WO2016167317A1 - Procédé de détection d'une mutation génétique - Google Patents

Procédé de détection d'une mutation génétique Download PDF

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WO2016167317A1
WO2016167317A1 PCT/JP2016/062004 JP2016062004W WO2016167317A1 WO 2016167317 A1 WO2016167317 A1 WO 2016167317A1 JP 2016062004 W JP2016062004 W JP 2016062004W WO 2016167317 A1 WO2016167317 A1 WO 2016167317A1
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wild
type
nucleic acid
mutant
gene mutation
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明男 山根
僚子 今川
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凸版印刷株式会社
株式会社理研ジェネシス
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the present invention is a method for detecting a gene mutation using a PCR (polymerase chain reaction) -clamping method, and even if there is a large variation in the quality and quantity of the specimen,
  • the present invention relates to a method for collectively and more accurately detecting the mutant types.
  • hEGFR anti-human epidermal growth factor receptor
  • cancer cells having a mutated gene resistant to a drug coexist at an extremely small ratio that cannot be detected by conventional testing methods. For this reason, there is a problem in that cancer cells having a resistance mutant gene gradually increase due to drug treatment in a patient whose mutation gene has not been detected in a test before medication. If cancer cells having resistance gene mutations can be detected at a stage where the existing ratio is smaller than before, the emergence of drug resistance can be predicted at an early stage, which can lead to better treatment.
  • highly sensitive detection of cancer-related gene mutations detection of a very low proportion of mutant genes mixed in the wild type
  • the most typical method for genetic mutation testing is the Sanger sequencing method.
  • the Sanger sequencing method has been called the gold standard for gene mutation detection.
  • detection is difficult when the proportion of the mutant gene mixed in the wild-type gene falls below 10 to 20%. Therefore, in the oncology area, a method that exceeds the sensitivity of Sanger sequencing is required, and various methods have been devised, and some are used clinically (see Non-Patent Document 1).
  • PNA Peptide Nucleic Acid
  • PNA is an artificial nucleic acid also called a peptide nucleic acid, and has a structure in which a phosphodiester bond connecting sugar moieties of a nucleic acid is replaced with a peptide bond having glycine as a unit.
  • PNA has no charge, but binds (hybridizes) more strongly to nucleic acids having complementary sequences than DNA and RNA, and exhibits high specificity.
  • high specificity means that PNA shows a high binding force to DNA or RNA that is completely complementary, but if a base that is not complementary is contained even at one base, the binding power is greatly reduced.
  • a PNA oligonucleotide having a sequence complementary to the wild type gene is added during the PCR reaction, and the PNA oligonucleotide is more resistant to the wild type gene than the mutant gene. This is a method utilizing specific hybridization.
  • this is a method for obtaining a PCR product in which amplification of a mutant gene that does not hybridize with a PNA oligonucleotide proceeds preferentially and the ratio of the mutant gene to the wild type gene is increased.
  • LNA Locked Nucleic Acid
  • LNA is a compound that introduces a new cyclic structure by connecting the 2'-oxygen and 4'-carbon of the ribose ring of the ribonucleoside with a methylene chain, and restricts the change in the conformation of the ribose ring. is there.
  • BNA Bandd Nucleic Acid
  • BNA Bridged Nucleic Acid
  • LNA Long RNA nucleic Acid
  • the bridge contains a nitrogen atom and has a ring structure. It is a six-membered ring. Furthermore, it is easy to introduce a functional group through a nitrogen atom.
  • PCR-clamping method using BNA oligonucleotide is performed to hydrolyze the PCR amplification product which is the mutant type to be detected. It has been reported that detection was performed using a probe (see, for example, Patent Document 2). The hydrolysis probe specifically detects the mutant type.
  • PCR-clamping method using PNA oligonucleotide is performed, and PCR amplification products are hydrolyzed by introducing a hydrolysis probe that detects both mutant type and wild type, and LNA that specifically detects the mutant type. It has been reported that an EGFR gene mutation was detected by detection using a probe (see, for example, Non-Patent Document 4).
  • a wild type nucleic acid gene If the PCR amplification of the nucleic acid whose mutation site is a wild type) is completely suppressed, the obtained PCR amplification products are all mutant nucleic acids (nucleic acid whose gene mutation site is a mutation type). Therefore, the probe for detecting the PCR amplification product does not need to be specific for the mutant type, and may be a hydrolysis probe common to the wild type and the mutant type.
  • a threshold value of a signal emitted from the hydrolysis probe is set in advance, and the specimen is determined based on the threshold value. It is necessary to determine whether or not the mutant nucleic acid is included.
  • genomic DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue prepared for pathological examination has a very large variation from sample to sample in terms of quality and quantity.
  • FFPE formalin-fixed paraffin-embedded
  • the present invention relates to a PCR-clamping method using a BNA oligonucleotide as an oligonucleotide for suppressing PCR amplification of a wild-type gene, and even in the case where the quality and quantity of the specimen vary greatly,
  • An object of the present invention is to provide a method for detecting all mutation types contained in the above together and more accurately.
  • the present inventors have found that in the PCR-clamping method, in addition to the total amount of wild-type nucleic acid and mutant nucleic acid in the PCR amplification product, By quantitatively detecting the amount of the protein, and comparing the results of both, it was found that all the mutant types in the sample can be accurately detected together without being affected by variations in the quality of the sample, etc. Was completed.
  • the method for detecting a gene mutation according to the first aspect of the present invention is a method of PCR using a template DNA having a double-stranded DNA containing the same base sequence as the genome region containing the target gene mutation site, and the region containing the gene mutation site.
  • a wild-type oligo comprising a forward primer and a reverse primer configured to amplify, a base sequence complementary to a genomic region where the gene mutation site is a wild-type, and comprising at least one or more unnatural nucleic acid
  • a mutant / wild-type common probe configured to detect both a nucleotide, a nucleic acid in which the gene mutation site is a wild type, and a nucleic acid in which the gene mutation site is a mutation type; and the gene mutation site is a wild type PCR obtained by performing real-time PCR using a wild-type specific probe configured to specifically detect the type of nucleic acid Based on the difference between the PCR amplification product amount detected by the mutant / wild-type common probe and the PCR amplification product amount detected by the wild-type specific probe among the width products, the template DNA in the template DNA A double-stranded DNA whose gene mutation site is mutant is detected.
  • the mutant / wild-type common probe and the wild-type specific probe may be probes that emit fluorescence when hydrolyzed.
  • the non-natural nucleic acid may be BNA or LNA.
  • the target gene mutation site may be a mutation site of two or more types of gene mutations concentrated in a specific genomic region.
  • the target gene mutation site may be a deletion mutation site.
  • the target gene mutation site may be a point mutation site.
  • the gene mutation detection kit is a PCR method comprising a wild-type oligonucleotide having a base sequence complementary to a genomic region whose target gene mutation site is a wild type and a region containing the gene mutation site.
  • a forward primer and a reverse primer configured to amplify by, and a mutant / configured to detect both a nucleic acid in which the gene mutation site is a wild type and a nucleic acid in which the gene mutation site is a mutation type
  • a wild-type common probe, and a wild-type specific probe configured to specifically detect a nucleic acid whose gene mutation site is wild-type.
  • the method for detecting a genetic mutation according to the above aspect of the present invention includes all the extremely small amounts of mutant genes contained in a large excess of wild-type genes, regardless of variations in specimen quality and quantity, and the degree of mutation rate. Can be collectively detected. Further, by using the gene mutation detection kit according to the above aspect of the present invention, the method for detecting a gene mutation according to the above aspect of the present invention can be more easily performed.
  • FIG. 3 is a schematic diagram showing expected amplification curves of a mutant / wild-type common probe and a wild-type specific probe in real-time PCR in the method for detecting a gene mutation according to the first embodiment of the present invention.
  • FIG. 3 is a schematic diagram showing expected amplification curves of a mutant / wild-type common probe and a wild-type specific probe in real-time PCR in the method for detecting a gene mutation according to the first embodiment of the present invention.
  • FIG. 3 is a schematic diagram showing expected amplification curves of a mutant / wild-type common probe and a wild-type specific probe in real-time PCR in the method for detecting a gene mutation according to the first embodiment of the present invention.
  • Example 3 is a schematic diagram showing expected amplification curves of a mutant / wild-type common probe and a wild-type specific probe in real-time PCR in the method for detecting a gene mutation according to the first embodiment of the present invention.
  • Example 1 it is the figure which showed the result of real-time PCR.
  • Example 2 it is the figure which showed the result of real-time PCR.
  • Example 2 it is the figure which showed the result of real-time PCR.
  • real-time PCR is performed in the presence of a wild-type oligonucleotide having a base sequence complementary to a genomic region whose gene mutation site is wild-type.
  • PCR-clamping method which suppresses PCR amplification of wild-type nucleic acid and increases detection sensitivity of mutant nucleic acid, the total amount of wild-type nucleic acid and mutant nucleic acid in the PCR amplification product and the amount of wild-type nucleic acid.
  • the template DNA used as the sample contains only the wild-type gene.
  • the amount of wild-type nucleic acid is substantially smaller than the total amount of wild-type nucleic acid and mutant nucleic acid, the template DNA as a sample contains a mutant gene in addition to the wild-type gene. I can judge. Furthermore, it can be determined that the greater the difference between the total amount of wild-type nucleic acid and mutant nucleic acid and the amount of wild-type nucleic acid, the greater the proportion of mutant genes.
  • the specific genotype of the mutant gene contained in the template DNA is not specified.
  • the gene mutation detection method uses a double-stranded DNA as a template DNA, a forward primer, a reverse primer, a wild-type oligonucleotide, a mutant / wild-type common probe, and a wild-type specific probe.
  • the double-stranded DNA serving as the template DNA contains the same base sequence as the genomic region containing the target gene mutation site.
  • the forward primer and the reverse primer amplify a partial region including the gene mutation site by PCR.
  • Mutant / wild type common probes detect both wild type and mutant nucleic acids. Wild type specific probes specifically detect wild type nucleic acids.
  • the total amount of wild-type nucleic acid and mutant nucleic acid in the PCR amplification product is determined by using the mutant / wild-type common probe for detecting both the wild-type nucleic acid and the mutant nucleic acid.
  • the mutant / wild-type common probe is a probe that does not include the target gene mutation site and has a base sequence complementary to the genomic region having the same base sequence in the wild-type gene and the mutant gene. is there. Therefore, it can hybridize with both wild-type nucleic acid and mutant nucleic acid. That is, both the wild type nucleic acid and the mutant nucleic acid in the PCR amplification product are detected without being distinguished by the common mutant / wild type probe.
  • the amount of wild-type nucleic acid in the PCR amplification product is detected by a wild-type specific probe that specifically detects the wild-type nucleic acid.
  • the wild type specific probe is a probe having a base sequence complementary to a genomic region whose gene mutation site is a wild type. Therefore, in the nucleic acid chain extension reaction of real-time PCR, it hybridizes with the wild type nucleic acid but does not hybridize with the mutant nucleic acid. That is, only the wild type nucleic acid in the PCR amplification product is specifically detected by the wild type specific probe.
  • a gene mutation can be detected by detecting the amplification product using only the mutant / wild-type common probe.
  • PCR amplification of the wild type gene cannot be completely suppressed. Therefore, in the gene mutation detection method according to the present embodiment, gene mutation is detected more accurately by correcting the detection result of the mutant / wild-type common probe using the detection result of the wild-type specific probe. . That is, in the method for detecting a genetic mutation according to this embodiment, detection of a wild-type nucleic acid in a PCR amplification product using a wild-type specific probe is used as a “control reaction”.
  • control reaction is a reaction for obtaining information as to whether the detection of the target gene mutation to be detected has been correctly performed. Furthermore, it refers to a reaction for correcting the result of the reaction to be detected based on the result of the control reaction and leading to a correct result determination. For example, if a sample shows a negative mutation type (no mutant gene is included), the result may indicate that the test technique is inappropriate, the quality of the DNA contained in the sample, It is necessary to indicate whether it was a false negative due to a factor such as an insufficient amount or a true negative.
  • One of the roles of the control reaction is to ensure the reliability of such tests. In many measurement methods, the determination of positive or negative is often made based on a threshold value set for a measurement value.
  • the mutant / wild-type common probe and the wild-type specific probe are amplified by the same PCR primer. Reacts to the amplified product.
  • the template DNA is not added from the outside but is derived from the specimen itself. Therefore, even if the amount of template DNA, the quality of template DNA, the amount of contaminants that inhibit PCR, and the like differ from sample to sample, both probes exhibit reactivity that is equally affected by the variation. For this reason, it is clear that detection of wild-type nucleic acids in PCR amplification products with a wild-type specific probe is a very robust control reaction. This makes it possible to detect a more precise minute amount of gene mutation that is not affected by various conditions.
  • the gene mutation to be detected is not particularly limited, and may be a mutation in which one or a plurality of bases are substituted with respect to the wild-type base sequence, and deleted. It may be a mutation or an inserted mutation.
  • a wild-type oligo comprising a double-stranded DNA containing the same base sequence as a genomic region containing multiple types of gene mutations as a template DNA and a base sequence complementary to a genomic region in which the multiple types of genetic mutations are all wild-type Use nucleotides.
  • a wild-type specific probe that specifically recognizes a wild-type nucleic acid in which all of the plurality of types of gene mutations are all wild-type, and a base that does not recognize all of the plurality of types of gene mutation sites, By using a mutant / wild-type common probe that recognizes a genomic region having a common sequence, multiple types of gene mutations can be detected together.
  • somatic mutation detection such as cancer.
  • somatic mutations the types of mutations can be broadly divided into two types: point mutations such as substitution of one base and deletion / insertion, and mutations of deletion or insertion of several to tens of bases. . Even in the former point mutation, there may be a plurality of different point mutations in a specific genomic region. In addition, in the case of the latter deletion mutation or insertion mutation, a large number of deletion mutations with slightly different deletion parts or similar insertion mutations with different insertion locations or insertion sequences were found within the same genomic region. ing.
  • a representative example of a gene mutation in which different point mutations are concentrated in a specific region is a ras family gene.
  • KRAS and NRAS a plurality of mutations are found in the regions of codons 12 and 13, codons 59 and 61, codon 117, and codon 146.
  • codons 12 and 13 of KRAS there are 16 types of mutations at the base sequence level.
  • Some deletion mutations have a plurality of mutations in a specific genomic region.
  • a typical deletion mutation is a deletion mutation in exon 19 of EGFR. More than 30 deletion mutations have been found between approximately 45 bases of exon 19.
  • a typical insertion mutation is the mutation found in exon 20 of EGFR. More than 50 types of insertion mutations have been found in the range of approximately 45 bases of exon 20.
  • the method for detecting a gene mutation according to the present embodiment can detect a mutant gene that is very slightly mixed with an excessive amount of a wild-type gene with high sensitivity and accuracy. For this reason, in particular, it is preferably used for detection of somatic mutations in cancer cells and mitochondrial DNA mutations.
  • the biological tissues actually collected often contain not only cancer cells but also normal cells.
  • the gene containing the target gene mutation to be detected is contained in both cancer cells and normal cells, and the mutant form of the gene is contained only in the cancer cells. Therefore, if most of the collected specimens are normal cells and the percentage of cancer cells is small, the detection target gene is a mixture of normal genes (wild type) and mutated genes (mutant types) The proportion of mutated genes is very small compared to wild type genes. In addition, even if the proportion of cancer cells in the collected sample is large, if the proportion of cancer cells with the target gene mutation is small, the overall proportion of the mutant gene is small relative to the wild-type gene. Become.
  • mutant genes from cancer cell-derived genomic fragments released into the blood wild-type genes released from normal cells coexist, and only a small percentage of wild-type genes are mixed. Mutant genes must be detected.
  • the gene mutation detection method according to this embodiment is suitable for detecting gene mutations in mitochondrial DNA.
  • the wild-type oligonucleotide used in the gene mutation detection method according to the present embodiment has a base sequence complementary to a genomic region where the target gene mutation site to be detected is a wild-type. That is, a base that is not complementary to a mutant nucleic acid whose target gene mutation site is a mutant is included. For this reason, the wild-type oligonucleotide specifically hybridizes to the wild-type nucleic acid rather than the mutant nucleic acid.
  • a wild-type oligonucleotide contains at least one or more non-natural nucleic acids.
  • the non-natural nucleic acid may be a PNA having no phosphate bond or an artificial nucleic acid having a phosphate bond.
  • Examples of the artificial nucleic acid having a phosphate bond include LNA and BNA.
  • LNA and BNA have a structure in which nucleosides are connected by a phosphodiester bond. Therefore, it can be said that the structure is very close to that of natural nucleic acid compared to PNA.
  • PNA oligonucleotides have problems such as difficulty in synthesizing those having a long chain length due to the specificity of the structure, and remarkably lower solubility in water depending on the base sequence. On the other hand, there is no such problem with LNA oligonucleotides and BNA oligonucleotides.
  • the nucleoside structure of BNA is shown in the following formulas (1) and (2).
  • Wild type oligonucleotides are oligonucleotides containing non-natural nucleic acids such as BNA. Therefore, compared with oligonucleotides formed only from natural nucleic acids such as DNA and RNA, it has a strong binding force with template DNA and high specificity. For this reason, wild-type oligonucleotides bind tightly (hybridize) with wild-type nucleic acids, but hardly hybridize with mutant nucleic acids, and specifically suppress PCR amplification using wild-type nucleic acids as templates. be able to.
  • the wild-type oligonucleotide used in the gene mutation detection method according to the present embodiment may be an LNA oligonucleotide, a BNA oligonucleotide, or an oligo containing two or more of LNA and BNA. It may be a nucleotide.
  • a BNA oligonucleotide is preferable because it has a higher binding force and specificity to the template DNA and a higher wild-type amplification suppression effect.
  • BNA oligonucleotides two types of BNA nucleosides with different structures and properties can be incorporated. Therefore, highly specific oligonucleotides can be selected by optimization.
  • the wild-type oligonucleotide may be composed only of a non-natural nucleic acid, or may be composed of a non-natural nucleic acid and a natural nucleic acid (at least one of DNA and RNA). Moreover, when it comprises a non-natural nucleic acid and a natural nucleic acid, the number of non-natural nucleic acids contained in the wild-type oligonucleotide may be one or two or more.
  • a nucleoside of BNA can introduce a protecting group for nucleic acid synthesis by the phosphoramidate method and an activated phosphate group (amidate) in the same manner as a natural nucleoside.
  • BNA oligonucleotides can be synthesized.
  • LNA is only one type having a structure in which the 2'-oxygen and 4'-carbon of the ribose ring of the ribonucleoside are connected by a methylene chain.
  • An LNA oligonucleotide mixed with a natural nucleoside in the same manner as BNA can be synthesized using a nucleoside unit obtained by binding this structure and four types of bases.
  • the site that hybridizes with the target gene mutation site that is, the site that is non-complementary to the mutant nucleic acid is sufficiently inhibited from non-specific hybridization with the mutant nucleic acid. It can be determined appropriately in consideration of the type of gene mutation, the base sequence of the genomic region containing the target gene mutation site, and the like. For example, assuming that the wild-type oligonucleotide is an oligonucleotide formed entirely from DNA, the Tm value between the oligonucleotide and the wild-type nucleic acid is sufficiently higher than the Tm value between the oligonucleotide and the mutant nucleic acid.
  • the base sequence of the wild-type oligonucleotide is designed using known primer design software or the like. By replacing one or more bases in the designed base sequence with a non-natural nucleic acid, a wild-type oligonucleotide can be designed.
  • the wild-type oligonucleotide may be protected from the hydroxyl group at the 3 'end with a substituent so as not to cause an elongation reaction.
  • substituent include a phosphate group.
  • some DNA polymerases used for PCR have a 3 ' ⁇ 5' nuclease activity that is responsible for the repair function. Therefore, some substituents are decomposed and the extension reaction may proceed. Therefore, the substituent to be introduced into the hydroxyl group at the 3 'end of the wild-type oligonucleotide is preferably determined in consideration of the DNA polymerase to be used.
  • the site that hybridizes with the target gene mutation site in the wild-type oligonucleotide used in this embodiment is preferably not the 5 ′ end or 3 ′ end of the wild-type oligonucleotide, and is located near the center of the wild-type oligonucleotide. More preferably.
  • the gene mutation is a single nucleotide mutation, it is preferable that there is a site that hybridizes with the gene mutation site near the center of the wild-type oligonucleotide.
  • the region where the wild-type oligonucleotide hybridizes is in the region amplified by the forward primer and the reverse primer.
  • the forward primer and the reverse primer used in the present embodiment may be oligonucleotides formed from nucleosides constituting natural DNA, and oligos containing one or more kinds of unnatural bases in a part thereof. It may be a nucleotide.
  • Non-natural bases include LNA and BNA.
  • the binding with the template DNA can be strengthened, or the base specificity can be increased.
  • the non-natural base may inhibit the extension reaction by DNA polymerase, it is preferably on the 3 ′ end side of the primer.
  • a configuration in which a marker substance is bound and labeled for detection of an amplification product obtained by PCR amplification may be used.
  • the marker substance may be a substance that specifically binds to a specific protein such as biotin, or may be a directly detectable substance such as a fluorescent substance.
  • the forward primer and reverse primer can be designed using known software based on the base sequence of the genomic region containing the target gene mutation site so that the nucleic acid fragment containing the target gene mutation site can be amplified. it can. Specifically, Tm value, which is an index of the binding strength between primer and template DNA, secondary structure formation within primer, binding between primers, binding strength of primer to non-specific region of genome, etc. Designed.
  • the base length of the forward primer and the reverse primer can be set to, for example, 15 to 30 bases as in the case of primers generally used in PCR.
  • the length of the nucleic acid fragment that is PCR-amplified by the forward primer and the reverse primer is not particularly limited, and can be determined in consideration of how to use the amplified nucleic acid fragment.
  • the length can be 40 to several thousand bases, and preferably 40 to 500 bases.
  • the specimen is often derived from FFPE or a body fluid such as blood, and the DNA in the specimen as the template DNA may be fragmented. Many. In FFPE, fragmentation occurs due to formalin fixation, and in body fluids, DNA is often contained in exosomes, and the length is said to be about 180 bases.
  • the region to be amplified by the forward primer and the reverse primer is preferably as short as possible, preferably 200 bases or less, more preferably 150 bases or less.
  • the nucleic acid chain elongation reaction by DNA polymerase is a method in which a wild-type oligonucleotide hybridizes with a template DNA whose gene mutation site is a wild type, and a template DNA whose gene mutation site is a mutation type. Perform at a temperature that does not hybridize.
  • the extension reaction of thermostable DNA polymerase used for PCR is most efficiently performed at about 68 to 72 ° C.
  • the wild-type oligonucleotide used in this embodiment is designed so that the Tm value with the wild-type template DNA is 68 ° C. or higher and the Tm value with the mutant-type template DNA is less than 68 ° C. It is preferable. The larger the difference between the Tm value for the wild-type template DNA and the Tm value for the mutant-type template DNA, the higher the specificity and the more preferable.
  • the Tm value is an indicator of the stability of double strand formation, that is, the binding force with the template DNA.
  • the Tm value of an oligonucleotide formed from a natural nucleoside can be predicted by calculation based on a value obtained from an empirical value, and is generally obtained by calculation.
  • the Tm value between the LNA oligonucleotide and the template DNA single-stranded DNA formed from a natural nucleoside
  • the Tm value can be predicted.
  • the Tm value obtained in this way is used as a reference in designing primers and the like.
  • the calculated Tm value is only a predicted value, and the actual Tm value does not necessarily match the predicted value for any oligonucleotide or LNA oligonucleotide formed only from natural nucleosides.
  • BNA has a different structure from LNA and has two types of nucleosides of formula (1) or formula (2), and calculation of the formula is extremely difficult. For this reason, there is no method for examining the Tm value of a BNA oligonucleotide other than making a very rough prediction and actually conducting experiments and measurements.
  • the wild-type specific probe When the target gene mutation is a point mutation, the wild-type specific probe needs to be designed to distinguish between a mutant nucleic acid that differs by only one base and a wild-type nucleic acid and hybridize only to the wild-type nucleic acid. is there. For this reason, as a wild type specific probe, a TaqMan (registered trademark) probe bound with a minor groove binder that generally obtains high specificity, or an oligonucleotide portion that hybridizes with a template DNA, is highly specific and robust. A probe into which a non-natural nucleic acid that hybridizes is introduced. Examples of the non-natural nucleic acid include LNA and BNA, and BNA is particularly preferable.
  • the stability of the entire probe is enhanced, and at the same time, the specificity of the non-natural nucleic acid is exhibited in the base pair formation of the mutated portion, and the probe as a whole has a higher specificity.
  • the target gene mutation is a deletion mutation or insertion mutation with 3 or more consecutive deletion or insertion bases, the base between the wild type gene and the mutant gene There is a big difference in the arrangement. For this reason, it is relatively easy to increase the specificity of the wild-type specific probe, and sufficient specificity can be obtained even with an oligonucleotide probe formed only from a natural nucleic acid.
  • the mutant / wild type common probe hybridizes to a region where the nucleotide sequence of the mutant type and the wild type are common, there is no need to increase the specificity, and the oligonucleotide part that hybridizes to the template DNA is naturally present. It may be an oligonucleotide probe formed only from the nucleic acid. However, depending on the positional relationship between the wild-type oligonucleotide and the primer, a base sequence having a sufficient length for designing a mutant / wild-type common probe may not be ensured. In that case, non-natural nucleosides such as LNA and BNA can be introduced to enhance the stability, and the desired function can be provided even if the probe length is short. The same effect can be expected by introducing a molecule such as a groove binder.
  • the base sequence of the region that hybridizes with the template DNA in the wild type specific probe is designed based on the information on the base sequence of the wild type gene and the base sequence of the mutant gene. That is, the Tm value for the wild-type nucleic acid is equal to or higher than the temperature at the time of nucleic acid chain extension reaction of the real-time PCR to be performed, and the Tm value for the mutant nucleic acid is the temperature at the time of nucleic acid chain extension reaction of the real-time PCR to be performed.
  • the base sequence is designed to be sufficiently lower than that.
  • the design may be performed by a conventional method using known software or the like, or may be performed based on the actually measured Tm value.
  • the base sequence of the region where the mutant / wild type specific probe hybridizes with the template DNA is designed based on the information of the base sequence of the portion common to the wild type gene and the mutant gene. That is, the base sequence is designed so that the Tm value for the wild-type nucleic acid and the mutant nucleic acid is equal to or higher than the temperature at the time of nucleic acid chain extension reaction of real-time PCR to be performed.
  • the design may be performed by a conventional method using known software or the like, or may be performed based on the actually measured Tm value.
  • the wild-type specific probe and the mutant / wild-type common probe used in the present embodiment can be appropriately selected from known probes used in a method capable of detecting a PCR amplification product in real time.
  • a hydrolysis probe is an oligonucleotide probe that is labeled with a fluorescent substance and a quenching substance and includes a base sequence complementary to a PCR amplification product to be detected.
  • the hydrolysis probe does not emit fluorescence as it is by FRET (fluorescence resonance energy transfer).
  • the amount of amplification product produced can be monitored by measuring the fluorescence intensity.
  • the fluorescent substance and quenching substance for labeling the hydrolysis probe can be appropriately selected from combinations of fluorescent substances and quenching substances that can be used for FRET.
  • the fluorescent substance various substances having different fluorescent properties such as fluorescein, FAM, Yakima Yellow (registered trademark), FITC, VIC (registered trademark) are available.
  • quenching substances there are two types of quenching substances: a substance that does not emit light when it receives fluorescence energy from the donor side (releases energy as heat) and a substance that receives fluorescence energy and emits fluorescence of another wavelength. In general, a quenching substance that does not emit fluorescence is considered to be advantageous.
  • TAMRA is often used as a quencher that emits fluorescence.
  • the combination of a fluorescent substance and a quenching substance for labeling both probes is the fluorescence wavelength of the fluorescence emitted by the degradation of the mutant / wild type common probe and the fluorescence emitted by the degradation of the wild type specific probe.
  • a fluorescent substance and a quenching substance for labeling both probes is the fluorescence wavelength of the fluorescence emitted by the degradation of the mutant / wild type common probe and the fluorescence emitted by the degradation of the wild type specific probe.
  • HEX which is a fluorescein derivative
  • TAMRA can be used as the quencher.
  • FIG. 1 shows PCR primers (forward primer 2 and reverse primer 3), wild-type oligonucleotide 4, mutation when detecting a plurality of gene mutations concentrated in a specific genomic region 1a in genome 1
  • the set points of the type / wild type common probe 5 and the wild type specific probe 6 are schematically shown.
  • the PCR primer must be capable of amplifying a base sequence containing all mutations in the region.
  • the wild-type oligonucleotide should not selectively suppress only the amplification of the wild-type nucleic acid and inhibit the amplification of the mutant-type nucleic acid.
  • the mutant / wild-type common probe must be able to detect all mutant nucleic acids and wild-type nucleic acids without discrimination.
  • the wild type specific probe detects only wild type nucleic acid and should not detect any mutant nucleic acid.
  • the plurality of variants shown here are gene variants that are concentrated in a specific genomic region, and may be single-base mutations, deletion mutations, or insertion mutations. These may be mixed.
  • the ideal design as shown in FIG. 1 is not always possible, and the wild-type oligonucleotide and the base sequence of the wild-type specific probe, or the mutant / wild-type common
  • the base sequence of the probe and the wild-type specific probe may overlap with the base sequence of the wild-type oligonucleotide and the mutant / wild-type common probe.
  • the length and base composition of the overlapping parts, and whether the overlapping nucleotides or probes are the same template strands or complementary template strands are designed, and each is as described above. Select combinations that demonstrate specificity.
  • FIG. 2A to FIG. 2C show expected amplification curves when real-time PCR was performed using a mutant / wild-type common probe and a hydrolysis probe as a wild-type specific probe.
  • FIG. 2A shows an amplification curve when the specimen (template DNA) contains only the wild type gene.
  • FIG. 2B shows an amplification curve when the specimen contains very few mutant genes.
  • FIG. 2C shows an amplification curve when the specimen contains a sufficiently large number of mutant genes.
  • the amplification product obtained is only a wild-type nucleic acid that was not amplified and suppressed by the wild-type oligonucleotide. Therefore, both the mutant / wild type common probe and the wild type specific probe bind to all amplification products. Therefore, the amplification curves obtained from the mutant / wild-type common probe and the wild-type specific probe are the same (FIG. 2A).
  • the obtained amplification product includes a mutant nucleic acid in addition to the wild-type nucleic acid that was not amplified and suppressed by the wild-type oligonucleotide.
  • the mutant / wild-type common probe reacts with both the wild-type nucleic acid that has been amplified leaking from the amplification suppression by the wild-type oligonucleotide and the mutant nucleic acid that has been amplified without being suppressed. Accordingly, the amplification curve obtained is the sum of the wild-type nucleic acid and the mutant nucleic acid obtained by amplification.
  • wild type specific probes react only with wild type nucleic acids. Therefore, when even a slight amount of the mutant gene is present in the specimen, the rise of the mutant / wild-type common probe is faster than that of the wild-type specific probe in the amplification curve of real-time PCR (FIG. 2B).
  • the ratio of mutant genes When the ratio of mutant genes is large, the amount of mutant nucleic acids to be amplified is also large, and the mutant / wild type common probe will be launched at an extremely early stage of the PCR cycle.
  • the proportion of the wild type gene is small compared to the case where the specimen contains only the wild type gene or a few mutant genes, and amplification is strongly suppressed. Therefore, the rise of the wild type specific probe is further delayed compared to the other two specimens (FIG. 2C).
  • the reactivity of the probe varies from probe to probe, and even if the same amount of amplification product is obtained, the number of cycles of rising is not always the same.
  • the comparison of the response of the mutant / wild-type common probe and the response of the wild-type specific probe is performed in advance, and the actual data is reflected to reflect the result. May be corrected.
  • Real-time PCR buffered forward and reverse primers wild-type oligonucleotides, mutant / wild-type common probes, wild-type specific probes, template DNA, 4 types of deoxynucleotide triphosphates, and heat-resistant DNA polymerase
  • the reaction mixture is used as a reaction solution, and the liquid temperature of the reaction solution is raised or lowered according to the temperature cycle.
  • the deoxynucleotide triphosphate, heat-resistant DNA polymerase, and buffer used can be appropriately selected from those generally used in PCR.
  • Various thermostable DNA polymerases with different characteristics are on the market, but general Taq DNA polymerase is often used.
  • a DNA polymerase having a calibration function that reduces the error rate of the extension reaction may be used.
  • the concentration of the forward primer and the reverse primer in the reaction solution can be the same as that of a primer used in general PCR.
  • the amount of the wild-type oligonucleotide in the reaction solution is not particularly limited as long as the amount of the PCR-clamping effect can be obtained. For example, it can be set to 1/3 equivalent of the forward primer or reverse primer, and can be optimized with 1/10 equivalent as a guide.
  • PCR General temperature conditions for PCR can be used as the temperature conditions for real-time PCR. That is, a denaturation reaction near 95 ° C., a primer annealing reaction near 60 ° C., and an extension reaction near 72 ° C.
  • PCR may be performed by setting only two points, a denaturation reaction and an annealing reaction, without setting the extension reaction time.
  • it is important that the wild-type oligonucleotide is firmly hybridized to the wild-type template DNA during the extension reaction. For this reason, the above three points of temperature setting may increase the specificity.
  • the hot start method which introduces a mechanism that prevents the extension reaction from proceeding at low temperatures, is a very important technique for increasing the specificity of PCR primer extension, and is also used in the implementation of real-time PCR in this embodiment. It is preferable to do.
  • the double-stranded DNA containing the same base sequence as the genomic region containing the target gene mutation site used as the template DNA may be the genomic DNA itself, and includes the target gene mutation site using the genomic DNA as a template.
  • An amplification product obtained by previously amplifying the genomic region by PCR or the like may be used.
  • Genomic DNA can be extracted from a biological sample such as a cell or tissue by a conventional method.
  • the cell or tissue collected from the subject for obtaining genomic DNA is not particularly limited as long as it contains genomic DNA, and may include any cell or tissue.
  • cDNA synthesized by reverse transcription reaction using total RNA extracted from a cell or tissue collected from a subject as a template can be used as a template DNA.
  • the reverse transcription reaction and the real-time PCR performed in this embodiment may be performed continuously in the same reaction container.
  • Extraction of DNA and RNA from a biological sample is to obtain substantially only nucleic acid components by decomposing cells and fine vesicles and excluding components such as proteins other than DNA and RNA.
  • it means separating DNA or RNA floating in a body fluid from other components.
  • the separation of other components is not necessarily strict, but it is necessary to strictly remove or deactivate the RNase when detecting DNA and the RNase when detecting RNA.
  • substances that are present in body fluids and that inhibit the reaction of heat-resistant DNA polymerase must be removed to the extent that the reaction is not inhibited.
  • the substance includes hemoglobin contained in blood.
  • the kit for detecting a genetic mutation according to the second embodiment of the present invention includes the wild-type oligonucleotide, the forward primer, the reverse primer, the mutant / wild-type common probe, and the wild-type specific probe. Have.
  • the gene mutation detection kit By using the gene mutation detection kit, the gene mutation detection method according to the first embodiment can be more easily carried out.
  • the gene mutation detection kit includes a buffer solution for preparing a PCR reaction solution, a heat-resistant DNA polymerase, four types of deoxynucleotide triphosphates, and instructions for using the gene mutation detection kit. Etc. may be provided.
  • the base sequence of the BNA oligonucleotide used was described according to the following notation. That is, among the compounds having the BNA structure represented by the formula (2), those containing an adenine base are A (E), those containing a guanine base are G (E), and those containing a 5-methylcytosine base are 5 (E), those containing a thymine base are denoted as T (E). Of those having the BNA structure represented by the formula (1), those containing thymine are represented as T (H), and those containing 5-methylcytosine are represented as 5 (H). Furthermore, when a phosphate group was introduced into the 3 'hydroxyl group at the 3' end of the BNA oligonucleotide, "p" was added. Natural nucleosides were designated as A, G, C, and T.
  • the PCR template DNA used in the following experiments was a linearized product obtained by cutting an artificially synthesized plasmid with the restriction enzyme Hind III, and 20,000 to 30,000 copies were used per PCR reaction.
  • the plasmid was obtained by inserting the hEGFR fragment sequence into the restriction enzyme EcoRV site of pMD20T, and was purchased from Takara Bio Inc.
  • Table 1 shows the name of the plasmid, the base sequence of the inserted hEGFR fragment, and its SEQ ID NO.
  • the plasmid name “EG_” is followed by the genotype of the EGFR fragment inserted into the plasmid.
  • CFX96 real-time PCR detection system manufactured by BioRad
  • the reaction conditions were 95 ° C. (30 seconds), 95 ° C. (20 seconds), 58 ° C. (30 seconds), and 72 ° C. (30 seconds) in order of 50 cycles.
  • SEQ ID NO: 11 is the number of the base sequence before various modifications of the BNA oligonucleotide.
  • EG_19wt (Ex19w) which is a wild-type plasmid, plasmid EG_del746_750 (2236) (2236), EG_del746_750 (2235) (2235), EG_delL747_A750insP (A750_S75Ps), EG_delL747_A750insP (A750PS) (P753S), EG_del747_T751insS (T751S), and EG_delL747_S752E746V (E746V) were used.
  • FIG. 3A The result of measuring the fluorescence intensity of FAM in real-time PCR is shown in FIG. 3A, and the result of measuring the fluorescence intensity of HEX is shown in FIG. 3B.
  • FAM signal when the template DNA is a mutant plasmid, the amplification curve rises quickly, and when the template DNA is a wild-type plasmid, the rise is clearly delayed (FIG. 3A).
  • HEX signal the rise of the amplification curve was seen only when the wild type plasmid was used as the template DNA, and when the mutant plasmid was used as the template DNA, no rise of the amplification curve was seen at all. (FIG. 3B).
  • the amplification curve in FIG. 3A and the amplification curve in FIG. 3B both show an increase curve of the same wild-type nucleic acid amplification product.
  • the rise of the amplification curve varies depending on the amount and quality of DNA contained in the specimen.
  • the signal of HEX shows only the amount of amplification product of the wild type nucleic acid.
  • the FAM signal is only an amplification product of the wild-type nucleic acid, and if the sample contains a mutant-type nucleic acid, the FAM signal is an amplification curve that combines the amplification products of both wild-type and mutant-type nucleic acids. . Therefore, if the rise of the amplification curve by the FAM signal is earlier than the rise of the HEX amplification curve, the template DNA contains the mutant nucleic acid. Actually, the rise of the FAM signal and the HEX signal is not exactly the same even if the specimen is a wild-type nucleic acid because of the difference depending on the nature of the probe. Therefore, correction may be performed based on the respective values when the specimen is only the wild-type nucleic acid.
  • SEQ ID NO: 16 is the number of the base sequence before various modifications of the BNA oligonucleotide.
  • EG_19wt which is a wild type plasmid and plasmid EG_del752_759 (2253) (2253) having a deletion mutant type sequence of exon 19 of hEGFR were used.
  • FIG. 4A The result of measuring the fluorescence intensity of FAM in real-time PCR is shown in FIG. 4A, and the result of measuring the fluorescence intensity of HEX is shown in FIG. 4B.
  • the FAM signal when the template DNA was a mutant plasmid, the rise of the amplification curve was fast, and when the template DNA was a wild-type plasmid, the rise was clearly delayed (Fig. 4A).
  • the HEX signal the rise of the amplification curve was seen only when the wild type plasmid was used as the template DNA, and when the mutant plasmid was used as the template DNA, no rise of the amplification curve was seen at all. .
  • the gene mutation detection method according to the present invention is particularly useful in clinical examinations.

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Abstract

La présente invention concerne un procédé de détection d'une mutation génétique, l'amplification en chaîne par polymérase (PCR) en temps réel est effectuée en utilisant : un ADN matrice qui présente un ADN double brin contenant une séquence de base qui est identique à une région génomique contenant un site de mutation génétique cible ; une amorce motrice et une amorce inverse qui sont configurées pour amplifier, par l'intermédiaire de la PCR, la région contenant le site de mutation génétique ; un oligonucléotide de type sauvage qui contient au moins un acide nucléique non d'origine naturelle et qui présente une séquence de base qui est complémentaire d'une région génomique dans laquelle le site de mutation génétique est de type sauvage ; une sonde de jonction de type mutant/type sauvage qui est configurée pour détecter à la fois un acide nucléique auquel le site de mutation génétique est de type sauvage et un acide nucléique au niveau duquel le site de mutation génétique est de type mutant ; et une sonde spécifique de type sauvage configurée pour détecter spécifiquement un acide nucléique au niveau duquel le site de mutation génétique est de type sauvage. Un ADN double brin au niveau duquel le site de mutation génétique est de type mutant est détecté parmi l'ADN modèle sur la base de la différence, parmi les produits obtenus amplifiés par PCR, entre la quantité de produits amplifiés par PCR détectés par la sonde de jonction de type mutant/de type sauvage et la quantité de produits amplifiés par PCR détectés par la sonde spécifique de type sauvage.
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JP2018113898A (ja) * 2017-01-18 2018-07-26 国立大学法人広島大学 遺伝子の変異検出キット及び遺伝子の変異検出方法
WO2019004335A1 (fr) * 2017-06-28 2019-01-03 東洋鋼鈑株式会社 Procédé d'évaluation d'une mutation génétique, et kit d'évaluation d'une mutation génétique
CN110343747A (zh) * 2019-07-09 2019-10-18 北京市医疗器械检验所 一种校正egfr基因突变频率的定值引物和探针组
WO2020067388A1 (fr) * 2018-09-27 2020-04-02 東洋鋼鈑株式会社 Kit d'évaluation de mutations génétiques pertinentes pour la prédiction pronostique du carcinome papillaire de la thyroïde
CN114369597A (zh) * 2022-01-11 2022-04-19 重庆迪安医学检验中心有限公司 一种通用型探针检测芯片及其应用
WO2024048608A1 (fr) * 2022-09-01 2024-03-07 東洋鋼鈑株式会社 Kit de test pour le syndrome myélodysplasique

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018113898A (ja) * 2017-01-18 2018-07-26 国立大学法人広島大学 遺伝子の変異検出キット及び遺伝子の変異検出方法
WO2019004335A1 (fr) * 2017-06-28 2019-01-03 東洋鋼鈑株式会社 Procédé d'évaluation d'une mutation génétique, et kit d'évaluation d'une mutation génétique
JPWO2019004335A1 (ja) * 2017-06-28 2020-05-07 東洋鋼鈑株式会社 遺伝子変異評価方法、遺伝子変異評価用キット
JP7248982B2 (ja) 2017-06-28 2023-03-30 東洋鋼鈑株式会社 遺伝子変異評価方法、遺伝子変異評価用キット
WO2020067388A1 (fr) * 2018-09-27 2020-04-02 東洋鋼鈑株式会社 Kit d'évaluation de mutations génétiques pertinentes pour la prédiction pronostique du carcinome papillaire de la thyroïde
CN110343747A (zh) * 2019-07-09 2019-10-18 北京市医疗器械检验所 一种校正egfr基因突变频率的定值引物和探针组
CN114369597A (zh) * 2022-01-11 2022-04-19 重庆迪安医学检验中心有限公司 一种通用型探针检测芯片及其应用
CN114369597B (zh) * 2022-01-11 2024-02-09 重庆迪安医学检验中心有限公司 一种通用型探针检测芯片及其应用
WO2024048608A1 (fr) * 2022-09-01 2024-03-07 東洋鋼鈑株式会社 Kit de test pour le syndrome myélodysplasique

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