

  As described above, according to the present invention, it was proved that a heteroduplex nucleic acid fragment containing a target mismatch can be prepared with an ultimate efficiency of 100% or almost 100%.

<Verification as a single nucleotide polymorphism screening model>
(the purpose)
A single nucleotide polymorphism screening model is created to verify the usefulness of the present invention in single nucleotide polymorphism screening.

(Materials and methods)
1. Preparation of Library Model Two types of DNA libraries were prepared using the mutation-introduced plasmid pBR322M prepared in Example 1 and the wild-type plasmid pBR322.

  First, the two plasmids were separately digested with the same two types of 4-base recognition restriction enzymes. Sau3AI (TAKARA) and NlaIII (New England Bio Labs.) Were used as restriction enzymes. To tube 1, 15.0 μl of 666 ng / μl pBR322M, 10 μl of 10 U / μl Sau3AI, 10 μl of 10 × H buffer attached to Sau3AI at the time of purchase were added, and the volume was made up to 100 μl with MQ water. Further, 6.4 ng / μl of pBR322M, 16.4 μl, 10 μl of 10 U / μl Sau3AI, 10 μl of 10 × H buffer attached to Sau3AI at the time of purchase were added to tube 2, and the volume was increased to 100 μl with MQ water. Both tubes were incubated at 37 ° C. for 2 hours to carry out the enzyme reaction. The sample was then purified by ethanol precipitation. Specifically, 10 μl of 3M sodium acetate and 275 μl of ethanol were added to the solution, allowed to stand at −80 ° C. for 30 minutes, and then centrifuged at 15000 rpm for 5 minutes at 10 ° C. After centrifugation, the supernatant was removed, and the precipitate was washed with 500 μl of 70% ethanol, and then the precipitate was dried by suction. Subsequently, digestion with NlaIII, which is the second restriction enzyme, was performed. First, 10 μl of 10 U / μl NlaIII and 10 μl of 10 × NEB4 buffer attached to NlaIII at the time of purchase were added to the total amount of the dried precipitate (Sau3AI digested product) in each tube, and the volume was increased to 100 μl with MQ water. . Both tubes were incubated at 37 ° C. for 2 hours to carry out the enzyme reaction. The sample was then purified by ethanol precipitation in the same manner as above.

Next, a library plasmid vector was prepared in order to insert the digested fragment.
PUC19 was used as a plasmid vector for the library. BamHI and SphI (both from TAKARA) were used as restriction enzymes. Add 32.5 μl of 154 ng / μl pUC19, 5 μl of 10 U / μl BamHI, 5 μl of 10 U / μl SphI to the tube, add 10 μl of 10 × H buffer attached to either enzyme at the time of purchase, and 100 μl with MQ water I made a female up. The tube was incubated at 37 ° C. for 2 hours to carry out the enzyme reaction.

  The obtained BamHI / SphI digested pUC19 (library plasmid vector) and the Sau3AI / NlaIII digested pBR322M and pBR322 fragments (insert fragment) were each dissolved in MQ water, and then the DNA concentration was measured and used for ligation. It was. Ligation Kit Ver2.0 (TAKARA) was used for the ligation reaction. The reaction was performed by adding 1 μl of 113.5 ng / μl BamHI / SphI digested pUC19, 415 ng / μl of Sau3AI / NlaIII digested pBR322M (or) fragment 0.5 μl, and Ligation Kit soln I 1.5 μl and mixing at 16 ° C. Incubated for 30 min. After incubation, the ligation sample was used directly for transformation of E. coli. First, 3 μl of 0.5 ml ligation sample was dispensed into a microtube, and 30 μl of Escherichia coli Competent High E. coli DH5 (manufactured by TOYOBO) was added. Next, after incubation on ice for 30 minutes, a heat shock was performed at 42 ° C. for 40 seconds. 300 μl of SOC Medium was added thereto and incubated at 37 ° C. for 1 hour, then the entire amount was inoculated into 10 ml of Circle Grow culture solution (BIO101) supplemented with 100 μg / ml ampicillin and cultured at 37 ° C. for 16 hours. . Thereafter, plasmid DNA was extracted from the culture broth using 9 ml of the culture broth using QIAprep Spin Miniprep Kit (QIAGEN). The extraction method followed the attached instruction manual. The plasmid solutions obtained for the respective inserts of pBR322M and pBR322 fragments were designated as pBR322M library and pBR322 library.

In order to confirm whether or not both of the prepared libraries could be prepared as intended, the following test was performed. That is, PCR was performed using a plasmid pair of the above library as a template and a primer pair that hybridizes to a base sequence located at a position sandwiching the insertion region on the vector. The primers are DNAs described in SEQ ID NOs: 5 and 6 (respectively PUCMCSF and PUCMCSR: corresponding to the forward / reverse sequence in the multicloning site of pUC19), and AmpliTaq Gold Polymerase (Applied Biosystems) is used as the DNA polymerase. did. In order to confirm that the region mutated in Example 1 in pBR322M was included in the prepared library, a PBRF and PBRR primer pair was also used. Each primer pair was mixed so that the forward / reverse primer was 5 pmol / μl. All of the PCR reactions were performed using 1 μl library, 5 μl 10 × PCR buffer (attached to AmpliTaq Gold Polymerase), 3 μl 25 mM MgCl ₂ , 4 μl 2.5 mM dNTP mixture, 5 μl 5 pmol / μl primer pair, 0.25 μl of 5U / μl AmpliTaq Gold DNA polymerase was diluted to 50 μl and mixed. The reaction conditions are as follows: heat treatment at 95 ° C. for 10 minutes, 30 cycles of 95 ° C. for 30 seconds, 60 ° C. for 30 seconds and 72 ° C. for 2 minutes, followed by treatment at 72 ° C. for 8 minutes. , Placed at 4 ° C. These reactions were performed using DNAengine (BIORAD).

  Each obtained PCR product was analyzed by agarose gel electrophoresis. As a result, it was confirmed that the inserted fragment was smeared by SEQ ID NOs: 5 and 6 (not shown), and the desired polyclonal library was prepared. In addition, a band corresponding to the region was also confirmed in the electrophoresis result for the region into which mutation was introduced by using the PBRF and PBRR primer pair (not shown).

2. Screening model experiment of heteroduplex nucleic acid fragment containing mismatched base pair The present invention is supposed to be used for the purpose of screening unknown single nucleotide polymorphisms. Therefore, the following test was performed as a model closer to a state in which it is actually used. That is, only a fragment having the substitution was selectively screened according to the present invention from a library (pBR322M library) in which only one place in several thousand base pairs was substituted by one base.

First, magnetic beads were carried on both the PUCMCSF and PUCMCSR primers represented by SEQ ID NOs: 5 and 6. Magnetic beading followed the method described in Example 2. The completed magnetized primers were named Magnet-PUCMCSF and Magnet-PUCMCSR, respectively. A PCR reaction was performed for the purpose of confirming that the magnetized primer supported magnetic beads. As the template nucleic acid, a plasmid mixture prepared from the prepared library was used. AmpliTaq Gold Polymerase (Applied Biosystems) was used as the DNA polymerase. In addition, the primer pairs were Magnet-PUCMCSF / PUCMCSR and PUCMCSF / Magnet-PUCMCSR2 pairs, and the reaction was performed in separate tubes (tubes A and B). In each tube, 1 μl of template nucleic acid 5 μl 10 × PCR buffer (provided with AmpliTaq Gold Polymerase), 4 μl 2.5 mM dNTP mixture, 3 μl 25 mM MgCl ₂ , 5 μl 100 pmol / μl magnetic bead primer Then, 2.5 μl of 10 pmol / μl normal primer and 0.25 μl of 5 U / μl AmpliTaq Gold DNA polymerase were mixed to a total volume of 50 μl. The reaction conditions are as follows: heat treatment at 95 ° C. for 10 minutes, 30 cycles of 95 ° C. for 30 seconds, 60 ° C. for 30 seconds and 72 ° C. for 2 minutes, followed by treatment at 72 ° C. for 8 minutes. , Placed at 4 ° C. These reactions were performed using DNAengine (BIORAD). After the nucleic acid amplification reaction, the double-stranded DNA fragment obtained in each tube was incubated at 96 ° C. for 5 minutes to make a single strand. The tube lid was opened and the magnet was immediately pressed against the wall of the tube to adsorb the beads, and then all the liquid phase was removed by suction. Subsequently, the single-stranded DNA solution obtained from tubes A and B was mixed. When the mixture was electrophoresed, a PCR product having a target size was detected (not shown), and it was confirmed that the primer was magnetized normally.

  Next, using these magnetized primers, a nucleic acid amplification reaction by PCR was performed using each of the prepared two libraries (pBR322M library and pBR322 library) as a template nucleic acid. The PCR reaction followed the reaction conditions used in the confirmation of the magnetized beads. For the primer pair, Magnet-PUCMCSF / PUCMCSR was used for the tube (tube I) using the pBR322M library as a template, and PUCMCSF / Magnet-PUCMCSR was used for the tube (tube II) using the pBR322 library as a template. After the nucleic acid amplification reaction by PCR, the double-stranded DNA fragments obtained in each tube were each incubated at 100 ° C. for 3 minutes to be single-stranded. The tube lid was opened and the magnet was immediately pressed against the wall of the tube to adsorb the beads, and then all the liquid phase was removed by suction. Subsequently, 50 μl of each single-stranded DNA solution obtained from tubes I and II was mixed. At this time, many kinds of nucleic acid amplification products derived from the two libraries are mixed in the mixed sample. Annealing was carried out over a long period of time so that they formed exactly two strands with the complementary strands. That is, the mixture was denatured at 96 ° C. for 10 minutes using a thermal cycler (DNAengine: BIORAD), cooled to 96-67 ° C. at 0.1 ° C./second, and then kept at 67 ° C. for 18 hours. Thereafter, it was cooled to 67-25 ° C. at 0.1 ° C./second and completely annealed by placing it at 25 ° C. In the case of the present example which is a screening model, it is known that the length of the nucleic acid amplification fragment containing the target mismatch base pair is 240 bp.

Subsequently, double-stranded nucleic acid fragments containing mismatched base pairs were separated from the annealed samples by affinity chromatography using MBL described in Embodiment 3. Specifically, after pre-washing the column with 6 ml of 0.5 M NaCl / 20 mM Na—PO ₄ (pH 7.0) /0.01% NaN ₃ , 100 μl of the annealing sample was applied to the column. After washing with 6 ml of 0.5 M NaCl / 20 mM Na—PO ₄ (pH 7.0) /0.01% NaN ₃ , alkali elution was performed with 6 ml of 0.5 M NaCl / 50 mM NaOH. The eluate was neutralized with 12 μl acetic acid. Since the double-stranded nucleic acid fragment containing the desired mismatched base pair in the eluate is expected to be small, it was amplified by PCR. This is an operation for confirming the presence or absence of the obtained double-stranded nucleic acid fragment by electrophoresis. As the primer pair, a primer that was not made into magnetic beads (that is, PUCMCSF / PUCMCSR) was used. PCR reaction conditions were the same as in Example 4. The obtained amplification product was analyzed by agarose gel electrophoresis.

(result)
The results are shown in FIG. As shown in the figure, the obtained amplification product matched the expected size of 240 bp of the nucleic acid fragment containing the desired mismatched base pair. From the above, it was demonstrated that only fragments having a single base substitution can be selectively screened from the library using the present invention.

Conceptual diagram of Embodiment 1 of the present invention Conceptual diagram of Embodiment 2 of the present invention Results from the method for preparing heteroduplex nucleic acid fragments containing mismatched base pairs of the present invention Results of preparation of heteroduplex nucleic acid fragments containing mismatched base pairs by conventional methods Results in a single nucleotide polymorphism screening model using the present invention

Explanation of symbols

1: Nucleic acid for template 2: Selection molecule (magnetic bead)
31: Forward primer 1
32: Forward primer 2
41: Reverse primer 1
42: Reverse primer 2
5: Double-stranded amplified nucleic acid fragment 6: Single-stranded amplified nucleic acid fragment
7: Magnet 8: Hetero double-stranded nucleic acid fragment 9: Detection reagent

Claims (10)

A method for preparing a heteroduplex nucleic acid fragment containing mismatched base pairs, comprising:
(1) a nucleic acid amplification step of performing a nucleic acid amplification reaction using a template nucleic acid and a primer pair that hybridizes to the template nucleic acid in each of the two reaction systems,
The template nucleic acid for each reaction system is derived from two different nucleic acid molecules of the same origin containing the base sequences of the corresponding regions, and in either reaction system, either the forward / reverse primer carries the selection molecule. In the other reaction system, the nucleic acid amplification step in which the primer in the opposite direction carries the selected molecule,
(2) a single-strand preparation step of denaturing the nucleic acid fragment amplified in each reaction system into a single strand;
(3) a selection step of selecting a single-stranded amplified nucleic acid fragment carrying the selection molecule using the characteristics of the selection molecule;
(4) A mixing step of mixing the single-stranded amplified nucleic acid fragments obtained in the respective reaction systems,
(5) a double-stranded preparation step in which single-stranded amplified nucleic acid fragments derived from the different sources are annealed to form a double-stranded nucleic acid fragment, and (6) a detection reagent specifically associated with the mismatched base pair is used. The preparation method comprising a separation step of separating a hetero double-stranded nucleic acid fragment associated with the detection reagent. A method for preparing a heteroduplex nucleic acid fragment containing mismatched base pairs, comprising:
(1) Two template nucleic acids derived from different sources and containing the base sequence of the corresponding region, and hybridizing to each of the template nucleic acids, and in either primer pair, one of the forward / reverse primers A nucleic acid amplification step in which a nucleic acid amplification reaction is carried out using two pairs of primers that carry a selection molecule, and in the other primer pair, a primer in the opposite direction carries a selection molecule;
(2) a single strand preparation step for denaturing the amplified nucleic acid fragment into a single strand;
(3) a selection step of selecting a single-stranded amplified nucleic acid fragment carrying the selection molecule using the characteristics of the selection molecule;
(4) a double-stranded preparation step in which single-stranded amplified nucleic acid fragments derived from the different sources are annealed to form a double-stranded nucleic acid fragment, and (5) a detection reagent specifically associated with the mismatched base pair is used. The preparation method comprising a separation step of separating a hetero double-stranded nucleic acid fragment associated with the detection reagent.   The preparation method according to claim 1 or 2, wherein the selected molecule is a magnetic bead.   The preparation method according to any one of claims 1 to 3, wherein a hetero double-stranded nucleic acid fragment associated with a detection reagent is separated by an affinity separation method.   The preparation method according to any one of claims 1 to 4, wherein the detection reagent is a mismatch-binding ligand NC-link (MBL) or a mismatch repair enzyme.   The preparation method according to any one of claims 1 to 5, wherein the origin is a homogenous genome.   The preparation method according to any one of claims 1 to 5, wherein the origin is the same gene.   A single-base substitution detection method for determining the base sequence of each strand of a hetero double-stranded nucleic acid fragment obtained by the preparation method according to claim 1 to identify a single-base substitution existing between the nucleic acid fragments. A point mutation detection kit comprising two sets of primer pairs and a detection reagent,
The two sets of primer pairs are configured to hybridize with two different template nucleic acids of the same or different origin including the base sequences of regions corresponding to each other, and the two sets of primer pairs One of the primer pairs has one of the forward / reverse primers carrying a selection molecule, the other primer pair has a primer in the opposite direction carrying a selection molecule, and
The detection reagent is a mismatch-binding ligand NC-link (MBL) or a mismatch repair enzyme
Said kit . The kit according to claim 9, wherein the selection molecule is a magnetic bead.

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