JP2008048603A - Method of amplifying and detecting nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid amplification method whereby a mutant gene contained in a minor amount in a sample, which contains a large amount of a wild type gene, can be specifically amplified. <P>SOLUTION: The method of specifically amplifying either a wild type gene or a mutant gene which are both contained in a sample comprises (a) the step of preparing a primer set containing at least one primer having sequences being homologous or complementary to two or more regions on the gene to be amplified and at least one primer having sequence(s) being homologous or complementary to one or more regions on the gene, wherein a nucleotide residue concerning the mutation is contained in at least one of the above-described regions, (b) the step of performing a nucleic acid amplification reaction by using the above-described primer set with the use of a nucleic acid molecule contained in the above-described sample as a template, and (c) the step of performing the subsequent nucleic acid amplification reaction by using the above-described primer set with the use of the amplification product obtained by the former nucleic acid amplification reaction as a template. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

Background of the Invention

The present invention relates to a method for specifically amplifying and detecting a small amount of nucleic acid molecules contained in a sample.

Background Art In various diseases, cells having properties specific to the disease are known. For example, cancer cells having various properties are known even in cancer of the same tissue. Furthermore, it is known that the effects of therapeutic drugs, cancer progression, prognosis, etc. differ depending on the nature of such cancer cells. Therefore, by specifically detecting each of these disease-related cells having various properties, the disease can be diagnosed, and further, selection of a therapeutic drug, selection of a treatment method, and operation at the time of surgery. Important information related to formula selection etc. is provided.

  In recent years, research on molecular biology of diseases such as cancer has progressed, and many gene mutations specific to each of disease-related cells such as cancer cells have been found. A mutant gene having such a mutation can be used as a marker indicating the presence of disease-related cells in the diagnosis of each disease.

  In order to detect a mutated gene, the base sequence of the gene is determined and the presence or absence of the mutation is examined. However, the determination of the base sequence requires a lot of time and cost because the purity of the mutant gene is high and a nucleic acid sample containing almost no other type gene such as wild type is required.

  Recently, a method for amplifying only a mutated gene specific to a disease using a nucleic acid amplification method and detecting the disease based on the presence or absence of an amplification product has been developed.

  In general, the PCR method (Patent Document 1: US Pat. No. 4,683,195; Patent Document 2: US Pat. No. 4,683,202; and Patent Document 3: US Pat. No. 4,800,159) is the best nucleic acid amplification method. Used. However, in normal PCR methods, primers may anneal to sequences that are not perfectly complementary, resulting in false amplification reactions, so PCR methods are specific for detecting mutations, especially single nucleotide mutations. It cannot be said that the method has

  Moreover, in the detection of mutant genes in disease diagnosis, the sample specimen often contains both wild type genes and mutant genes, and further contains mutant genes having other mutations. Sometimes. Furthermore, the amount of the mutant gene contained in the sample may be extremely small compared to the wild type gene or other mutant genes. For example, in the diagnosis of cancer, it is necessary to detect a very small amount of cancer cells contained in a normal tissue. Therefore, it is necessary to detect a very small amount of a mutant gene from a specimen containing a large amount of a wild-type gene.

  Therefore, the specificity of the nucleic acid amplification method to be used is particularly important when detecting mutant genes by the nucleic acid amplification method for the purpose of disease diagnosis.

  On the other hand, various methods other than the PCR method have been developed as a nucleic acid amplification method. For example, a strand displacement amplification method (SDA method; Patent Document 4: Japanese Patent Publication No. 7-114718), self-sustained sequence amplification Method (3SR method), Qβ replicase method (Patent Document 5: Japanese Patent No. 2710159), NASBA method (Patent Document 6: Japanese Patent No. 2650159), LAMP method (Patent Document 7: International Publication No. 00) / 28082 pamphlet), ICAN method (Patent document 8: WO 02/16639 pamphlet), rolling circle method, method described in WO 2004/040019 pamphlet (Patent document 9), and the like are known. .

  Furthermore, as a method for detecting a gene mutation using a nucleic acid amplification method, the above-described mutation detection method using the LAMP method (Patent Document 10: JP-A-2003-159100), WO 01/34838 (Patent Document) The method described in 11) is known.

US Pat. No. 4,683,195 US Pat. No. 4,683,202 U.S. Pat. No. 4,800,159 Japanese Patent Publication No.7-111418 Japanese Patent No. 2710159 Japanese Patent No. 2650159 International Publication No. 00/28082 Pamphlet WO 02/16639 pamphlet International Publication No. 2004/040019 Pamphlet JP 2003-159100 A International Publication No. 01/34838 Pamphlet

Summary of the Invention

  The present inventors use a primer containing a sequence specific to two or more regions in a target nucleic acid sequence and a primer containing a sequence specific to one or more regions in the target nucleic acid sequence in a nucleic acid amplification reaction. In addition, it has been found that by designing these primers so that one or more regions of the region include nucleotide residues involved in mutation, either the wild-type gene or the mutant gene can be specifically amplified. It was. Furthermore, the present inventors include a large amount of wild-type gene by repeating the nucleic acid amplification reaction using a reaction solution containing the amplification product obtained by the nucleic acid amplification reaction as a template and newly adding the reagent such as the primer. It was found that a very small amount of mutant gene in a sample can be specifically amplified. The present invention is based on these findings.

  Therefore, an object of the present invention is to provide a nucleic acid amplification method capable of specifically amplifying a trace amount of a mutant gene in a sample containing a large amount of wild type gene or a trace amount of a wild type gene in a sample containing a large amount of mutant type gene. Is to provide.

  The nucleic acid amplification method according to the present invention is a method for specifically amplifying any one gene from a sample containing both a wild type gene and a mutant gene, and (a) on the gene to be amplified. A primer set comprising at least one primer containing a sequence homologous or complementary to two or more regions and at least one primer containing a sequence homologous or complementary to one or more regions on the same gene, A step of preparing a primer set in which nucleotide residues involved in mutation are contained in one or more of the regions, and (b) a nucleic acid amplification reaction by the primer set using the nucleic acid molecule contained in the sample as a template. And (c) a nucleic acid produced by the primer set using the amplification product obtained by the nucleic acid amplification reaction as a template. It is those which comprise the step of performing a wide reaction.

  According to the present invention, it is possible to specifically amplify either one of a sample containing both a wild type gene and a mutant gene, and in particular, the gene to be amplified is only in a very small amount in the sample. Even if it does not exist, it can be specifically amplified. This makes it possible to detect a very small amount of disease-related cells having a mutated gene from a specimen containing a large amount of normal cells having a wild-type gene.

Detailed description of the invention

  The nucleic acid amplification method according to the present invention is a method of specifically amplifying either gene from a sample containing both a wild type gene and a mutant type gene. The specificity is extremely high, and even if the sequence difference between the wild-type gene and the mutant-type gene is a single base substitution, only one of these can be amplified. Furthermore, even if any type of gene is present in a trace amount in the sample, the gene can be specifically amplified. These effects are achieved by the structure of the primer to prevent the generation of erroneous amplification products due to primer mispriming, and the repetition of the nucleic acid amplification reaction.

  In the nucleic acid amplification method according to the present invention, at least one primer containing a sequence homologous or complementary to two or more regions on the gene (wild type gene or mutant gene) to be amplified and one or more primers on the same gene A primer set comprising at least one primer containing a sequence homologous or complementary to a region is prepared. This primer set is designed so that nucleotide residues involved in mutation are included in one or more of the regions.

  The two kinds of primers included in the primer set can typically be a forward primer and a reverse primer designed for a target nucleic acid sequence to be amplified. Thus, one primer can contain a sequence homologous to the 5 'terminal region of the target nucleic acid sequence, and the other primer can contain a sequence complementary to the 3' terminal region of the target nucleic acid sequence. Furthermore, at least one of these primers includes a sequence homologous or complementary to a region different from the 5 'terminal region and the 3' terminal region of the target nucleic acid sequence. For example, one of the primers hybridizes to a target nucleic acid sequence or a complementary sequence thereof, and after the extension reaction of the primer occurs, it hybridizes to a region on the extended strand to form a loop structure. Thus, it can be incorporated on the 5 ′ end side of the same primer. Thus, at least one primer is designed based on the sequence of two or more regions on the target nucleic acid sequence (gene to be amplified), and the upper limit of the number of the regions is not particularly limited, but preferably 2 It is designed based on the arrangement of -5 regions, more preferably 2-3 regions, and even more preferably 2 regions. The other kind of primer is designed based on the sequence of one or more regions on the target nucleic acid sequence (gene to be amplified), and the upper limit of the number of the regions is not particularly limited. It is designed based on an arrangement of 5 regions, more preferably 1 to 3 regions, and still more preferably 1 to 2 regions. Furthermore, the primer set may further include other primers involved in the nucleic acid amplification reaction in addition to the two kinds of primers.

  The region on the target nucleic acid sequence (gene to be amplified) used for designing the two kinds of primers included in the primer set is selected so that at least one of them contains a nucleotide residue to be mutated. A plurality of regions containing nucleotide residues related to such mutations can also be used for primer design, which is particularly advantageous when nucleotide residues related to a plurality of mutations are present in the mutant gene.

  Other design criteria for the primer set are determined according to the mechanism of action of the nucleic acid amplification reaction used. Such a nucleic acid amplification reaction may be any method known to those skilled in the art, but is preferably capable of amplifying the target nucleic acid by a reaction under isothermal conditions. Therefore, according to a preferred embodiment of the present invention, the primer set enables amplification of a target nucleic acid by an isothermal reaction, in which case the nucleic acid amplification reaction can be performed isothermally. . Examples of such isothermal nucleic acid amplification methods include the LAMP method (International Publication No. 00/28082 pamphlet), the method described in International Publication No. 2004/040019 pamphlet, and the like.

  According to a particularly preferred embodiment of the present invention, the nucleic acid amplification method developed by the present inventors is used as the isothermal nucleic acid amplification method. In this embodiment, the primer set is a primer set comprising at least two primers capable of amplifying the target nucleic acid sequence, and the first primer included in the primer set is 3 ′ of the target nucleic acid sequence. A sequence (B) comprising a sequence (Ac ') complementary to the sequence (A) of the terminal portion at the 3' terminal portion and existing 5 'to the sequence (A) in the target nucleic acid sequence A homologous sequence (B ′) is included on the 5 ′ side of the sequence (Ac ′), and the second primer included in the primer set is a 3 ′ end portion of a complementary sequence of the target nucleic acid sequence. A folded sequence (D-Dc ′) comprising a sequence (Cc ′) complementary to the sequence (C) in the 3 ′ terminal portion and two nucleic acid sequences hybridizing to each other on the same strand. Containing on the 5 ′ side of the sequence (Cc ′) It is.

  In the present invention, “hybridize” means that a region in a primer hybridizes to a target region under stringent conditions and does not hybridize to a region other than the target region. Stringent conditions can be determined depending on the melting temperature Tm (° C.) of the duplex between the region in the primer and its complementary strand, the salt concentration of the hybridization solution, etc., for example, J. Sambrook, Reference can be made to EF Frisch, T. Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory (1989). For example, when hybridization is performed at a temperature slightly lower than the melting temperature of the primer region to be used, the region in the primer can be specifically hybridized to the target region. Such a primer can be designed using commercially available primer construction software, such as Primer 3 (manufactured by Whitehead Institute for Biomedical Research). According to a preferred embodiment of the present invention, the region in the primer that hybridizes to a target region comprises a sequence complementary to that target region.

  The action mechanism of nucleic acid synthesis by the first primer is schematically shown in FIG. First, a target nucleic acid sequence in a nucleic acid to be a template is determined, and a sequence (A) at the 3 ′ end portion of the target nucleic acid sequence and a sequence (B) existing on the 5 ′ side of the sequence (A) are determined. . The first primer includes a sequence (Ac ′) and further includes a sequence (B ′) on the 5 ′ side thereof. The sequence (Ac ′) is a sequence complementary to the sequence (A), and the sequence (B ′) is a sequence homologous to the sequence (B). Here, the first primer may include an intervening sequence that does not affect the reaction between the sequence (Ac ′) and the sequence (B ′). When such a primer is annealed to the template nucleic acid, the sequence (Ac ′) in the primer is hybridized to the sequence (A) of the target nucleic acid sequence (FIG. 1 (a)). When a primer extension reaction occurs in this state, a nucleic acid containing a complementary sequence of the target nucleic acid sequence is synthesized. Then, the sequence (B ′) present on the 5 ′ end side of the synthesized nucleic acid hybridizes to the sequence (Bc) present in the nucleic acid, and thereby the stem in the 5 ′ end portion of the synthesized nucleic acid. -A loop structure is formed. As a result, the sequence (A) on the template nucleic acid becomes a single strand, and another primer having the same sequence as the first primer hybridizes to this portion (FIG. 1 (b)). Thereafter, an extension reaction from the newly hybridized first primer occurs by the strand displacement reaction, and at the same time, the previously synthesized nucleic acid is separated from the template nucleic acid (FIG. 1 (c)).

  In the above action mechanism, the phenomenon that the sequence (B ′) hybridizes to the sequence (Bc) occurs due to the presence of complementary regions on the same strand. In general, when a double-stranded nucleic acid is dissociated into a single strand, partial dissociation starts from its terminal or other relatively unstable part. In the double-stranded nucleic acid generated by the extension reaction using the first primer, the base pair of the terminal portion is in an equilibrium state of dissociation and binding at a relatively high temperature, and the double strand is maintained as a whole. In such a state, when a sequence complementary to the dissociated portion of the terminal is present on the same strand, a stem-loop structure can be formed as a metastable state. Although this stem-loop structure does not exist stably, the same other primer binds to the complementary strand part (sequence (A) on the template nucleic acid) exposed by the formation of the structure, and the polymerase immediately extends. By performing the above, a previously synthesized strand is replaced and released, and at the same time, a new double-stranded nucleic acid can be generated.

  The design criteria for the first primer in the preferred embodiment of the present invention are as follows. First, in order for a new primer to efficiently anneal to the template nucleic acid after the complementary strand of the template nucleic acid is synthesized by extension of the primer, the template is formed by forming a stem-loop structure at the 5 ′ end of the synthesized complementary strand. The part of the sequence (A) on the nucleic acid needs to be a single strand. For this purpose, the difference (X−Y) between the base number X of the sequence (Ac ′) and the base number Y of the region sandwiched between the sequence (A) and the sequence (B) in the target nucleic acid sequence is expressed as X The ratio (X−Y) / X to is important. However, it is not necessary to make a single strand up to the portion which is present 5 ′ from the sequence (A) on the template nucleic acid and which is not related to primer hybridization. In addition, in order for a new primer to efficiently anneal to a template nucleic acid, it is also necessary to efficiently perform the above-described stem-loop structure formation. For efficient stem-loop structure formation, that is, efficient hybridization between the sequence (B ′) and the sequence (Bc), the distance between the sequence (B ′) and the sequence (Bc) (X + Y) is important. In general, the optimum temperature for the primer extension reaction is at most around 72 ° C., and at such a low temperature, it is difficult for the extended strand to dissociate over a long region. Therefore, in order for the sequence (B ′) to efficiently hybridize to the sequence (Bc), it is considered preferable that the number of bases between both sequences is small. On the other hand, in order for the sequence (B ′) to hybridize to the sequence (Bc) and to make the portion of the sequence (A) on the template nucleic acid a single strand, the sequence (B ′) and the sequence (Bc) It is considered that a larger number of bases between is preferred.

  From the above viewpoints, the first primer according to a preferred embodiment of the present invention is the (X−) in the case where no intervening sequence exists between the sequence (Ac ′) and the sequence (B ′) constituting the primer. Y) / X is −1.00 or more, preferably 0.00 or more, more preferably 0.05 or more, more preferably 0.10 or more, and 1.00 or less, preferably 0.75 or less, It is designed to be preferably 0.50 or less, more preferably 0.25 or less. Further, (X + Y) is preferably 15 or more, more preferably 20 or more, more preferably 30 or more, and preferably 50 or less, more preferably 48 or less, and further preferably 42 or less.

  Further, when there is an intervening sequence (base number Y ′) between the sequence (Ac ′) and the sequence (B ′) constituting the primer, the first primer according to a preferred embodiment of the present invention is {X- (YY ')} / X is -1.00 or more, preferably 0.00 or more, more preferably 0.05 or more, more preferably 0.10 or more, and 1.00 or less, It is designed to be preferably 0.75 or less, more preferably 0.50 or less, and still more preferably 0.25 or less. Further, (X + Y + Y ′) is preferably 15 or more, more preferably 20 or more, more preferably 30 or more, and is preferably 100 or less, more preferably 75 or less, and even more preferably 50 or less.

  The first primer has a chain length that allows base pairing with a target nucleic acid while maintaining the required specificity under given conditions. The primer has a chain length of preferably 15 to 100 nucleotides, more preferably 20 to 60 nucleotides. Moreover, the lengths of the sequence (Ac ′) and the sequence (B ′) constituting the first primer are preferably 5 to 50 nucleotides, more preferably 7 to 30 nucleotides, respectively. If necessary, an intervening sequence that does not affect the reaction may be inserted between the sequence (Ac ′) and the sequence (B ′).

  As described above, the second primer is complementary to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence (the strand opposite to the strand to which the first primer hybridizes). A folded sequence (D-Dc ′) comprising two nucleic acid sequences that hybridize to each other on the same strand and 5 ′ of the sequence (Cc ′). On the side. The structure of such a second primer is, for example, as shown in FIG. 2, but is not limited to the sequence or the number of nucleotides shown in FIG. The length of the sequence (Cc ′) constituting the second primer is preferably 5 to 50 nucleotides, more preferably 10 to 30 nucleotides. The length of the folded sequence (D-Dc ′) is preferably 2 to 1000 nucleotides, more preferably 2 to 100 nucleotides, still more preferably 4 to 60 nucleotides, and even more preferably 6 to 40 nucleotides. The number of nucleotides of base pairs formed by hybridization within the sequence is preferably 2 to 500 bp, more preferably 2 to 50 bp, still more preferably 2 to 30 bp, and further preferably 3 to 20 bp. The nucleotide sequence of the folded sequence (D-Dc ′) may be any sequence and is not particularly limited, but is preferably a sequence that does not hybridize to the target nucleic acid sequence. If necessary, an intervening sequence that does not affect the reaction may be inserted between the sequence (Cc ′) and the folded sequence (D-Dc ′).

  A possible mechanism of action for the nucleic acid amplification reaction by the first primer and the second primer will be described with reference to FIG. 3 (FIGS. 3a and 3b). First, the first primer hybridizes to the sense strand of the target nucleic acid, and the primer extension reaction occurs (FIG. 3 (a)). Next, a stem-loop structure is formed on the extended strand (−), and thereby a new first primer hybridizes to the sequence (A) on the target nucleic acid sense strand that has become a single strand (FIG. 3 ( b)), the extension reaction of the primer occurs, and the previously synthesized extended strand (−) is eliminated. Next, the second primer hybridizes to the sequence (C) on the released extended strand (−) (FIG. 3 (c)), the extension reaction of the primer occurs, and the extended strand (+) is synthesized. (FIG. 3 (d)). A stem-loop structure is formed at the 3 ′ end of the generated extended strand (+) and the 5 ′ end of the extended strand (−) (FIG. 3 (e)), and the extended strand (+) which is the free 3 ′ end. At the same time, an extension reaction occurs from the loop tip of the above, and the extension chain (-) is detached (FIG. 3 (f)). By the extension reaction from the loop tip, a hairpin-type double-stranded nucleic acid in which the extension strand (−) is bonded to the 3 ′ side of the extension strand (+) via the sequence (A) and the sequence (Bc) is generated, The first primer hybridizes to the sequence (A) and the sequence (Bc) (FIG. 3 (g)), and an extended chain (−) is generated by the extension reaction (FIG. 3 (h) and (i)). . In addition, a free 3 ′ end is provided by the folded sequence present at the 3 ′ end of the hairpin type double-stranded nucleic acid (FIG. 3 (h)), and by an extension reaction therefrom (FIG. 3 (i)), A single-stranded nucleic acid having a folded sequence at both ends and alternately containing an extended strand (+) and an extended strand (−) through the sequences derived from the first and second primers is generated (FIG. 3 (j )). In this single-stranded nucleic acid, since the free 3 ′ end (starting point of complementary strand synthesis) is provided by the folded sequence present at the 3 ′ end (FIG. 3 (k)), the same extension reaction is repeated, The chain length is doubled per extension reaction (FIGS. 3 (l) and (m)). In addition, in the extended strand (−) from the first primer desorbed in FIG. 3 (i), the free 3 ′ end (the complementary strand synthesis origin) is provided by the folded sequence present at the 3 ′ end. For this reason (FIG. 3 (n)), stem-loop structures are formed at both ends by the extension reaction therefrom, and the extension strand (+) and the extension strand (-) are alternately included via the sequence derived from the primer. Single-stranded nucleic acid is produced (FIG. 3 (o)). In this single-stranded nucleic acid as well, the complementary strand synthesis starting point is sequentially provided by the loop formation at the 3 'end, so that elongation reactions occur one after another. The single-stranded nucleic acid thus automatically extended contains sequences derived from the first primer and the second primer between the extended strand (+) and the extended strand (−). Therefore, it is possible for each primer to hybridize to cause an extension reaction, and thereby the sense strand and the antisense strand of the target nucleic acid are significantly amplified.

  In addition to the first primer and the second primer, the primer set may further include a third primer that hybridizes to the target nucleic acid sequence or a complementary sequence thereof. A primer should not compete with other primers for hybridization to a target nucleic acid sequence or its complementary sequence.

  In the present invention, “non-competing” means that the primer does not interfere with the provision of the complementary strand synthesis starting point by hybridizing to the target nucleic acid.

  When the target nucleic acid is amplified by the first primer and the second primer, as described above, the amplification product has the target nucleic acid sequence and its complementary sequence alternately. A folded sequence or a loop structure exists at the 3 'end of the amplified product, and extension reactions occur one after another from the complementary strand synthesis starting point provided thereby. The third primer can be annealed to the target sequence present in the single-stranded portion when such an amplification product is partially in a single-stranded state. As a result, a new complementary strand synthesis starting point is provided in the target nucleic acid sequence in the amplification product, and an extension reaction takes place therefrom, so that the nucleic acid amplification reaction is performed more rapidly.

  The third primer is not necessarily limited to one type, and two or more types of third primers may be used simultaneously in order to improve the speed and specificity of the nucleic acid amplification reaction. These third primers typically comprise a different sequence from the first primer and the second primer, but may hybridize to partially overlapping regions as long as they do not compete with these primers. . The chain length of the third primer is preferably 2 to 100 nucleotides, more preferably 5 to 50 nucleotides, and even more preferably 7 to 30 nucleotides.

  The third primer mainly serves as an auxiliary function for proceeding the nucleic acid amplification reaction by the first primer and the second primer more rapidly. Therefore, the third primer preferably has a Tm lower than the Tm of each 3 'end of the first primer and the second primer. Moreover, it is preferable that the addition amount of the third primer to the amplification reaction solution is smaller than the addition amounts of the first primer and the second primer.

  In order to determine the presence or absence of a gene mutation using the primer set, the primer set is used so that the nucleotide residues involved in the mutation are included in the sequence (A), the sequence (B) or the sequence (C). Can be designed. In addition, when the third primer hybridizes to a nucleic acid sequence in the nucleic acid sample or a complementary sequence thereof, one or more mismatches between the nucleic acid sequence or the complementary sequence due to the presence or absence of the mutation A third primer can also be designed to occur. Thereby, it is possible to determine the presence or absence of the mutation by confirming the presence or absence of the amplification product, and it is possible to determine the amount of the gene having the mutation by quantifying the amplification product.

  According to one embodiment of the present invention, the primer set is designed so that nucleotide residues involved in mutation are included in the sequence (A). In this embodiment, when the target nucleic acid sequence in the nucleic acid sample serves as a template, the first primer anneals to the sequence (A) in the nucleic acid amplification reaction, so that an amplification product is obtained. When a nucleic acid sequence different from the target nucleic acid sequence at the mutation site in the nucleic acid sample is used as a template, it becomes difficult for the first primer to anneal to the sequence (A) in the nucleic acid amplification reaction. The amount of amplification product not obtained or obtained is significantly reduced. The nucleotide residue involved in the mutation is preferably contained in the 5 'end of the sequence (A) (corresponding to the 3' end in the first primer).

  According to another embodiment of the present invention, the primer set is designed such that nucleotide residues involved in mutation are included in the sequence (C). In this embodiment, when the target nucleic acid sequence in the nucleic acid sample serves as a template, the second primer anneals to the sequence (C) in the nucleic acid amplification reaction, so that an amplification product is obtained. When a nucleic acid sequence different from the target nucleic acid sequence at the mutation site in the nucleic acid sample is used as a template, it becomes difficult for the second primer to anneal to the sequence (C) in the nucleic acid amplification reaction. The amount of amplification product not obtained or obtained is significantly reduced. The nucleotide residue involved in the mutation is preferably contained in the 5 'end of the sequence (C) (corresponding to the 3' end in the second primer).

  According to a preferred embodiment of the present invention, the primer set is designed so that nucleotide residues involved in mutation are included in the sequence (B). In this embodiment, when the target nucleic acid sequence in the nucleic acid sample is a template, the nucleic acid amplification reaction includes the first primer after annealing to the sequence (A) and the extension reaction. Since the sequence (B ′) hybridizes to the sequence (Bc) on the extended strand, a stem-loop structure is efficiently formed. Formation of this efficient stem-loop structure allows other first primers to anneal to the template, and the mechanism of action shown in FIG. 1 proceeds efficiently, resulting in an amplification product. On the other hand, when a nucleic acid sequence different from the target nucleic acid sequence at the mutation site in the nucleic acid sample is used as a template, it is difficult to form the stem-loop structure in the nucleic acid amplification reaction. The mechanism is impeded and no amplification product is obtained or the amount of amplification product obtained is significantly reduced.

  The detection of the mutation in the above-mentioned sequence (B) will be described in more detail. In the mechanism of action shown in FIG. 1, the phenomenon that the sequence (B ′) hybridizes to the sequence (Bc) occurs due to the presence of complementary regions on the same strand. In general, when a double-stranded nucleic acid is dissociated into a single strand, partial dissociation starts from its terminal or other relatively unstable part. In the double-stranded nucleic acid produced by the extension reaction with the first primer, the base pair of the terminal portion is in an equilibrium state of dissociation and binding at a relatively high temperature, and the double-stranded nucleic acid is maintained as a whole. In such a state, when a sequence complementary to the dissociated portion of the terminal is present on the same strand, a stem-loop structure can be formed as a metastable state. However, this stem-loop structure does not exist stably, especially if there are non-complementary nucleotides between the sequence (B ′) and the sequence (Bc) part forming the stem. Or the stem is not formed at all. In this case, the hybridization between the sequence (A) on the template and the sequence (Ac ') in the primer is superior, and the sequence (A) does not become a single strand. Primer cannot be annealed. Therefore, it is extremely difficult to cause the continuous reaction shown in FIG.

  In the nucleic acid amplification method according to the present invention, a nucleic acid amplification reaction using the primer set is performed using a nucleic acid molecule contained in the sample as a template. Those reaction conditions can be appropriately determined by those skilled in the art according to the nucleic acid amplification reaction used.

  When the primer set enables amplification of a target nucleic acid by a reaction under isothermal conditions, the nucleic acid amplification reaction is preferably performed under isothermal conditions. Here, “isothermal” refers to maintaining an approximately constant temperature condition such that the enzyme and the primer can substantially function. Furthermore, the “substantially constant temperature condition” means that not only the set temperature is accurately maintained but also a temperature change that does not impair the substantial functions of the enzyme and the primer is allowed. To do. The nucleic acid amplification reaction under a certain temperature condition can be carried out by keeping the temperature at which the activity of the enzyme used can be maintained. In this nucleic acid amplification reaction, in order for the primer to anneal to the target nucleic acid, for example, the reaction temperature is preferably set to a temperature near or below the melting temperature (Tm) of the primer, The level of stringency is preferably set in consideration of the melting temperature (Tm) of the primer. Therefore, this temperature is preferably about 20 ° C to about 75 ° C, more preferably about 35 ° C to about 65 ° C.

  The polymerase used in the nucleic acid amplification reaction is preferably one having strand displacement activity (strand displacement ability), and any of normal temperature, intermediate temperature, and heat resistance can be suitably used. Further, this polymerase may be either a natural body or a mutant with artificial mutation. Examples of such a polymerase include DNA polymerase. Furthermore, it is preferable that the DNA polymerase has substantially no 5 '→ 3' exonuclease activity. Examples of such a DNA polymerase include thermophilic Bacillus bacteria such as Bacillus stearothermophilus (hereinafter referred to as “B.st”) and Bacillus caldotenax (hereinafter referred to as “B.ca”). Examples include mutants lacking the 5 ′ → 3 ′ exonuclease activity of the derived DNA polymerase, Klenow fragment of E. coli derived DNA polymerase I, and the like. Examples of the DNA polymerase used in the nucleic acid amplification reaction include Vent DNA polymerase, Vent (Exo-) DNA polymerase, DeepVent DNA polymerase, DeepVent (Exo-) DNA polymerase, Φ29 phage DNA polymerase, MS-2 phage DNA polymerase, Z -Taq DNA polymerase, Pfu DNA polymerase, Pfu turbo DNA polymerase, KOD DNA polymerase, 9 ° Nm DNA polymerase, Therminater DNA polymerase and the like.

  Other reagents used in the nucleic acid amplification reaction include, for example, catalysts such as magnesium chloride, magnesium acetate, magnesium sulfate, substrates such as dNTP mix, Tris-HCl buffer, tricine buffer, sodium phosphate buffer, potassium phosphate buffer, etc. Can be used. Furthermore, additives such as dimethyl sulfoxide and betaine (N, N, N-trimethylglycine), acidic substances described in International Publication No. 99/54455 pamphlet, cation complexes and the like may be used.

  In the nucleic acid amplification reaction, a melting temperature adjusting agent can be added to the reaction solution in order to increase the amplification efficiency of the nucleic acid. The melting temperature (Tm) of a nucleic acid is generally determined by the specific nucleotide sequence of the double stranded portion in the nucleic acid. By adding a melting temperature adjusting agent to the reaction solution, this melting temperature can be changed. Therefore, under a certain temperature, the intensity of double strand formation in the nucleic acid can be adjusted. A general melting temperature adjusting agent has an effect of lowering the melting temperature. By adding such a melting temperature adjusting agent, the melting temperature of the duplex forming part between two nucleic acids can be lowered, in other words, the strength of the duplex formation can be lowered. It becomes. Therefore, when such a melting temperature adjusting agent is added to the reaction solution in the nucleic acid amplification reaction, it is efficiently used in a GC-rich nucleic acid region that forms a strong double strand or a region that forms a complex secondary structure. The double-stranded portion can be made into a single strand, and this makes it easy for the next primer to hybridize to the target region after the extension reaction with the primer is completed, so that the nucleic acid amplification efficiency can be increased. The melting temperature adjusting agent used in the present invention and its concentration in the reaction solution are appropriately selected by those skilled in the art in consideration of other reaction conditions that affect the hybridization conditions, such as salt concentration, reaction temperature, etc. The Therefore, the melting temperature adjusting agent is not particularly limited, but is preferably dimethyl sulfoxide (DMSO), betaine, formamide or glycerol, or any combination thereof, more preferably dimethyl sulfoxide (DMSO). .

  Furthermore, in the nucleic acid amplification reaction, an enzyme stabilizer can be added to the reaction solution. Thereby, since the enzyme in the reaction solution is stabilized, the amplification efficiency of the nucleic acid can be increased. The enzyme stabilizer used in the present invention may be any known in the art, such as glycerol, bovine serum albumin, saccharides, etc., and is not particularly limited.

  Furthermore, in the nucleic acid amplification reaction, a reagent for enhancing the heat resistance of an enzyme such as DNA polymerase or reverse transcriptase can be added to the reaction solution as an enzyme stabilizer. As a result, the enzyme in the reaction solution is stabilized, so that the nucleic acid synthesis efficiency and amplification efficiency can be increased. Such a reagent may be any known in the art and is not particularly limited, but is preferably a saccharide, more preferably a mono- or oligosaccharide, more preferably trehalose, sorbitol or mannitol, or these It is set as the mixture of 2 or more types.

  According to a preferred embodiment of the present invention, the nucleic acid amplification reaction is carried out in the presence of a mismatch recognition protein, which enables more specific specific amplification of a mutant gene or a wild type gene.

  It is already known that bacteria, yeast, and the like have a mechanism for repairing a base pair that cannot be partially matched (mismatched) in a double strand of DNA. This repair is performed by a protein called “mismatch binding protein” (also referred to as “mismatch recognition protein”). MutS protein (Japanese Patent Publication No. 9-504699), MutM protein (Japanese Patent Laid-Open No. 2000-300265) The use of various mismatch binding proteins such as MutS protein (International Publication No. 99/06591 pamphlet) bound to GFP (Green Fluorescence Protein) has been reported. Furthermore, in recent years, genetic diagnostic methods for detecting mismatches using mismatch binding proteins have been developed (M. Gotoh et al., Genet. Anal., 14, 47-50, 1997). As a method for detecting polymorphisms and mutations at specific nucleotides in nucleic acids, for example, a control nucleic acid having no mutation is hybridized with a test nucleic acid suspected of having the mutation, and a mismatch recognition protein is added thereto. A method of detecting a mismatch by introducing it is known.

  In the present invention, “mismatch” means that a pair of base pairs selected from adenine (A), guanine (G), cytosine (C), and thymine (T) (uracil (U) in the case of RNA) is normal. It means that it is not a correct base pair (a combination of A and T, or a combination of G and C). Mismatches include not only one mismatch, but also multiple consecutive mismatches, mismatches caused by insertion and / or deletion of one or more bases, and combinations thereof.

  Even in a nucleic acid amplification reaction for specific amplification of a mutant gene or a wild-type gene, the specificity (accuracy) can be improved by using these mismatch binding proteins. In nucleic acid amplification reactions, a small amount of heteroduplex structure may occur between two nucleotide sequences that are not complementary to each other. In the present invention, the “heteroduplex structure” means a duplex structure that is substantially a complementary duplex structure but includes a non-complementary region by having one or more mismatches. To do. Such a heteroduplex structure results in a false amplification product that would not otherwise be produced. Therefore, if a mismatch binding protein is added to the reaction solution used for the nucleic acid amplification reaction, the mismatch binding protein binds to the heteroduplex structure as described above, and the subsequent amplification reaction is hindered. Therefore, by using a mismatch binding protein, it is possible to prevent generation of an erroneous amplification product.

  The mismatch binding protein used in the present invention may be any protein that recognizes a mismatch in a double-stranded nucleic acid and can bind to the mismatch site, such as any known to those skilled in the art. Also good. In addition, in the mismatch binding protein used in the present invention, one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of the wild-type protein as long as the mismatch in the double-stranded nucleic acid can be recognized. Alternatively, it may be a protein (variant) having an amino acid sequence. Such mutants may occur in nature but can also be made artificially. Many methods are known for introducing amino acid mutations into proteins. For example, site-directed mutagenesis methods include WP Deng and JA Nickoloff (Anal. Biochem., 200, 81, 1992), KL Makamaye and F. Eckstein (Nucleic Acids Res., 14, 9679-9698). , 1986) are known, and random mutagenesis methods include the use of E. coli XL1-Red strain (Stratagene) deficient in the basic repair system, and the use of sodium nitrite and other chemical bases. A modification method (J.-J. Diaz et al., BioTechnique, 11, 204-211, 1991) is known. Many such mismatch binding proteins are known, such as MutM, MutS and their analogs (Radman, M. et al., Annu. Rev. Genet. 20: 523-538 (1986)). Radaman, M. etal., Sci. Amer., August 1988, pp40-46; Modrich, P., J. Biol. Chem. 264: 6597-6600 (1989); Lahue, RS et al., Science 245: 160-164 (1988); Jiricny, J. et al ,. Nucl. Acids Res. 16: 7843-7853 (1988); Su, SSet al., J. Biol. Chem. 263; 6829-6835 (1988) Lahue, RSet al., Mutat. Res. 198: 37-43 (1988); Dohet, C. et al., Mol. Gen. Gent. 206: 181-184 (1987); Jones, M. et al , Gentics 115: 605-610 (1987); Salmonella typhimurium Muts (Lu, AL, Genetics 118: 593-600 (1988); HaberL.T. Et al., J. Bacteriol. 170: 197-202 (1988) Pang, PP et al., J. Bacteriol. 163: 1007-1015 (1985)); and Priebe SD et al., J. Bacterilo. 170: 190-196 (1988)). The mismatch binding protein used in the present invention is preferably derived from MutS, MutH, MutL, or yeast, more preferably MutS, MutH, or MutL.

  It is known that mismatch binding proteins may also bind to single-stranded nucleic acids, and binding of such mismatch binding proteins to single-stranded nucleic acids is inhibited by single-stranded binding proteins. Therefore, when a mismatch binding protein is used in the nucleic acid amplification reaction, it is preferable to use a single-stranded binding protein in combination. Mismatch binding proteins may also bind to double-stranded nucleic acids that do not contain mismatches. Such misbinding of mismatch binding proteins must be activated using an activator in advance. It is known to be inhibited by Therefore, when using a mismatch binding protein in a nucleic acid amplification reaction, it is preferable to use a protein activated in advance by an activator.

  The single stranded binding protein (SSB) used to inhibit the binding of the mismatch binding protein to the single stranded nucleic acid can be any SSB known in the art. Preferred SSBs include single chain binding proteins from Escherichia coli, Drosophila, and Xenopus, and the gene 32 protein from T4 bacteriophage, and their equivalents from other species. Examples of the mismatch binding protein used in this case include MutS, MutH, MutL, HexA, MSH1-6, Rep3, RNaseA, uracil-DNA glycosidase, T4 endonuclease VII, resolvase, etc., preferably MutS, MSH2 or MSH6, or a mixture of two or more of these, more preferably MutS.

  The activator for activating the mismatch binding protein can be appropriately selected by those skilled in the art and is not particularly limited. However, preferably, ATP (adenosine 5′-triphosphate), ADP (adenosine 5 ′) -Diphosphate), ATP-γ-S (adenosine 5′-O- (3-thiotriphosphate)), AMP-PNP (adenosine 5 ′-[β, γ-imide] triphosphate) and the like Or one of the nucleotides that can bind to the mismatch binding protein. Activation of the mismatch binding protein can be performed by incubating the mismatch binding protein and the active agent at room temperature for several seconds to several minutes.

  In the nucleic acid amplification method according to the present invention, the nucleic acid amplification reaction using the primer set is performed using the amplification product obtained by the nucleic acid amplification reaction as a template. This nucleic acid amplification reaction can be carried out under the same conditions as described above, using as a sample all or part of the reaction solution obtained by the nucleic acid amplification reaction. More specifically, a reagent such as primer, polymerase, dNTP mix, and buffer solution is added to the reaction solution obtained by the nucleic acid amplification reaction as necessary to prepare a new reaction solution, which is used for nucleic acid amplification. The reaction can be performed. According to a preferred embodiment of the present invention, the nucleic acid amplification reaction using such an amplification product as a template is repeated two or more times.

  The presence of the amplification product obtained by the nucleic acid amplification method can be detected by many various methods. One method is the detection of amplification products of a specific size by general gel electrophoresis. In this method, for example, it can be detected by a fluorescent substance such as ethidium bromide or cyber green. As another method, it is also possible to detect by using a labeled probe having a label such as biotin and hybridizing it to the amplification product. Biotin can be detected by binding to fluorescently labeled avidin, avidin bound to an enzyme such as peroxidase, and the like. As yet another method, there is a method using an immunochromatograph. In this method, it has been devised to use a chromatographic medium utilizing a label detectable with the naked eye (immunochromatography method). If the amplified fragment and the labeled probe are hybridized, and a capture probe capable of hybridizing with a further different sequence of the amplified fragment is fixed to the chromatographic medium, the trapped portion can be trapped. Detection with is possible. As a result, it is possible to perform simple detection with the naked eye. Furthermore, in the nucleic acid amplification method by the present inventors, the amplification efficiency in the nucleic acid amplification reaction is very high, so that the amplification product can be indirectly detected by utilizing the fact that pyrophosphate is generated as a byproduct of amplification. As such a method, for example, there is a method of visually observing the white turbidity of the reaction solution by utilizing the fact that pyrophosphoric acid is bonded to magnesium in the reaction solution to cause white precipitation of magnesium pyrophosphate. As another method, there is a method utilizing the fact that the concentration of magnesium ions in the reaction solution is remarkably reduced by forming an insoluble salt by strongly binding pyrophosphate to metal ions such as magnesium. In this method, a metal indicator (for example, Eriochrome Black T, Hydroxy Naphthol Blue, etc.) whose color tone changes according to the magnesium ion concentration is added to the reaction solution, and the color change of the reaction solution is visually observed. This makes it possible to detect the presence or absence of amplification. Furthermore, by using Calcein or the like, it is possible to visually observe the increase in fluorescence accompanying the amplification reaction, so that the amplification product can be detected in real time.

  The presence of the amplification product obtained by the nucleic acid amplification method according to the present invention can also be detected by observing the aggregation of the solid phase carrier resulting from the generation of the amplification product. When performing such detection, it is assumed that at least one primer contained in the primer set used for the nucleic acid amplification reaction comprises a solid phase carrier or a site (group) capable of binding to the solid phase carrier. The The solid phase carrier or the site capable of binding to the solid phase carrier may be introduced into any part such as the 3 ′ end portion, 5 ′ end portion, central region of the primer, but preferably the 5 ′ end portion. It is assumed that it was introduced. Alternatively, the substrate used in the nucleic acid amplification reaction may comprise a solid phase carrier or a site capable of binding to the solid phase carrier.

  The solid phase carrier used in the present invention is a carrier that is insoluble in the reaction solution used for the nucleic acid amplification reaction, or a property from the liquid phase to the solid phase (gel phase) or from the solid phase (gel phase) to the liquid phase before and after amplification. Any phase transition carrier that changes can be used. Preferred solid phase carriers include water-insoluble organic polymer carriers, water-insoluble inorganic polymer carriers, synthetic polymer carriers, phase transition carriers, metal colloids, magnetic particles, and the like, and further solvent-insoluble organic polymer carriers. , Solvent-insoluble inorganic polymer carriers, solvent-soluble polymer carriers, gel polymer carriers and the like. Furthermore, examples of the water-insoluble organic polymer include silicon-containing materials such as porous silica, porous glass, diatomaceous earth, and celite, and cross-linked polysaccharides such as nitrocellulose, hydroxyapatite, agarose, dextran, cellulose, and carboxymethylcellulose. , Cross-linked protein such as methylated albumin, gelatin, collagen, casein, gel particles, dye sol and the like. Examples of the water-insoluble inorganic polymer include aluminum oxide, titanium oxide, and ceramic particles. Examples of the synthetic polymer include polystyrene, poly (meth) acrylate, polyvinyl alcohol, polyacrylonitrile or a copolymer thereof, a styrene-styrene sulfonic acid copolymer, a vinyl acetate-acrylic acid ester copolymer, and the like. . Examples of the metal colloid include gold colloid. Magnetic particles include magnetic iron oxide beads, monodispersed magnetic iron oxide finely pulverized particles on the surface, superparamagnetic particles (Japanese Patent Publication No. 4-501959), and superparamagnetic oxidation covered with a polymerizable silane coating. Examples thereof include magnetically responsive particles having iron (Japanese Patent Publication No. 7-6986), and finely magnetizable particles encapsulated in an organic polymer. The magnetized solid phase carrier can be easily separated from solid and liquid using magnetic force. Examples of the shape of the solid support include particles, membranes, fibers, and filters. Particles are particularly preferred as the shape of the solid support, and the surface thereof may be either porous or non-porous. Particularly preferred solid phase carriers include latex in which a synthetic polymer carrier is uniformly dispersed in water, metal colloid particles such as gold colloid, magnetic particles such as magnet beads, and the like.

  Immobilization of a primer or a substrate to a solid phase carrier can be performed by a method known to those skilled in the art, and may be a method by either physical bonding or chemical bonding. Immobilization of a primer or a substrate on a solid phase carrier is generally performed by combining, for example, a substance capable of labeling an oligonucleotide such as a primer or a probe with a solid phase carrier to which a substance capable of binding thereto is bound. be able to. As a combination of substances used for such purpose, those well known in the art can be used, for example, a combination of biotin and avidin or streptavidin, a combination of an antigen and an antibody capable of binding thereto, a ligand, And a combination of two nucleic acids which hybridize with each other. Specifically, for example, a primer or a substrate labeled with biotin is bound to a solid phase carrier whose surface is coated with avidin or streptavidin, whereby the primer or the substrate can be immobilized on the solid phase carrier. Examples of the antigen include haptens such as FITC, DIG, and DNP. Examples of antibodies that can bind to these include antibodies such as anti-FITC antibody, anti-DIG antibody, and anti-DNP antibody. In addition, these antibodies may be either monoclonal antibodies or polyclonal antibodies. In particular, the combination of biotin and streptavidin has high specificity and good binding efficiency, so that these combinations are particularly preferable. Labeling substances such as biotin, hapten, and ligand may be used alone or in a plurality of combinations if necessary, by known means (Japanese Patent Laid-Open Nos. 59-93099, 59-148798, and No. 59-204200) can be introduced at the 5 ′ end of the primer.

  The site (or group) capable of binding to the solid phase carrier used in the present invention can be selected according to the above-described method used for immobilization of the primer or the substrate to the solid phase carrier, and thus the solid phase carrier. Any of those that allow physical binding to or chemical binding to each other may be used, but specific binding is preferable. Examples of the site capable of binding to such a solid phase carrier include biotin, avidin, streptavidin, antigen, antibody, ligand, receptor, nucleic acid, protein and the like, and preferably biotin or streptavidin. More preferably, it is biotin. By using a primer or a substrate containing such a site, it is possible to bind the solid phase carrier to the amplification product after performing a nucleic acid amplification reaction. The solid phase carrier used in this case may include a binding partner of the site contained in the primer or the substrate, if necessary. Such a binding partner is present in a form capable of binding to the site contained in the primer or the substrate, preferably present on the surface of the solid support, more preferably the solid support. It was assumed that it was applied on the surface of

  Since the nucleic acid amplification method according to the present invention has high specificity and detection sensitivity, the sample is a specimen derived from a subject, and the amplification target is a mutant gene having a mutation associated with a disease or a gene contained in a pathogen. It becomes possible to detect a trace amount of disease-related cells or pathogens contained in the specimen.

  Therefore, according to the present invention, there is provided a method for detecting a cell associated with a disease from a specimen derived from a subject, the method comprising (a) two or more mutant genes having a mutation associated with the disease. A primer set comprising at least one primer containing a sequence homologous or complementary to a region and at least one primer containing a sequence homologous or complementary to one or more regions on the same gene, A step of preparing a primer set in which nucleotide residues involved in mutation in a gene are contained in one or more of the regions; (b) nucleic acid amplification by the primer set using a nucleic acid molecule contained in the specimen as a template; (C) performing a nucleic acid amplification reaction with the primer set using the amplification product obtained by the nucleic acid amplification reaction as a template. Step, and (d) comprising the step of detecting the amplified product. In this method, when an amplification product is detected in step (d), it is indicated that cells related to the disease have been detected.

  A cell associated with a disease has a gene mutation associated with the disease, preferably a gene mutation specific to the disease, and is appropriately selected by those skilled in the art depending on the target disease. Genetic mutations related to this are known for various diseases, and those skilled in the art can appropriately use combinations of these diseases and genetic mutations. Examples of such gene mutation include a mutation in β-globin gene specific for sickle cell disease, a mutation in p53 gene specific for cancer, and the like.

  The mutation of β globin gene specific to sickle cell disease known as a genetic disease includes a GAG to GTG mutation at the 7th codon, and this single nucleotide mutation is the sixth of β-globin expressed. Are replaced from glutamic acid to valine. Therefore, cells associated with sickle cell disease can be detected by specifically amplifying a mutant β-globin gene having this mutation.

  Many gene mutations specific to cancer cells are known, but the most common are mutations in the p53 gene. The p53 protein expressed from this gene is one of transcription factors, and controls the cell cycle, apoptosis, and DNA repair, and plays a role in preventing the accumulation of mutations (such as canceration) at the individual level. In human cancers, mutations in the p53 gene are seen in more than 50% of the cases, 80% of which are point mutations with amino acid substitutions. This mutation rate is considerably higher than other known oncogenes. The p53 protein is a DNA binding protein, and many of the mutations found in cancer are concentrated in the DNA binding domain. In addition, a hot spot in which mutations are markedly concentrated in the p53 gene has been reported (Lopez M et al., Use of cytological specimens for p53 gene alteration detection in oral squamous cell carcinoma risk patients. Clin. Oncol. (R Coll 2004 Aug; 16 (5): 366-70; Soussi T et al., Reassessment of the TP53 mutation database in human disease by data mining with a library of TP53 missense mutations, Hum. Mutat. 25 (1): 6-17, 2004; Sudhir Srivastava, et al., Biomarkers for Early Detection of Colon Cancer, Clinical Cancer Research vol. 7, 1118, 2001; Thierry Soussi, et al., Significance of TP53 Mutation in Human Cancer: A Critical Analysis of Mutations at CpG Dinucleotides, Human. Mutation Vol. 21, 192, 2003). Moreover, the frequency of mutation and the position where the mutation is likely to occur vary depending on the organ. Among human cancers, those having a high frequency of mutations in the p53 gene are lung cancer, colon cancer, pancreatic cancer and the like, and mutations are found in nearly 70% of these cancers. For example, mutations in the p53 gene that are common in colorectal cancer are mutations in CG found at codons 175, 248, and 273, followed by codons 196, 213, 245, There is a tendency for mutations to concentrate at the 282nd codon and the like. There is also a clear tendency for the type of point mutation, and there are overwhelmingly many transversions from GC to AT. In the method for detecting a disease-related cell according to the present invention, cancer cells can be detected by specifically amplifying a mutant gene having any one or a combination of two or more of such mutations.

  Furthermore, according to the present invention, there is provided a method for specifically detecting a pathogen from a specimen derived from a subject, the method comprising (a) two or more specifics on a gene contained in the target pathogen to be detected. A primer set comprising at least one primer containing a sequence homologous or complementary to a target region and at least one primer containing a sequence homologous or complementary to one or more specific regions on the same gene (B) a step of performing a nucleic acid amplification reaction using the primer set using the nucleic acid molecule contained in the specimen as a template, and (c) a nucleic acid amplification using the primer set using the amplification product obtained by the nucleic acid amplification reaction as a template. And (d) detecting the amplification product. In this method, when an amplification product is detected in step (d), it is indicated that the target pathogen strain has been detected.

  Examples of pathogens include pathogenic bacteria, fungi, viruses and the like. The specific region on the gene contained in the target pathogen can be easily selected by comparing the sequence of the gene with the sequence of the nucleic acid contained in the sample. In addition, pathogens may include not only wild strains having wild-type genes but also mutant strains having mutant genes. As such mutant strains, various strains are known along with differences in gene sequences from wild strains. According to the method of the present invention, all or a part of such wild strains and mutant strains can be detected simultaneously, or only one of them can be specifically detected. For example, when multiple strains of pathogens are detected simultaneously, a region having a sequence that is common to these strains and that is not found in nucleic acids contained in other strains or specimens should be used as the specific region. Can do. In the case of specifically detecting only a single strain of a pathogen, the region having a sequence that is not found in nucleic acids contained in other strains or specimens and is found only in the target pathogen strain is described above. It can be used as a specific region.

  In particular, in order to specifically detect only one of a wild strain and a mutant strain of a pathogen, a primer set so that nucleotide residues involved in the mutation are included in one or more of the specific regions. Can be designed. For example, when the nucleic acid amplification method developed by the present inventors is used, the primer set is such that nucleotide residues involved in mutation are included in the sequence (A), the sequence (B) or the sequence (C). Can be designed. In addition, when the third primer hybridizes to a nucleic acid sequence in the nucleic acid sample or a complementary sequence thereof, one or more mismatches between the nucleic acid sequence or the complementary sequence due to the presence or absence of the mutation A third primer can also be designed to occur. Thus, it is possible to determine the presence or absence of the target pathogen strain by confirming the presence or absence of the amplification product, and it is possible to determine the amount of the pathogen strain by quantifying the amplification product.

  Each step of the method for detecting a disease-related cell or pathogen according to the present invention can be performed as described above for the nucleic acid amplification method according to the present invention.

  When the disease-related cell or pathogen is detected in the disease-related cell or pathogen detection method according to the present invention, it can be determined that the subject suffers from the disease or an infection caused by the pathogen strain. Thus, according to the present invention, a method for diagnosing a disease in a subject is provided, which method comprises all steps of the method for detecting disease-related cells or pathogens according to the present invention.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the scope of the present invention is not limited to these examples.

Example 1: Detection of the mutant gene from a sample in which wild-type and mutant β-globin genes coexist In this example, a wild-type human β-globin gene and a mutant human β-globin gene containing a single base substitution are obtained. The mutant gene was specifically amplified from the contained sample and detected.

  In order to create a single-base mutation model, a long-chain synthetic oligonucleotide containing a single-base mutation in a specific region in the human β-globin gene and a long-chain synthetic oligonucleotide containing no single-base mutation were synthesized. Further, these long-chain synthetic oligonucleotides were amplified by the PCR method, and amplification products of wild-type DNA containing no single-base mutation and mutant-type DNA containing a single-base mutation were obtained. After sequencing and confirming the nucleotide residues of the mutated portion, these amplified products were used as templates in the following experiments.

  The nucleotide sequence (SEQ ID NO: 1) of the wild-type amplification product is shown in FIG. In FIG. 4, a residue with a box is a residue to be mutated, and this residue is “t” in the mutant amplification product.

  As a primer, a wild type DNA detection primer pair and a mutant DNA detection primer pair having the following sequences were used. The region on the template used for the design of these primers is shown as the underlined portion in FIG.

Wild type DNA detection primer:
F1-W: 5′-gatgctccat acaactgtgttcactagcaa- 3 ′ (SEQ ID NO: 2);
R1: 5'-ggatatatatatatcc cttcaccacgttcaccttg- 3 '(SEQ ID NO: 4).

Mutant DNA detection primer:
F1-M: 5′-gatgcaccat acaactgtgttcactagcaa- 3 ′ (SEQ ID NO: 3);
R1: 5'-ggatatatatatatcc cttcaccacgttcaccttg- 3 '(SEQ ID NO: 4).

  In the forward primer F1-W, the sequence on the 3 ′ end side (20 mer: underlined portion) is annealed to the template, and the sequence on the 5 ′ end side (10 mer: other than the underlined portion) is on the extended strand by the primer. , Designed to hybridize to a region beginning 16 bases downstream of the 3 ′ terminal residue of the primer. The forward primer F1-M has the same nucleotide sequence as that of the primer F1-W except that the forward primer F1-M has a sixth T residue for mutation from the 5 'end. The reverse primer R1 has a structure in which the sequence on the 3 ′ end side (19mer: underlined portion) is annealed to the template, and the sequence on the 5 ′ end side (16mer: other than the underlined portion) is folded within the region. Designed to.

Using the above wild type DNA, mutant DNA, or a mixture thereof as a template, a nucleic acid amplification reaction using each of the templates was performed using a wild type DNA detection primer pair or a mutant DNA detection primer pair. Specifically, a reaction solution (25 μL) having the following composition: Tris-HCl (20 mM, pH 8.8), KCl (10 mM), (NH ₄ ) ₂ SO ₄ (10 mM), MgSO ₄ (8 mM), DMSO (3%), Triton X-100 (1%), dNTP (1.4 mM), MutS (1 μg), 2000 nM each of the above primer pairs, the above template (10 ⁻¹⁹ mol / tube (about 60000 molecules)) , Cyber Green (0.01 μl / ml), and 16 U of Bst DNA polymerase (NEW ENGLAND BioLabs) were prepared and incubated at 60 ° C. for 20 minutes. Thereafter, 1 μl was taken from the reaction solution, and this was used as a template solution to prepare 25 μl of a reaction solution having the same composition as above (dilution step), and incubated again at 60 ° C. for 20 minutes. Thereafter, the same dilution step and incubation step were performed once more. The template was allowed to react with double strands. These reactions were carried out using a real-time PCR apparatus (Stratagene).

  FIG. 5 shows changes with time in the amplification amount in the third nucleic acid amplification reaction. Table 1 summarizes the results of nucleic acid amplification. In this table, the results of the amplification reaction are ++, ++, +, and ± in descending order of the increase rate of the amplification product (in the graph where the rise of the curve is fast), and no amplification was observed at all. -. The sample numbers described in FIG. 5 correspond to the sample numbers described in Table 1.

  According to FIG. 5 and Table 1, it was confirmed that the wild-type DNA detection primer pair specifically detects only wild-type DNA, and the mutant-type DNA detection primer pair specifically detects only mutant-type DNA. It was. It was also shown that the mutant DNA can be detected from a sample in which the mutant DNA is only 1/5000 of the wild type DNA.

FIG. 1 is a diagram schematically showing the action mechanism of a nucleic acid amplification reaction using a first primer. FIG. 2 is a diagram illustrating the structure of the second primer. FIG. 3a is a diagram schematically showing the action mechanism of the nucleic acid amplification reaction using the first primer and the second primer. FIG. 3b is a diagram schematically showing the action mechanism of the nucleic acid amplification reaction using the first primer and the second primer. FIG. 4 is a diagram showing the nucleotide sequence (SEQ ID NO: 1) contained in the human β-globin gene, the mutation site used in Example 1, and the region used for primer design. FIG. 5 is a graph showing the time course of the third nucleic acid amplification reaction in Example 1.

Claims (30)

A method of specifically amplifying either gene from a sample containing both a wild type gene and a mutant gene,
(A) at least one primer comprising a sequence homologous or complementary to two or more regions on the gene to be amplified and at least one sequence comprising a sequence homologous or complementary to one or more regions on the gene A step of preparing a primer set comprising the primers of the above, wherein the nucleotide residues involved in the mutation are contained in one or more of the regions,
(B) performing a nucleic acid amplification reaction using the primer set using the nucleic acid molecule contained in the sample as a template; and (c) performing a nucleic acid amplification reaction using the primer set using the amplification product obtained by the nucleic acid amplification reaction as a template. A method comprising the step of performing.   The method of claim 1, wherein step (c) is repeated two or more times.   The method according to claim 1, wherein the primer set enables amplification of a target nucleic acid by a reaction under isothermal conditions, and the nucleic acid amplification reaction is performed under isothermal conditions. The primer set comprises at least two primers capable of amplifying a target nucleic acid sequence,
The first primer included in the primer set comprises a sequence (Ac ′) complementary to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and in the target nucleic acid sequence Comprising a sequence (B ′) homologous to the sequence (B) present 5 ′ to the sequence (A) on the 5 ′ side of the sequence (Ac ′),
The second primer included in the primer set comprises a sequence (Cc ′) complementary to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and The method according to claim 1, comprising a folded sequence (D-Dc ′) containing two nucleic acid sequences that hybridize to the same strand on the 5 ′ side of the sequence (Cc ′).   The method according to claim 4, wherein the primer set is designed so as to include a nucleotide residue involved in the mutation in the sequence (A), the sequence (B), or the sequence (C).   The method according to claim 4, wherein the primer set is designed to include a nucleotide residue related to the mutation in the sequence (B).   The primer set further includes a third primer that hybridizes to the target nucleic acid sequence or its complementary sequence, and does not compete with other primers for hybridization to the target nucleic acid sequence or its complementary sequence. The method of claim 4, comprising:   When a third primer hybridizes to a nucleic acid sequence in the nucleic acid sample or a complementary sequence thereof, the presence or absence of the mutation causes one or more mismatches with the nucleic acid sequence or the complementary sequence thereof. The method according to claim 7, which is designed.   The method according to claim 1, wherein a polymerase having strand displacement ability is used in the nucleic acid amplification reaction.   The method according to claim 1, wherein the nucleic acid amplification reaction is performed in the presence of a mismatch binding protein.   11. The method of claim 10, wherein the mismatch binding protein is MutS, MSH2 or MSH6, or a mixture of two or more thereof. A method for detecting cells related to a disease from a specimen derived from a subject,
(A) at least one primer containing a sequence homologous or complementary to two or more regions on a mutant gene having a mutation associated with the disease, and a sequence homologous or complementary to one or more regions on the same gene A step of preparing a primer set comprising at least one kind of primer, wherein nucleotide residues involved in mutation in the mutant gene are contained in one or more of the regions,
(B) performing a nucleic acid amplification reaction with the primer set using a nucleic acid molecule contained in the specimen as a template;
(C) using the amplification product obtained by the nucleic acid amplification reaction as a template, performing a nucleic acid amplification reaction using the primer set, and (d) detecting the amplification product. A method, wherein if detected, indicates that cells associated with the disease have been detected.   The method of claim 12, wherein step (c) is repeated two or more times.   The method according to claim 12, wherein the primer set enables amplification of a target nucleic acid by a reaction under isothermal conditions, and the nucleic acid amplification reaction is performed under isothermal conditions. The primer set comprises at least two primers capable of amplifying a target nucleic acid sequence,
The first primer included in the primer set comprises a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and The sequence (B ′) hybridizes to the complementary sequence (Bc) of the sequence (B) existing 5 ′ to the sequence (A) on the 5 ′ side of the sequence (Ac ′). ,
The second primer included in the primer set comprises a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and The method according to claim 12, comprising a folded sequence (D-Dc ′) containing two nucleic acid sequences that hybridize to the same strand on the 5 ′ side of the sequence (Cc ′).   The method according to claim 15, wherein the primer set is designed so as to include a nucleotide residue involved in the mutation in the sequence (A), the sequence (B) or the sequence (C).   The method according to claim 15, wherein the primer set is designed to include a nucleotide residue related to the mutation in the sequence (B).   The primer set further includes a third primer that hybridizes to the target nucleic acid sequence or its complementary sequence, and does not compete with other primers for hybridization to the target nucleic acid sequence or its complementary sequence. The method according to claim 15, comprising:   When a third primer hybridizes to a nucleic acid sequence in the nucleic acid sample or a complementary sequence thereof, the presence or absence of the mutation causes one or more mismatches with the nucleic acid sequence or the complementary sequence thereof. The method of claim 18, wherein the method is designed.   The method according to claim 12, wherein a polymerase having strand displacement ability is used in the nucleic acid amplification reaction.   The method according to claim 12, wherein the nucleic acid amplification reaction is performed in the presence of a mismatch binding protein.   The method of claim 21, wherein the mismatch binding protein is MutS, MSH2 or MSH6, or a mixture of two or more thereof. A method for specifically detecting a pathogen from a specimen derived from a subject,
(A) at least one primer containing a sequence homologous or complementary to two or more specific regions on a gene contained in the target pathogen to be detected, and homologous to one or more specific regions on the same gene Or providing a primer set comprising at least one primer comprising a complementary sequence;
(B) performing a nucleic acid amplification reaction with the primer set using a nucleic acid molecule contained in the specimen as a template;
(C) using the amplification product obtained by the nucleic acid amplification reaction as a template, performing a nucleic acid amplification reaction using the primer set, and (d) detecting the amplification product. A method that, if detected, indicates that the pathogen of interest has been detected.   24. The method of claim 23, wherein step (c) is repeated two or more times.   The method according to claim 23, wherein the primer set enables amplification of a target nucleic acid by a reaction under isothermal conditions, and the nucleic acid amplification reaction is performed under isothermal conditions. The primer set comprises at least two primers capable of amplifying a target nucleic acid sequence,
The first primer included in the primer set comprises a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and The sequence (B ′) hybridizes to the complementary sequence (Bc) of the sequence (B) existing 5 ′ to the sequence (A) on the 5 ′ side of the sequence (Ac ′). ,
The second primer included in the primer set comprises a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and The method according to claim 23, comprising a folded sequence (D-Dc ′) containing two nucleic acid sequences that hybridize to the same strand on the 5 ′ side of the sequence (Cc ′).   The primer set further includes a third primer that hybridizes to the target nucleic acid sequence or its complementary sequence, and does not compete with other primers for hybridization to the target nucleic acid sequence or its complementary sequence. 27. The method of claim 26, comprising:   The method according to claim 23, wherein a polymerase having strand displacement ability is used in the nucleic acid amplification reaction.   24. The method of claim 23, wherein the nucleic acid amplification reaction is performed in the presence of a mismatch binding protein.   30. The method of claim 29, wherein the mismatch binding protein is MutS, MSH2 or MSH6, or a mixture of two or more thereof.

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