JP2003159100A - Method for detecting mutation of improved gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting mutation by which the mutation is more universally and accurately detected in good sensitivity while precisely preventing the synthesis of a complemental strand on an untargeted base sequence even in any nucleic acid sequence in the method for detecting the mutation of a gene by using a LAMP method. <P>SOLUTION: The synthesis of the complemental strand on the untargeted base sequence is prevented by using an oligonucleotide capable of specifically annealing to a mutation site on the untargeted base sequence. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an improved method for detecting a mutation in a gene. More specifically, in a mutation detection method using the LAMP method, by using an oligonucleotide that can specifically anneal to a mutation site on a non-target base sequence, what kind of complementary strand synthesis can be performed from the non-target base sequence? The present invention relates to a method for reliably preventing mutations in nucleic acid sequences and more generally enabling more accurate detection of mutations and polymorphisms.

[0002]

2. Description of the Related Art Mutations in the gene region cause structural / functional changes in proteins and dysregulation of their expression. Therefore, mutation detection provides useful information for diagnosis and treatment of genetic diseases. Among the mutations, mutations that occur at a frequency of 1% or more in a certain population are referred to as polymorphisms. The susceptibility to diabetes and allergies and the ability to metabolize drugs depend on gene polymorphisms, especially single nucleotide polymorphisms (hereinafter, SNPs). single nucleo
abbreviated as tide polymorphisms).

On the other hand, HCV, influenza virus,
Subtypes of bacteria and viruses such as Helicobacter pylori are also considered to be a type of polymorphism, but the therapeutic effects of various drugs differ depending on these subtypes. Therefore,
Examination of polymorphisms such as these viruses also gives important information in selecting a treatment method.

As a method for detecting a mutation in a gene, PCR is carried out using a primer designed to have a sequence complementary to the sequence containing the mutation to be detected (allele-sp).
ecific PCR, Nucleic Acids Res 17: p25031989, Genomi
cs 5: p535 1989, J. Lab. Clin. Med 114: p105 1989)
It has been known. However, PCR-based methods for confirming nucleotide sequences are always accompanied by problems due to non-specific annealing of primers (S. Kwok et.al., Nucleic Acids Res 18: p9
99 1990). That is, even if the base sequence is not completely complementary, the primer anneals to synthesize a complementary strand, and exponential amplification occurs. Therefore, 1 in allele-specific PCR
In order to distinguish between the bases, another artificial mismatch is added, but this is not perfect. That is,
Under normal conditions, it is difficult to distinguish single nucleotide differences such as SNPs by the PCR method.

The second problem of the PCR method is that the primer can be set only in two regions in principle. Therefore, when a plurality of genes having similar nucleotide sequences are present in the same sample, it is extremely difficult to confirm the mutation or polymorphism in each gene using the presence or absence of an amplification product based on the PCR method as an index.

In order to solve the above problems, the present inventors have
LAMP method (Notomi et.al., Nucleic Acids Research (200
0), Vo1.28, e63, WO00 / 28082, etc.) was reported (WO01 / 34838). In this method, a sequence for discriminating a nucleotide sequence is first introduced by synthesis of a complementary strand from a primer, and this sequence forms a loop in the strand to serve as a starting point for complementary strand synthesis using itself as a template. Therefore, a stricter check mechanism for the target base sequence can be realized (described later).
However, even if this method is used, it is difficult to completely prevent complementary strand synthesis due to non-specific annealing of the primer on the non-target base sequence depending on the base sequence of the nucleic acid, although it is slight. .

[0007]

The present invention is based on the conventional LAMP.
In a method for detecting a mutation in a gene using the method, it is possible to reliably prevent non-specific complementary strand synthesis in any nucleic acid sequence, and to provide a more universal, accurate and sensitive mutation detection method. To aim.

[0008]

Means for Solving the Problems As a result of intensive studies by the present inventors, when an oligonucleotide capable of specifically annealing to a mutation site on a non-target base sequence is used, complementary strand synthesis from the non-target base sequence is performed. The present invention has been completed by finding that it is possible to detect mutations more accurately and with higher sensitivity.

That is, the present invention provides the following (1) to (13):
Is provided. (1) In the method for detecting a mutation of a gene using the LAMP method, by using an oligonucleotide that can specifically anneal to a mutation site on a non-target base sequence, synthesis of a complementary strand from the non-target base sequence is prevented. A method for detecting a mutation, which comprises: (2) The method according to (1) above, wherein the oligonucleotide is a modified oligonucleotide. (3) The method according to (2) above, wherein the modified oligonucleotide is modified from its 3 ′ end so that an elongation reaction does not occur. (4) The method according to (2) or (3) above, wherein the modification is amination or phosphorylation of the 3′-terminal hydroxyl group. (5) The method according to (3) or (4) above, which comprises in the center of the oligonucleotide a sequence complementary to the mutation site on the non-target base sequence. (6) The method according to any one of (1) to (5) above, wherein the oligonucleotide has a length of 5 to 50 bases. (7) The method according to (1) above, wherein the oligonucleotide is a peptide nucleic acid. (8) The method according to (1) above, wherein the oligonucleotide is contained in a part of the primer for the LAMP method. (9) The method according to (8) above, wherein the oligonucleotide is contained in a part of the following inner primer (A) and / or (B) for the LAMP method. (A) on one strand constituting the target base sequence, when selecting an arbitrary sequence F2c existing on the 3 ′ side and an arbitrary sequence F1c containing a mutation site to be detected, the complementary sequence F2 to the F2c is Primer containing the same sequence as the F1c at the 5'side at the end (B) Arbitrary sequence R2c existing on the 3'side on the other strand constituting the target base sequence and a mutation site to be detected When the sequence R1c is selected, the sequence R2 complementary to the R2c is at its 3 ′ end, a primer containing the same sequence as the R1c on the 5 ′ side (10) The oligonucleotide is F2 and F1c of the inner primer (A). And / or between R2 and R1c of the inner primer (B), the method according to (9) above. (11) The method according to (8) above, wherein the oligonucleotide is contained in part of the following loop primer for the LAMP method. LAM
When complementary sequences generated on the same strand of the amplification product by the P method anneal to each other to form a loop, a primer containing a nucleotide sequence complementary to the sequence in the loop at its 3'end (12) should be detected The mutation is a single nucleotide polymorphism, and the above (1) to (1
The method according to any one of 1). (13) In the method for detecting a mutation of a gene using the LAMP method,
A method for preventing synthesis of a complementary strand from a non-target base sequence by using an oligonucleotide capable of specifically annealing to a mutation site on the non-target sequence.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. 1. Definition of terms LAMP method: “LAMP method” is a method for amplifying a nucleic acid developed by the present inventors, which includes an inner primer pair or an outer primer pair, and a loop primer pair, 2, 4, or 6 This is a method for rapidly and inexpensively amplifying DNA or RNA under isothermal conditions (around 65 ° C.) using a species-specific primer, a strand displacement type polymerase and a substrate nucleotide.

The inner primer is an essential primer for the LAMP method, and when an arbitrary sequence X2c existing on the 3'side of the template and an arbitrary sequence X1c on the 5'side thereof is selected, it is complementary to the X2c. The sequence X2 and the sequence X1c identical to the X1c are 3 ′
It refers to a primer (having a structure of X1c + X2) that is included in this order from the side to the 5'side. Functionally, X2 on the inner primer is the part that anneals specifically to the template and provides the starting point for complementary strand synthesis, and X1c is the complementary region for the amplification (extension) product described below to form a loop. Given the sequence, the loop serves as the origin of new complementary strand synthesis.

[0012] The outer primer means two kinds of primers (one for each of the two strands) which can anneal to the outside (3 'side of the template) of the inner primer. In addition, a loop primer means that, when complementary sequences generated on the same strand of an amplification product by the LAMP method anneal to each other to form a loop, a nucleotide sequence complementary to the sequence in the loop is added to its 3 ′ end. Two types of primers (one for each of the two strands). The outer primer and the loop primer are not essential for the LAMP method, but if they are present, the amplification (extension) reaction proceeds more efficiently.

In the LAMP method, sequences complementary to each other are produced at the upper ends of the same strand of the amplification product, and these are annealed to form a loop, and an elongation reaction by a polymerase starting from the loop occurs (FIG. 1). (8)). At the same time, a strand displacement type extension reaction occurs from the primer annealed in the loop, and the above extension product is dissociated into single strands (Fig. 1 (8) to (11)). Since the dissociated single strand also has a complementary sequence at the end, the above reaction is repeated (Fig. 1).
(11)). Thus, in the LAMP method, the extension reaction and the amplification reaction simultaneously proceed at a plurality of positions on the same strand of the amplification product, so that the amplification of the nucleic acid is achieved in a super-exponential manner and under isothermal conditions.

For details of the LAMP method, see Notomi et.al.,
Nucleic Acids Research (2000), Vo1.28, e63 and international application WO00 / 28082. Method for detecting mutation of gene using LAMP method: "Method for detecting mutation of gene using LAMP method" is an accurate and sensitive mutation detection method using the LAMP method, and details thereof are described in International Application " Method for detecting mutation and / or polymorphism (WO01 / 3
4838) ”.

Specifically, the method comprises the steps of incubating the following (a) to (e) under conditions in which complementary strand synthesis starting from the first and second primers is possible It is a method of detecting the presence or absence of a mutation based on the amount of. (a) Nucleic acid sample containing the target base sequence (b) Strand-displacing polymerase (c) Arbitrary sequence F2c existing on the 3'side and a mutation site to be detected on one strand constituting the target base sequence When the sequence F1c is selected, a sequence F2 complementary to the F2c at its 3'end, a second primer containing the same sequence as the F1c on the 5'side (d) on the other strand constituting the target base sequence In, when selecting an arbitrary sequence R2c present on the 3'side and an arbitrary sequence R1c containing a mutation site to be detected, a sequence R2 complementary to the R2c at its 3'end, a sequence identical to the R1c is 5 ' First primer included in the side (e) nucleotide substrate In the above method, in addition to the first and second primers (hereinafter, referred to as inner primer), an outer primer
It is preferable to use two kinds, and it is more preferable to use two kinds of loop primers. The outer primer and the loop primer are as described in the above LAMP method.

In the mutation detection method using the LAMP method, the usual method
Similar to the LAMP reaction, X2 at the 3'end of the inner primer (F2 above)
And the R2) portion specifically anneals to the template to provide the origin of complementary strand synthesis and the X1c (F1c and R1c above) portion provides the complementary sequences required for loop formation of the amplification product.

However, X1c is designed to include a mutation site on the target base sequence. Therefore, unless the template contains the mutation to be detected, the sequence X1 complementary to X1c will not be generated on the extension product from the primer.
Therefore, the extension product cannot form a loop, and the LAMP reaction stops there (FIG. 2). on the other hand,
If the template contains the mutation to be detected, a sequence X1 complementary to X1c is generated on the extension product from the primer, so that the extension product forms a loop and the LAMP reaction proceeds. After all, by measuring the amount of the synthetic product by the LAMP method, the presence or absence of the mutation to be detected can be known (see FIG. 2).

A feature of this method is that the primer checks whether or not the template contains a mutation to be detected by the presence or absence of loop formation of its extension product, and this loop serves as a starting point for synthesis of a new complementary strand. In point. In other words, the check mechanism operates every time the loop is formed in the extension or amplification product. Therefore, once the complementary strand is synthesized by misreading as in PCR, the risk of exponentially synthesizing the amplification product thereafter is low, and a more accurate detection result is given.

Target base sequence: "Target base sequence" means a base sequence containing a mutation or polymorphism to be detected. In other words, the base sequence existing between the regions to which the primer of the present invention anneals constitutes the target base sequence of the present invention. Non-target base sequence: “Non-target base sequence” means a base sequence that includes mutations or polymorphisms that are not targets of detection. In other words, the sequence of the portion corresponding to the above "target base sequence" in the nucleic acid sample that is not the detection target constitutes the non-target base sequence of the present invention.

That is, when the detection target of the present invention is a wild type, the base sequence containing the mutation site in the wild type nucleic acid sample is the “target base sequence” and includes the mutation site in the mutant nucleic acid sample. The base sequence becomes a "non-target base sequence". On the other hand, when the detection target is a mutant type, the base sequence containing the mutation site in the mutant nucleic acid sample is the “target base sequence”, and the base sequence containing the mutation site in the wild type nucleic acid sample is “non- It becomes the target base sequence ”.

The (non-) target base sequence in the present invention includes not only the base sequence of the sense strand but also the base sequence of its complementary strand, that is, the antisense strand. Mutation site: “Mutation site” is a base sequence of a site that can cause a mutation on a target base sequence and a non-target base sequence, and the mutation includes polymorphism and SNPs.

Template and complementary strand: As used herein, the term "template" refers to the nucleic acid that serves as the template for complementary strand synthesis. A "complementary strand" having a base sequence complementary to the template has a meaning as a strand corresponding to the template, but the relationship between the two is merely relative. That is, the strand synthesized as a complementary strand can function again as a template. That is, the complementary strand can serve as a template.

Nucleic acid synthesis and amplification
ion): “Synthesis of nucleic acid” in the present invention means extension of nucleic acid from an oligonucleotide serving as a starting point of synthesis. In addition to the synthesis, when the production of another nucleic acid and the extension reaction of the produced nucleic acid occur successively, the series of reactions are collectively referred to as “amplification”.

Annealing: "annealing" and "hybridization" means that a nucleic acid has a double helix structure due to base pair binding based on the Watson-Crick model.
re) is formed. Therefore, even if the nucleic acid chain forming the base pair bond is a single strand, it is annealed or hybridized if the complementary base sequence in the molecule forms the base pair bond. In the present invention, annealing and hybridization are synonymous with each other in that the nucleic acid forms a double helix structure by base pairing. Base paired 3 '
When the end serves as the starting point for complementary strand synthesis, it is sometimes referred to as annealing. However, it cannot be denied that hybridization is the starting point of complementary strand synthesis.

Nucleic acid: "Nucleic acid" or "nucleic acid sample" is a DN
A, RNA, or a chimeric molecule thereof may be used, and may be natural or artificially synthesized. Moreover, even if it is a nucleotide derivative partially or entirely consisting of an artificial structure,
It is included in the nucleic acid of the present invention as long as it can form a base pair bond or so long as it functions as a template for complementary strand synthesis. The number of bases constituting the nucleic acid in the present invention is not limited. Nucleic acid is synonymous with the term polynucleotide. Oligonucleotide: The term “oligonucleotide” is used as a term indicating a polynucleotide having a particularly small number of bases. Generally, a polynucleotide having a length of 2 to 100 bases, more generally a length of about 2 to 50 bases is referred to as an oligonucleotide, but is not limited to these numerical values.

2. Oligonucleotide of the present invention (block oligo) The oligonucleotide of the present invention (hereinafter referred to as "block oligo") contains a sequence complementary to the mutation site on the non-target base sequence, and the mutation site on the non-target base sequence. Can be annealed specifically. The block oligo contains a mutation site on the non-target base sequence due to specific annealing.
It is preferable to include a sequence complementary to a region of -50 bases in length, particularly a region of 10 to 30 bases in length.

The block oligo may have a modified base. The site of the modified base is not particularly limited, but is preferably the base at the 3'end. The method for modifying the base is not particularly limited, but amination or phosphorylation is preferable.

When the 3'end of the block oligo is modified, the mutation site is preferably present in the center of the block oligo. Here, "center" refers to the oligonucleotide
It means that the number of bases from the 3'end and the 5'end to the mutation site is the same or different by one.

[0029] 3. Prevention of non-specific complementary strand synthesis by block oligo In the method for detecting mutation of a gene of the present invention, the block oligo is annealed specifically at a mutation site on a non-target base sequence (blocking the mutation site) to Surely prevent complementary strand synthesis from the target base sequence.

As shown in FIG. 3, in the gene mutation detection method using the LAMP method, whether or not the template contains a mutation to be detected is checked depending on whether or not the extension product from the primer can form a loop. , This loop is the origin of new complementary strand synthesis. This check mechanism works every time a loop is formed in the extension or amplification product. Therefore, once the complementary strand is synthesized by misreading, as in the PCR-based method that detects mutations depending on whether or not the primer is annealed to the template, the amplification product is then exponentially synthesized. The risk is low.
Moreover, since the mutation site on the non-target base sequence is blocked by the block oligo in advance, it is possible to reliably prevent the complementary strand synthesis due to the erroneous loop formation of the extension product.

The mutation detection method of the present invention is achieved, for example, by incubating various elements used in the above-mentioned “method for detecting mutation of gene using LAMP method”, for example, the following 1) to 4) with the block oligo. It 1) Nucleic acid sample containing a target base sequence 2) Strand-displacing polymerase 3) Two inner primers, or two inner primers and two outer primers, or two inner primers, outer primer and loop primer 2 types 4) nucleotide substrate In the mutation detection method of the present invention, in addition to the above elements, betaine is further added within a range that does not impair the object and effects of the present invention,
A melting temperature adjusting agent such as dimethyl sulfoxide, formamide, or a tetraalkylammonium salt, a nucleic acid detecting agent such as an intercalator (intercalating agent), and various reagents can be used.

4. Block Oligo Comprising Peptide Nucleic Acid In one embodiment of the invention, peptide nucleic acid (PNA)
Can be used as a block oligo. PNA is
The deoxyribose backbone of the oligonucleotide has a structure in which the peptide backbone is replaced. As the peptide main chain, N- (2-aminoethyl) linked by an amide bond
An example is a repeating unit of glycine. As the base to be bonded to the peptide backbone of PNA, naturally occurring bases such as thymine, cytosine, adenine, guanine, inosine, uracil, 5-methylcytosine, thiouracil and 2,6-diaminopurine, bromothymine, azaadenine and azaguanine Artificial bases such as.

The PNA is manufactured by a known method [WO96 / 4068].
No. 5], a monomer having each nucleobase bound thereto is produced, and then the obtained monomer is reacted according to a known general peptide synthesis method. For example, according to the solid-phase synthesis method, the reaction may be performed using an automatic synthesizer so that the desired nucleotide sequence is obtained. In the above method, commercially available reagents and raw materials can be used. If necessary, commercially available reagents can be appropriately derivatized according to a conventional method, for example, N-terminal and C-terminal that do not participate in the reaction of the starting amino acid. , A side chain functional group and the like can be protected before use. The obtained nucleobase-bound PNA can be easily isolated and purified by a usual separation means such as high performance liquid chromatography, electrophoretic chromatography, solvent extraction, salting out and the like.

Since PNA has a stronger binding force to nucleic acid than ordinary oligonucleotides, the use of this allows more reliable blocking of mutation sites. PN
When A is used as a block oligo, the sequence complementary to the mutation site on the non-target base sequence may be at its 3'end, 5'end or near the center. The length of the block oligo composed of the PNA is preferably 5 to 20 bases.

[0035] 5. Mutation Detection Method Using Inner Primer Containing Block Oligo In another embodiment of the present invention, the block oligo is LAMP.
It may be linked to a method primer. The primer is preferably the inner primer or a loop primer described later. The site for linking the block oligo is not particularly limited as long as the primer functions as a primer on the target base sequence and blocks the LAMP reaction on the non-target base sequence. However, when the block oligo is linked to the inner primer, it is preferably between F1c and F2 or between R1c and R2.

Specifically, the primer containing the block oligo can be prepared, for example, as follows. When linked to the second inner primer: on one strand constituting the target base sequence, when an arbitrary sequence F2c existing on the 3'side and an arbitrary sequence F1c containing a mutation site to be detected are selected, complementary to the F2c The second primer having the specific sequence F2 at its 3'end and the sequence identical to the block oligo and the F1c in its 5'side in this order is linked to the first inner primer: constitutes the target base sequence On the other strand, when the arbitrary sequence R2c present on the 3'side and the arbitrary sequence R1c containing the mutation site to be detected is selected, it has a complementary sequence R2 to the R2c at its 3'end, and its 5 ' Block oligo on the side and the same sequence as the R1c
First primer included on the 5'side

[0037] 6. Detection of Synthetic Reaction Product In the mutation detection method of the present invention, detection of mutation is achieved by detecting the amount of amplification product by a known method. For example, when the reaction solution is analyzed by gel electrophoresis, the amplification product unique to the present invention can be confirmed as a clear band. The amplification product in the present invention comprises a repeating base sequence having a target base sequence and its complementary strand as one unit. When this is separated by electrophoresis, ladder-shaped bands appearing at regular intervals. Besides this,
A method of tracking the progress of complementary strand synthesis while reacting is also known. For example, with a double-stranded nucleic acid-specific fluorescent dye such as ethidium bromide or cyber green, the accumulation of complementary strand synthesis product can be traced as a change in fluorescence intensity.

In the mutation detection method of the present invention, the detection may be carried out by comparing with an internal standard. As the internal standard, for example, a nucleic acid that is derived from the same nucleic acid sample and that is clearly free of mutation or polymorphism can be used. For the purpose of analyzing the base sequence of the genome, a base sequence whose mutation or polymorphism is not known in the genome can be used as an internal standard. Alternatively, when the detection method of the present invention is performed using mRNA as a template, a gene whose constant expression level is observed in any cell can be used as an internal standard. Against these internal standards, the complementary sequence synthesis reaction was performed under the same conditions as the present invention using the base sequence as the target base sequence, and the amount of the product was used as a control and the result obtained from the base sequence to be detected for mutation Can be compared.

The synthetic reaction product in the mutation detection method of the present invention is a nucleic acid in which complementary base sequences are alternately linked on a single strand, but the nucleic acid does not necessarily have the same structure as a natural nucleic acid. . It is known that a nucleic acid derivative can be synthesized by using a nucleotide derivative as a substrate when synthesizing a nucleic acid by the action of a polymerase. As such a nucleotide derivative, a nucleotide labeled with a radioisotope or a nucleotide derivative labeled with a binding ligand such as biotin or digoxin is used. By using these nucleotide derivatives, labeling of the product nucleic acid derivative is achieved. Alternatively, the product nucleic acid can be made into a fluorescent derivative by using a fluorescent nucleotide as a substrate. By using these, the synthetic reaction product can be directly detected.

7. Use of Mutation Detection Method of the Present Invention The detection method of the present invention can be applied to any nucleic acid. Specifically, for example, genomes of prokaryotic cells and eukaryotic cells, genomic DNAs and RNAs of viruses, genomes of intracellular parasites such as mycoplasma and rickettsiae, cDNAs derived from mRNAs of these species, and further Library derived from the gene source of E. coli, clones isolated from the library, and the like.
If the gene whose mutation or polymorphism is to be detected is RNA, it can be converted into cDNA by the action of DNA polymerase having reverse transcriptase activity. The nucleic acid used as the sample of the present invention is generally contained in a biological sample. A biological sample is an animal,
It can be a tissue, cell, culture, excrement or extract of a plant or a microorganism. The present invention is
Nucleic acids derived from the organism or nucleic acids derived from infectious parasites, microorganisms, and viruses contained in these biological samples, or infectious parasites using the organism as a host, can be detected. The nucleic acid according to the present invention is
It may be derived from the nucleic acid contained in the biological sample. For example, cDNA synthesized from mRNA
Alternatively, a nucleic acid amplified on the basis of a nucleic acid derived from a biological sample can be used as a sample for the detection method according to the present invention. These genes can be used as a sample for the detection method according to the present invention by denaturing them into single strands or, as will be described later, leaving them as double strands.

The nucleic acid containing the target nucleotide sequence in the present invention is used as a sample in the double-stranded state when it is incubated under the condition that the annealing of the primer and the reaction of synthesizing the complementary chain starting from the primer proceed. Can (WO
01/77317). That is, the mutation detection method of the present invention proceeds isothermally. This method uses a polymerase that can catalyze complementary strand synthesis with strand displacement, and
It is based on the reaction principle that complementary strand synthesis is repeated under isothermal conditions by repeating annealing to itself and complementary strand synthesis, and annealing of a new primer to a loop and complementary strand synthesis. Therefore, a double-stranded nucleic acid can be used as the target base sequence in the present invention.

According to the mutation detection method of the present invention, a certain level of amplification product cannot be generated unless the nucleotide sequences in a plurality of regions are predicted, and furthermore, nonspecific complementary strand synthesis is reliably prevented. Therefore, accurate and sensitive detection is possible. Therefore, the mutation detection method of the present invention brings the possibility of freely constructing a detection system that enables strict discrimination of similar base sequences. For example, among genes that contain similar nucleotide sequences to each other, such as human CYP2C19, SNPs existing in similar nucleotide sequences
Enables more accurate and sensitive detection of. Also, HL
It is also useful for detecting a minute base sequence difference between a plurality of genes having similar base sequences, such as A or platelet alloantigen, or typing of a pathogenic microorganism.

Others The present invention is also useful as a technique for analyzing drug metabolism genes that support tailor-made medicine. The drug metabolism gene is an important gene that influences drug sensitivity, and it is thought that the difference in its activity is due to the slight difference in the nucleotide sequence found in the gene encoding the enzyme involved in drug metabolism. There is. Therefore, the present invention has such a high utility value for analysis of drug metabolism genes brought about by the results of the Human Genome Project.

[0044]

The present invention will be described in more detail based on the following examples, but the invention is not intended to be limited to these examples. Example 1. Blocks in mutation detection of human CYP2C19 * 2 (m1)
Effect of Kuku Oligo 1. Preparation of human CYP2C19 * 2 (m1) allele amplification primer and block oligo As the human CYP2C19 * 2 (m1) allele amplification primer, the following primers were prepared. Furthermore, the following 23 bp oligonucleotides were prepared as block oligos for blocking wild type. The block oligo contains an SNP site at the 12th base and has an amination modification at the 3'end. Inner primer: FA: 5-CTGGGAAATAATCTTTTAATTTAATAAATTATTGTTTTCTCTTA
G-3 (SEQ ID NO: 1) RA: 5-CAGGAACCCGTGTTCTTTTACTTTCTCC-3 (SEQ ID NO: 2) Outer primer: F3: 5-CCAGAGCTTGGCATATTGTATC-3 (SEQ ID NO: 3) R3: 5-AGGGTTGTTGATGTCCATC-3 (SEQ ID NO: 4) Loop primer: FL: 5-GATAGTGGGAAAATTATTGC-3 (SEQ ID NO: 5) RL: 5-CAAATTACTTAAAAACCTTGCTT-3 (SEQ ID NO: 6) Block oligo: 5-TTATGGGTTCC C GGGAAATAATC (-NH ₂ ) -3 (SEQ ID NO: 7) (underlined part is SNP site) , And C (-NH ₂ ) at the 3'end indicates amination modification.)

2. Preparation of nucleic acid sample (human genomic DNA sample) m1 / m1 homozygous human genomic DNA or wt of 1 base difference
20 ng / μl of 50 mM Tris-HCl (ph8.8) was added to human genomic DNA (ml: mutant type, wt: wild type) of / wt homozygote.
Then, the nucleic acid sample was denatured by treatment at 95 ° C. for 3 minutes and ice-cooling for 1 minute.

3. LAMP reaction Add 2 μl of the nucleic acid sample (mutant sample and wild-type sample) to the reaction solution in Table 1 to make a total of 25 ul, and
Reaction was performed at 60 ° C. for 90 minutes in the presence and absence of nM block oligo. ABI PRISM7700 Sequence for detection of synthetic products
Followed using Detector (PE Applied Biosystems).

[0047]

[Table 1]

4. Results (Fig. 3) CYP2C19 * 2 (m1) allele amplification primer, when a mutant sample was amplified, regardless of the presence or absence of block oligo,
The amplification curve rose in about 20 minutes. The wild type sample was measured 20 times using the same primer. As a result, in the case where the block oligo was not used, the amplification curve stood up by 90 minutes in 9 cases, and in 1 case, the amplification curve stood up within 60 minutes. On the other hand, when a block oligo was used, there was no case where the amplification curve stood up by 90 minutes.

Example 2: Mutation detection of human CYP2C19 * 3 (m2)
Effect of primer containing block oligo in 1. Preparation of CYP2C19 * 3 (m2) alleles amplification primer Sequence of exon4 peripheral region (m2 region) where SNP of CYP2C19 * 3 is present (SEQ ID NO: 16) and CYP2C19 * 3 (m2) all by LAMP method
The sites corresponding to ele amplification are shown in FIG. Based on this, CYP2C19 * 3 (m2) alleles amplification primer and block primer incorporated inner primer (FA
+ b and RA + b primers) were prepared as follows. The underlined portion in the FA + b (RA + b) primer indicates the position of the block oligo. Inner primer: FA: 5-TTCAGGGGTCTTAACTTGATGGAAAAAT-3 (SEQ ID NO: 8) RA: 5-GAATCCAGGCCCAGAAAAAAAGACTGT-3 (SEQ ID NO: 9) Outer primer: F3: 5-TCCAGAAACGTTTCG-3 (SEQ ID NO: 10) R3: 5-AGGGCTTGGTCAATAT-3 ( SEQ ID NO: 11) Loop primer: LoopF: 5-GCTTACAATCCTGATGTT-3 (SEQ ID NO: 12) LoopR: 5-GTAAGGCCAAGTTTTTTG-3 (SEQ ID NO: 13) Inner primer containing block oligo: FA + b: TTCAGGGGT GGATCCAGT CTTAACTTGATGGAAAAAT (SEQ ID NO: 14) RA + b: GAATCCAGGCC TGGATCCCC CAGAAAAAAAGACTGT (SEQ ID NO: 15)

2. Preparation of nucleic acid sample (human genomic DNA sample) 50 mM Tris-HCl (ph8.8) was added to plasmid DNA in which the m2 region of CYP2C19 * 3 or the wt region with a difference of 1 base was cloned to obtain 3000 copies / ul, respectively, and then 95 ℃ 3 minutes, ice cooling 1
A nucleic acid sample was obtained by subjecting the sample to the denatured treatment.

[0051]

[Table 2]

3. LAMP reaction Add 2 μl of the m2 and Wt nucleic acid samples (mutant sample and wild-type sample) to the reaction solution in Table 2 to make a total of 25 ul, and in the presence or absence of 4000 nM block oligo at 60 ° C.
The reaction was carried out for 60 minutes. Detection of synthetic products is ABI PRISM7700 S
Followed using sequence detector (PE Applied Biosystems).

[0053] 4. Results (FIG. 5) When the mutant sample was amplified with the CYP2C19 * 3 (m2) allele amplification primer, the amplification curve stood up in about 20 minutes regardless of whether the block oligo was inserted or not. The same type of primer was used, and the measurement was performed 5 times for the wild type sample. As a result, in the case of using FA and RA primers containing no block oligo, there were 4 cases in which the amplification curve stood up by 60 minutes, and in 1 case, the amplification curve stood up within 40 minutes. On the other hand, in the case of using FA + b and RA + b primers in which a block oligo was inserted, there was no case where the amplification curve stood up by 60 minutes.

[0054]

EFFECTS OF THE INVENTION According to the present invention, in a method for detecting a mutation of a gene using the LAMP method, complementary strand synthesis from any non-target sequence on any base sequence is reliably prevented, and more universally, An accurate and sensitive mutation detection method can be provided.

[0055]

[Sequence list]                                SEQUENCE LISTING <110> Eiken Kagaku Kabusiki Kaisha <120> Improved Method of Detecting Gene Variation <130> P01-0695 <160> 16 <170> PatentIn Ver. 2.1 <210> 1 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 1 ctgggaaata atcttttaat ttaataaatt attgttttct cttag 45 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 2 caggaacccg tgttctttta ctttctcc 28 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 3 ccagagcttg gcatattgta tc 22 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 4 agggttgttg atgtccatc 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 5 gatagtggga aaattattgc 20 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 6 caaattactt aaaaaccttg ctt 23 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed oligonucleotides as a  blockoligo of the invention, 3'terminal cytosine is aminated <400> 7 ttatgggttc ccgggaaata atc 23 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 8 ttcaggggtc ttaacttgat ggaaaaat 28 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 9 gaatccaggc ccagaaaaaa agactgt 27 <210> 10 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 10 tccagaaacg tttcg 15 <210> 11 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 11 agggcttggt caatat 16 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 12 gcttacaatc ctgatgtt 18 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 13 gtaaggccaa gttttttg 18 <210> 14 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 14 ttcaggggtg gatccagtct taacttgatg gaaaaat 37 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 15 gaatccaggc ctggatcccc cagaaaaaaa gactgt 36 <210> 16 <211> 179 <212> DNA <213> Homo sapiens <220> <221> variation <222> (89) <223> CYP2C19 m2 region <400> 16 tccagaaacg tttcgattat aaagatcagc aatttcttaa cttgatggaa aaattgaatg 60 aaaacatcag gattgtaagc accccctgga tccaggtaag gccaagtttt ttgcttcctg 120 agaaaccact tacagtcttt ttttctggga aatccaaaat tctatattga ccaagccct 179

[0056]

[Sequence list free text]

SEQ ID NOS: 1-6: Primer SEQ ID NO: 7: Block oligo SEQ ID NOs: 8 to 15: primer SEQ ID NO: 16: human CYPC19 m2 region

[Brief description of drawings]

FIG. 1 shows the principle of the LAMP method.

FIG. 2 shows the principle of a method for detecting a gene mutation using a block oligo.

FIG. 3 shows the results of mutation detection of human CYP2C19 * 2 (m1) (A: when no block oligo is used, B: when a block oligo is used).

FIG. 4 is a human CYP2C19 * 3 m2 region sequence and its LAMP.
Amplification corresponding sites are shown.

FIG. 5 shows the results of mutation detection of human CYP2C19 * 3 (m2) (A: when no block oligo is used, B: when a block oligo is used).

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Hidetoshi Kanda             1381-3 Shimoishigami, Otawara City, Tochigi Prefecture Eiken Chemical             Nasu factory inside (72) Inventor Tsutomu Notomi             1381-3 Shimoishigami, Otawara City, Tochigi Prefecture Eiken Chemical             Nasu factory inside F-term (reference) 4B024 AA11 BA80 CA01 CA11 HA11                       HA19                 4B063 QA01 QA17 QQ42 QQ52 QR08                       QR42 QR62 QS25 QS31 QX02

Claims (13)

[Claims] 1. A method for detecting a gene mutation using the LAMP method, which comprises synthesizing a complementary strand from a non-target base sequence by using an oligonucleotide capable of specifically annealing to a mutation site on the non-target base sequence. A method for detecting a mutation, which comprises preventing the mutation. 2. The method of claim 1, wherein the oligonucleotide is a modified oligonucleotide. 3. The method according to claim 2, wherein the modified oligonucleotide is modified from its 3 ′ end so that an elongation reaction does not occur. 4. The method according to claim 2, wherein the modification is amination or phosphorylation of a 3′-terminal hydroxyl group. 5. The oligonucleotide containing a sequence complementary to a mutation site on a non-target base sequence in the center of the oligonucleotide.
Or the method described in 4. 6. The method according to claim 1, wherein the oligonucleotide has a length of 5-50 bases. 7. The method of claim 1, wherein the oligonucleotide is a peptide nucleic acid. 8. The method according to claim 1, wherein the oligonucleotide is contained as a part of a primer for the LAMP method. 9. The method according to claim 8, wherein the oligonucleotide is contained in a part of the following inner primers (A) and / or (B) for the LAMP method. (A) on one strand constituting the target base sequence, when selecting an arbitrary sequence F2c existing on the 3 ′ side and an arbitrary sequence F1c containing a mutation site to be detected, the complementary sequence F2 to the F2c is Primer containing the same sequence as the F1c at the 5'side at the end (B) Arbitrary sequence R2c existing on the 3'side on the other strand constituting the target base sequence and a mutation site to be detected When the sequence R1c is selected, a primer containing a sequence R2 complementary to the R2c at its 3 ′ end and a sequence identical to the R1c at the 5 ′ side 10. The method according to claim 9, wherein the oligonucleotide is contained between F2 and F1c of the inner primer (A) and / or between R2 and R1c of the inner primer (B). 11. The LAMP is the following LAMP:
The method according to claim 8, which is contained in a part of the loop primer for method. When complementary sequences generated on the same strand of the amplification product by the LAMP method anneal to each other to form a loop, a primer containing a base sequence complementary to the sequence in the loop at its 3'end 12. The mutation to be detected is a single nucleotide polymorphism,
The method according to any one of claims 1 to 11. 13. A method for detecting a gene mutation using the LAMP method, wherein an oligonucleotide capable of specifically annealing to a mutation site on a non-target sequence is used to prevent synthesis of a complementary strand from the non-target base sequence. Method.