WO2006082685A1 - Procede de detection d’un polymorphisme nucleotidique - Google Patents

Procede de detection d’un polymorphisme nucleotidique Download PDF

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WO2006082685A1
WO2006082685A1 PCT/JP2005/022985 JP2005022985W WO2006082685A1 WO 2006082685 A1 WO2006082685 A1 WO 2006082685A1 JP 2005022985 W JP2005022985 W JP 2005022985W WO 2006082685 A1 WO2006082685 A1 WO 2006082685A1
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nucleic acid
nucleotide polymorphism
single nucleotide
residue
evaluated
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PCT/JP2005/022985
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English (en)
Japanese (ja)
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Kazuhiko Nakatani
Hitoshi Suda
Akio Kobori
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Kyoto University
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Priority to JP2007501513A priority Critical patent/JP4752071B2/ja
Publication of WO2006082685A1 publication Critical patent/WO2006082685A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G01N21/80—Indicating pH value
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention relates to a method for detecting a single nucleotide polymorphism in a target nucleic acid and a kit thereof. More specifically, the present invention relates to a method for detecting a single nucleotide polymorphism in a target nucleic acid with high sensitivity and a kit thereof.
  • SNPs Single nucleotide polymorphisms
  • the detection of the SNPs is based on, for example, differences in mobility in electrophoresis, primer extension, hybridization using a probe labeled with a labeling substance such as a fluorescent substance, radioactive substance, amplification by PCR, etc. This is done by a detection method such as typing.
  • the separation of fragments in electrophoresis is largely performed using an increase in the difference in secondary structure due to polymorphism as an index for a DNA fragment to be analyzed and a reference DNA fragment.
  • the heterozygous reference DNA fragment and the DNA fragment to be analyzed are electrophoretically separated simultaneously with the same chirality under the following temperature conditions, and the peak is corrected with the reference DNA fragment 1 ⁇ ,
  • Detection method that examines the presence or absence of loss of heterozygosity of the DNA fragment to be analyzed (Patent Document 1); a chromosome containing a specific single nucleotide polymorphic site contained in a sample or a fragment thereof, and a wild-type primer and A detection method in which one or two types of mutant primers are allowed to act simultaneously or separately with DNA polymerase to examine the presence or absence of primer-based extension (Patent Document 2); multiple pairs of primers to be analyzed with genomic DNA And Detection method that amplifies a
  • Permissible literature 4 When the target nucleic acid region is not changed, the biological sample is contacted with a plurality of single-stranded peptide nucleic acids that continuously hybridize to the region, and then mismatched nucleic acid (single-stranded nucleic acid) Examples include a detection method (Patent Document 5) in which an agent that degrades (nucleic acid) is added and then the presence of degradation products is examined.
  • Patent Document 1 Japanese Patent Laid-Open No. 2002-78500
  • Patent Document 2 Pamphlet of International Publication No. 01Z042498
  • Patent Document 3 JP 2002-300894
  • Patent Document 4 JP 2003-52372
  • Patent Document 5 Special Table 2004— 521630
  • the gist of the present invention is as follows:
  • a signal based on the compound of (B) that recognizes cytosine bulge or thymine bulge formed when the nucleic acid probe and the nucleic acid to be evaluated are hybridized is detected, and the single nucleotide polymorphism is evaluated. Detection method of nucleotide polymorphism,
  • nucleic acid to be evaluated containing at least one single nucleotide polymorphism, and a cytosine residue at a position adjacent to the 5 ′ or 3 ′ end of the position where the single nucleotide polymorphism is paired
  • a cytosine residue or thymine residue is added at a position adjacent to the terminal side, and at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is further substituted with an inosine residue
  • [8] ⁇ ) comprising at least one single nucleotide polymorphism and capable of hybridizing complementarily to a nucleic acid to be evaluated containing a wild-type nucleotide at the position where the nucleotide polymorphism is present;
  • a nucleic acid probe having a base sequence to which a cytosine residue or a thymine residue is added at a position adjacent to the 5 ′ or 3 ′ end of the binding position, and Z or
  • ⁇ ) Comprising at least one single nucleotide polymorphism and complementary to the antisense strand of the nucleic acid to be evaluated containing the wild type nucleotide at the position where the nucleotide polymorphism is present.
  • a nucleic acid probe having a base sequence to which a cytosine residue or a thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position that matches the single nucleotide polymorphism on the antisense strand;
  • ⁇ ′ contains at least one single nucleotide polymorphism, and is capable of hybridizing complementarily to the nucleic acid to be evaluated containing the mutant nucleotide at the base polymorphism location, and matches the single nucleotide polymorphism.
  • a kit for use in the method for detecting a single nucleotide polymorphism in a nucleic acid according to any one of [1] to [7] V above,
  • i ′ comprising at least one single nucleotide polymorphism and capable of hybridizing complementarily to a nucleic acid to be evaluated containing a wild-type nucleotide at the position of the nucleotide polymorphism, wherein the single nucleotide polymorphism
  • a cytosine residue or thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the paired position, and the guanine residue present adjacent to the cytosine residue or thymine residue.
  • a nucleic acid probe having a base sequence in which at least one is replaced with an inosine residue;
  • a nucleic acid probe having a substituted base sequence comprising at least one single nucleotide polymorphism, and capable of complementary and antisense to the antisense strand of the nucleic acid to be evaluated containing the wild-type nucleotide at the position where the nucleotide polymorphism is present;
  • a cytosine residue or thymine residue is added at a position adjacent to the 5 'or 3' terminal side of the position where it is paired with a single nucleotide polymorphism on the chain, and is adjacent to the cytosine residue or thymine residue.
  • At least one of the guanine residues Furthermore, a nucleic acid probe having a substituted base sequence,
  • a cytosine residue or thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side, and at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is replaced with an inosine residue.
  • the antisense strand comprising at least one single nucleotide polymorphism, which can be complemented and hybridized to the antisense strand of the nucleic acid to be evaluated containing the mutant nucleotide at the position where the nucleotide polymorphism exists.
  • a cytosine residue or thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position where the single nucleotide polymorphism above is paired, and is present adjacent to the cytosine residue or thymine residue.
  • a nucleic acid probe having a base sequence in which at least one of guanine residues is further substituted with an inosine residue;
  • the detection method of the present invention single nucleotide polymorphism in a nucleic acid can be detected easily at a low cost and with high sensitivity without performing a complicated process such as labeling with a labeling substance. There is an excellent effect of being able to. Moreover, according to the kit of the present invention, there is an excellent effect that the detection method can be performed efficiently and simply at low cost.
  • FIG. 1 is a schematic diagram showing an example of the use of a nucleic acid probe for a target nucleic acid.
  • the underlined bold letters indicate the positions of single nucleotide polymorphisms to be evaluated.
  • the black circle indicates 2,7-diamino-1,8-naphthyridine.
  • FIG. 2 is a diagram showing the absorption spectrum and fluorescence spectrum of 10 / z M 2,7-diamino-1,8-naphthyridine under various conditions.
  • the left panel shows an absorption spectrum at 300 nm to 450 nm.
  • the right panel shows the fluorescence spectrum of 10 M 2,7-diamino-1,8-naphthyridine.
  • the excitation wavelength for fluorescence measurement has an absorption maximum in each absorption spectrum.
  • FIG. 3 is a diagram showing an absorption spectrum and a fluorescence spectrum of 10 / z M 2,7-diamino-1,8-naphthyridine bound to adenine bulge, thymine bulge, cytosine bulge and guanine bulge.
  • the left panel shows the absorption spectrum from 300 nm to 450 nm.
  • the right panel shows the fluorescence spectrum of 10 / z M 2,7-diamino-1,8-naphthyridine.
  • the excitation wavelength for fluorescence measurement is the absorption maximum in each absorption spectrum.
  • FIG. 4 shows a fluorescence spectrum based on the fluorescence of 2,7-diaminol 1,8-naphthyridine bound to a bulge in the presence of C or A allele.
  • a allele is a nucleic acid containing the base sequence shown in SEQ ID NO: 1
  • T probe is a nucleic acid containing the base sequence shown in SEQ ID NO: 2
  • G probe is shown in SEQ ID NO: 3.
  • Nucleic acid containing the base sequence shown below, C allele is the nucleic acid containing the base sequence shown in SEQ ID NO: 4
  • Iprobe is the nucleic acid containing the base sequence shown in SEQ ID NO: 5.
  • the present invention relates to a method for detecting a single nucleotide polymorphism in a nucleic acid.
  • the detection method of the present invention specifically comprises: (A) I) a position capable of complementary hybridization to a nucleic acid to be evaluated containing at least one single nucleotide polymorphism and paired with the single nucleotide polymorphism.
  • a nucleic acid probe having a base sequence to which a cytosine residue or thymine residue is added at a position adjacent to the 5 ′ or 3 ′ end of
  • nucleic acid probe having a base sequence to which a cytosine residue or thymine residue is added at a position adjacent to the terminal side;
  • a method comprising: detecting a signal based on the compound (B) that recognizes cytosine or thymine bulge formed when the nucleic acid probe and a nucleic acid to be evaluated are hybridized, and evaluating the single nucleotide polymorphism. It is.
  • a compound containing a naphthyridine ring is formed by nucleotides adjacent to a bulge base that shifts the wavelength power of the absorption maximum before binding by specifically binding to the noresi structure. This is based on the inventors' surprising knowledge that the fluorescence intensity varies depending on the type of base pair.
  • the present inventors can hybridize complementarily to a nucleic acid having a single nucleotide polymorphism, and have a probe containing a bulge base adjacent to the position where the single nucleotide polymorphism is paired with the single nucleotide polymorphism.
  • a duplex containing a bulge structure is formed by hybridizing with a nucleic acid, and the fluorescence of a compound containing a naphthyridine ring is bound to a bulge base in the next step, and the nucleotide at the position where the single nucleotide polymorphism exists is detected. It was found that single nucleotide polymorphisms can be detected from fluctuations in fluorescence intensity caused by differences.
  • base refers to deoxyribonucleotide unless otherwise specified. Therefore, in the present specification, “cytosine” (C), “thymine” (T), “adenine” ( ⁇ ) and “guanine” (G) are each deoxyribonucleotide, That is, it means “2′-deoxycytidine”, “2′-deoxythymidine”, “2′-deoxyadenosine” and “2′-deoxyguanosine”, respectively.
  • the nucleic acid to be evaluated containing at least one single nucleotide polymorphism means that at least one, more specifically, 1 to 5 single nucleotide polymorphisms exist.
  • the nucleotide present at the position of the single nucleotide polymorphism includes both wild-type nucleotides and mutant nucleotides.
  • wild-type nucleotide at the position of single nucleotide polymorphism is a nucleotide at a site where a single nucleotide polymorphism is confirmed, and is a strong portion in a so-called normal nucleotide sequence.
  • the nucleotide at the position of the single nucleotide polymorphism is the nucleotide at the site where the single nucleotide polymorphism has been confirmed, and in the so-called mutant nucleotide sequence. Nucleotides in the powerful parts! Uh.
  • bulge base means an unpaired base in double-stranded DNA
  • buldycytosine or “baldithymine” means that in double-stranded DNA, respectively.
  • a noble structure composed of a strong bulge base consists of a single-stranded DNA having a specific base sequence ability and a base sequence further including one extra nucleotide at an arbitrary position in the base sequence.
  • single-stranded DNA is subjected to hybridization, it can be generated as a duplex containing a bulge structure.
  • a cytosine residue or a thymine residue added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position where the single nucleotide polymorphism is paired corresponds to the bulge base.
  • cytosine bulge or "thymine bulge” means a bulge that is generated by the surplus of one cytosine or thymine with respect to the corresponding single-stranded DNA.
  • the term “compound capable of recognizing a bulge base” refers to, for example, forming a hydrogen bond with a bulge base present in the duplex to thereby stabilize the duplex having the bulge base.
  • examples include compounds having a naphthyridine ring, 2,6-diaminopyridine, and xanthine.
  • a compound having a naphthyridine ring that emits fluorescence as a signal is preferable because of easy detection and evaluation.
  • the detection method of the present invention is capable of detecting a single nucleotide polymorphism without using a labeling substance such as a fluorescent substance by using a compound having a naphthyridine ring as a compound capable of recognizing a bulge base. Exhibits excellent effects. Therefore, in the detection method of the present invention, extension reaction, amplification reaction, enzyme reaction (e.g., extension reaction, amplification reaction, mismatched nucleic acid degradation reaction, etc.), examination of electrophoresis conditions, labeling with a labeling substance, etc. A single nucleotide polymorphism can be easily detected without performing a complicated process such as the above. Furthermore, since the compound containing the naphthyridine ring specifically binds to the noresi, the level of the knock ground signal is reduced, and single nucleotide polymorphisms can be detected with high sensitivity.
  • a labeling substance such as a fluorescent substance
  • the compound containing the naphthyridine ring shifts the wavelength at which the absorption maximum of the compound containing the naphthyridine ring shifts due to binding to the bulge and is adjacent to the bulge base.
  • the fluorescence intensity varies depending on the type of nucleotide residue paired with the leotide residue (ie, allele). Therefore, the binding to the bulge can be evaluated by the shift of the wavelength showing the strong absorption maximum, and the single nucleotide polymorphism can be evaluated by the fluctuation of the fluorescence intensity.
  • Examples of the compound containing a naphthyridine ring include the following (I):
  • R 1 and R 2 each independently represent a primary amin residue, a secondary ammine residue or a tertiary ammine residue
  • Examples of the primary amine residue include an —NH group.
  • Examples of the secondary amin residue include:
  • examples of the tertiary ammine residue include a —N (CH 3) (CH 2) NH group and the like.
  • both R 1 and R 2 are secondary. It is preferably an amino acid residue.
  • 2,7-diamino-1,8-naphthyridine is shown.
  • the 2,7-diamino-1,8-naphthyridine can be synthesized, for example, by the method described in JP-A-2004-262827.
  • the wavelength that shifts by 20 nm and shows the maximum value of fluorescence intensity shifts from 398 ⁇ m to 430 nm by about 32 nm.
  • the same properties as 2,7-diamino-1,8-naphthyridine, that is, for example, generate fluorescence have binding ability to bulge, and bind to bulge.
  • Any derivative compound of 2,7-diamino-1,8-naphthyridine may be used as long as the absorption maximum wavelength is shifted and the fluorescence intensity is changed by the allele.
  • R 3 and R 4 are each independently a hydrogen atom or an amino group, and 1, m and n are
  • R 3 and R 4 are each independently a hydrogen atom or an amino group, which produces fluorescence and has a binding ability to a bulge.
  • one of them is preferably an amino group.
  • the m and n are each independently a natural number of 1-6.
  • the above 1 is preferably 2 or more, more preferably 3 or more, preferably 6 or less, more preferably 5 or less, even more preferably 4 or less, specifically, preferably 2 to 6 More preferably, it is 3-5, and more preferably 3-4.
  • the m is preferably 2 or more, more preferably 3 or more, preferably 6 or less, more preferably 5 or less, and still more preferably 4 or less. Specifically, it is preferably 2 to 6, more preferably 3 to 5, and still more preferably 3 to 4.
  • n is preferably 2 or more, more preferably 3 or more, preferably 6 or less, more preferably 5 or less, and further preferably 4 or less. Specifically, it is preferably 2-6, more preferably 3-5, and still more preferably 3-4.
  • R 5 and R 6 are each independently a hydrogen atom or an amino group, generate fluorescence, and have an ability to bind to a bulge. It is desirable that at least one is an amino group from the viewpoint of sufficiently exhibiting the property that the absorption maximum wavelength shifts due to the binding to the bulge and the fluorescence intensity changes due to the allele.
  • the o and p are each independently a natural number of 1-6.
  • the above o is Preferably, it is 2 or more, more preferably 3 or more, preferably 6 or less, more preferably Specifically, it is preferably 5 or less, more preferably 4 or less, preferably 2 to 6, more preferably 3 to 5, and further preferably 3 to 4. It is desirable that From the same viewpoint, the p is preferably 2 or more, more preferably 3 or more, preferably 6 or less, more preferably 5 or less, and still more preferably 4 or less. Specifically, it is preferably 2 to 6, more preferably 3 to 5, and still more preferably 3 to 4.
  • Hybridization when the nucleic acid probe of the present invention hybridizes complementarily to the nucleic acid to be evaluated containing at least one single nucleotide polymorphism or to the antisense strand of the nucleic acid to be evaluated is ImM to: LM, preferably Is preferably performed in a buffer solution containing 10 to 100 mM sodium chloride, pH 5 to 8, preferably pH 6 to 7, preferably phosphate buffer.
  • nucleic acid probe of the above-mentioned I) of the present invention has a base sequence corresponding to the antisense strand of the nucleic acid to be evaluated containing a single nucleotide polymorphism, and the single nucleotide polymorphism in the nucleic acid to be evaluated
  • a nucleic acid probe to which a cytosine residue or a thymine residue is added at a position adjacent to a nucleotide that forms a base pair with a nucleotide present at the site of A nucleotide sequence complementary to the nucleotide sequence that is, a nucleotide sequence corresponding to the sense strand of the nucleic acid to be evaluated containing a single nucleotide polymorphism, and a cytosine residue at a position adjacent to the nucleotide present in the site of the single nucleotide polymorphism.
  • one embodiment of the probe of the above I) or ⁇ ) is: I) a nucleotide sequence corresponding to an antisense strand of a continuous nucleotide sequence containing nucleotides having a single nucleotide polymorphism, and the single nucleotide polymorphism
  • a nucleic acid probe comprising a nucleotide sequence to which a cytosine residue or a thymine residue is added adjacent to the 5 ′ or 3 ′ terminal side of the position on the antisense strand corresponding to the existing position; or
  • a nucleic acid probe containing a base sequence to which a group or a thymine residue is added is added.
  • the nucleic acid probe used in the present invention includes a wild-type nucleotide at the position where the single nucleotide polymorphism exists.
  • Examples thereof include a nucleic acid probe containing a leotide and a nucleic acid probe containing a mutated nucleotide at the position where the single nucleotide polymorphism exists.
  • the nucleic acid probe to be used has a cytosine residue or a thymine residue at a position adjacent to the terminal 5 or 3, and a position adjacent to the terminal nucleic acid. Another major feature is that it contains an added base sequence. Therefore, in the detection method of the present invention, the nucleic acid probe is hybridized with the nucleic acid to be evaluated for single nucleotide polymorphism, thereby generating a bulge structure that recognizes and binds to the compound containing the naphthyridine ring.
  • cytosine bulge or thymine bulge-specific binding of the compound containing the naphthyridine ring can be induced, the knock signal level can be reduced, and detection with high sensitivity becomes possible.
  • the cytosine residue or thymine residue added adjacent to the 5 ′ or 3 ′ end corresponds to the “bulge base”.
  • the knowledge structure is generated when a nucleic acid probe has a bulge base when a nucleic acid probe is hybridized with a single nucleotide polymorphism nucleic acid to be evaluated.
  • a compound containing a naphthyridine ring recognizes and binds to a bulge structure by forming a hydrogen bond with a bulge base.
  • the nucleic acid probe has a basic guanine adjacent to the bulge base, i) at least from the viewpoint of suppressing the quenching of the signal based on the compound capable of recognizing the bulge base.
  • nucleic acid probe comprising a nucleotide sequence to which a thymine residue is added and at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is further substituted with an inosine residue, or
  • a cytosine residue or thymine residue is added at a position adjacent to the terminal side, and at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is further substituted with an inosine residue
  • a nucleic acid probe having the base sequence described above is preferred.
  • nucleic acid probe of i) or ii) is: i) a nucleotide sequence corresponding to an antisense strand of a continuous nucleotide sequence containing a nucleotide having a single nucleotide polymorphism, and the single nucleotide A cytosine residue or thymine residue is added adjacent to the 5 ′ or 3 ′ end of the position on the antisense strand corresponding to the polymorphic position, and adjacent to the cytosine residue or thymine residue.
  • a nucleic acid probe containing a base sequence in which at least one of the guanine residues present is further substituted with an inosine residue, or
  • a nucleic acid probe comprising a base sequence to which a group or thymine residue is added and at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is further substituted with an inosine residue;
  • nucleic acids i) and ii) are suitable when the base adjacent to the position where the single nucleotide polymorphism is present is cytosine.
  • the type of the nucleic acid probe is appropriately selected. Can be selected.
  • nucleic acid probe containing a wild-type nucleotide at the position where the single nucleotide polymorphism exists specifically includes ⁇ ) at least one single nucleotide polymorphism, and A cytosine residue or a thymine residue at a position adjacent to the 5 ′ or 3 ′ end of the position where the single nucleotide polymorphism is paired.
  • ⁇ comprising at least one single nucleotide polymorphism, and capable of complementary and antisense to the antisense strand of the nucleic acid to be evaluated containing the wild type nucleotide at the position where the nucleotide polymorphism exists, and said antisense strand
  • a nucleic acid probe having a base sequence to which a cytosine residue or a thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position that matches the single nucleotide polymorphism above;
  • i ′ contains at least one single nucleotide polymorphism, and the wild type nucleo It can be hybridized in a complementary manner to the nucleic acid to be evaluated containing tides, and a cytosine residue or a thymine residue is added at a position adjacent to the 5 ′ or 3 ′ end of the position where the single nucleotide polymorphism is paired.
  • a nucleic acid probe having a base sequence in which at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is substituted with an inosine residue
  • cytosine residue or thymine residue is added at a position adjacent to the 5 'or 3' terminal side of the position where it is paired with a single nucleotide polymorphism on the chain, and is adjacent to the cytosine residue or thymine residue.
  • a nucleic acid probe having a base sequence in which at least one of the guanine residues to be further substituted with an inosine residue;
  • nucleic acid probe containing a mutant nucleotide at the position where the single nucleotide polymorphism exists specifically includes ⁇ ′) at least one single nucleotide polymorphism, and the mutant nucleotide exists at the position where the nucleotide base polymorphism exists. And a cytosine residue or a thymine residue is added to a position adjacent to the 5 ′ or 3 ′ terminal side of the position where the single nucleotide polymorphism is paired.
  • nucleic acid to be evaluated which contains at least one single nucleotide polymorphism, and the mutant nucleotide is present at the position where the nucleotide polymorphism is present, and the antisense strand
  • a nucleic acid probe having a base sequence to which a cytosine residue or a thymine residue is added at a position adjacent to the 5 ′ or 3 ′ end of the single nucleotide polymorphism above,
  • a cytosine residue or thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side, and at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is replaced with an inosine residue.
  • a nucleic acid probe having a defined base sequence i) The antisense strand comprising at least one single nucleotide polymorphism, which can be complemented and hybridized to the antisense strand of the nucleic acid to be evaluated containing the mutant nucleotide at the position where the nucleotide polymorphism exists.
  • a cytosine residue or thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position where the single nucleotide polymorphism above is paired, and is present adjacent to the cytosine residue or thymine residue.
  • a single nucleotide polymorphism in the nucleic acid to be evaluated is identified based on the variation of the signal based on the compound containing the naphthyridine ring of (B) bound to each of the nucleic acids.
  • a schematic diagram showing an example of the use of the nucleic acid probe for the target nucleic acid is shown in FIG.
  • a guanine-cytosine (GC) base pair is formed with a nucleic acid probe at the position of the single nucleotide polymorphism of the nucleic acid to be evaluated
  • the evaluation is performed. Fluorescence based on the compound containing the naphthyridine ring (B) bound to the bulge formed between the target nucleic acid and the wild type probe or mutant probe is quenched to form an adenylate (AT) base pair.
  • AT adenylate
  • fluorescence based on the compound containing the naphthyridine ring (B) bound to the bulge formed between the nucleic acid to be evaluated and the wild type probe or the mutant type probe can be detected.
  • nucleotide having a single nucleotide polymorphism and Z or a nucleotide adjacent thereto are cytosine residues
  • the guanine residue at the corresponding position of the nucleic acid probe is used instead of the inosine residue.
  • CI cytosine inosine
  • the naphthyridine ring of (B) bound to the bulge formed between the nucleic acid to be evaluated and the wild type probe or the mutant type probe is used. Fluorescence based on the containing compound can be detected.
  • guanine When guanine is present at the position of the single nucleotide polymorphism, cytosine is adjacent to guanine, and ( ⁇ ') force (TA), cytosine bulge is replaced with guanine and YY' base at the position of single nucleotide polymorphism. Design the probe so that it is located between the pair and avoid fluorescence quenching by adjacent guanine bases.
  • guanine is present at the position of single nucleotide polymorphism, cytosine is adjacent to guanine, and ( ⁇ ') is (GC), cytosine bulge is placed 5' to the guanine at the position of single nucleotide polymorphism.
  • single nucleotide polymorphisms can be evaluated based on the behavior of fluorescence or quenching depending on the type of base pair at the site where these single nucleotide polymorphisms exist.
  • the length of the nucleic acid probe is 15 nucleotide residues or more, preferably 20 nucleotides, from the viewpoint of exerting sufficient stability and high stability and sequence specificity to bulge DNA. It is greater than or equal to a rhotide residue, less than or equal to 40 nucleotide residues, and preferably 30 nucleotide residues.
  • (Ii) and (C) are preferably (C) Z (A) [molar ratio] from 1Z5 to 1 from the viewpoint of sufficiently forming a double strand and obtaining sufficient fluorescence intensity.
  • Z (B) (molar ratio) is 1Z100 to 1Z20, more preferably (C) Z (A) (molar ratio) is 1Z12 to 1, and (C) Z (B) (molar ratio) ] Is 1/50 Desirable to mix, to be ⁇ 1Z30.
  • the pH conditions during the mixing include detection of a signal of a compound capable of recognizing a bulge base such as a compound containing a naphthyridine ring, shift of the absorption maximum wavelength based on the binding of the compound to the bulge, fluorescence intensity, etc.
  • the pH is preferably 5 or more, more preferably 6 or more, and even more preferably 6.5 or more, and a compound containing a naphthyridine ring and a bulge base
  • the pH is preferably 9 or less, more preferably 8 or less, and even more preferably 7.5 or less.
  • a phosphate buffer for example, a tris-HCl buffer, or the like can be used.
  • the mixture of the nucleic acid probe (A), the compound (B) containing a naphthyridine ring, and the nucleic acid to be evaluated for single nucleotide polymorphism (C) is as follows.
  • the nucleic acid probe of (A) and the nucleic acid to be evaluated for single nucleotide polymorphism of (C) are mixed to form a bulge structure, and then the compound containing the naphthyridine ring of (B) is further mixed.
  • the nucleic acid probe (A), the compound (B) containing a naphthyridine ring, and the nucleic acid to be evaluated for single nucleotide polymorphism (C) may be mixed at the same time.
  • Fluorescence intensity based on a compound containing a naphthyridine ring bonded to a nolesi base is sufficiently different from that derived from a compound derived from a compound containing a naphthyridine ring in (B) above.
  • the fluorescence intensity is preferably 400 to 480 nm, more preferably around 430 nm.
  • the present invention relates to a kit for use in the method for detecting a single nucleotide polymorphism in the nucleic acid of the present invention.
  • the kit of the present invention comprises the nucleic acid probe and the compound containing the naphthyridine ring. Since the kit of the present invention contains the nucleic acid probe and the compound containing the naphthyridine ring, the detection method of the present invention can be performed efficiently and simply at low cost, and a single base in the nucleic acid can be obtained. Demonstrates the excellent effect that polymorphisms can be detected with high sensitivity and efficiency by simple operation at low cost.
  • the kit of the present invention comprises nucleotides adjacent to the position of the single nucleotide polymorphism to be evaluated.
  • the type of nucleic acid probe can be appropriately selected according to the base sequence around the position where the single nucleotide polymorphism exists.
  • the nucleotide adjacent to the position of the single nucleotide polymorphism to be evaluated is a cytosine residue
  • the nucleotide adjacent to the position of the single nucleotide polymorphism to be evaluated is a nucleotide residue other than cytosine in combination with a nucleic acid probe
  • the nucleic acid probe of I) or ⁇ specifically, the nucleic acid probe of
  • the kit of the present invention appropriately contains a reagent for stably holding the nucleic acid probe, for example, a reagent for stably holding the compound containing the naphthyridine ring, such as a buffer solution. It may be.
  • the kit of the present invention provides a detection method of the present invention using all suitable reagents for performing a detection method such as an appropriate nucleic acid probe, a compound containing the naphthyridine ring, and other necessary reagents.
  • Nucleic acid probes, compounds containing a naphthyridine ring, other necessary reagents, etc. which may be provided in a single container containing a volume and container or form suitable for conducting, are provided in separate containers. It may be a form.
  • such a kit may contain instructions describing the procedures for performing the detection method of the present invention using the components contained in the kit.
  • 2,7-diamino-1,8-naphthyridine has an absorption maximum at pH 7.0 due to the binding with cytosine bulge.
  • the wavelength at which the fluorescence intensity reaches its maximum is shifted from 398 nm force by 430 nm to 32 nm.
  • nucleic acid forming bulge can be detected by 2,7-diamino-1,8-naphthyridine.
  • detection can be performed efficiently under neutral conditions.
  • 2,7-diamino-1,8-naphthyridine (10 / z M) represented by the above formula (I) is converted into a complete duplex (30 ⁇ ), adenine bulge, thymine bulge, cytosine bulge or guanine bulge.
  • duplex (30 mm)
  • it is mixed with 10 mM sodium phosphate buffer with pH 7.0, and the absorption intensity at wavelengths of 300 nm to 450 nm and the fluorescence intensity at wavelengths of 350 nm to 500 nm are measured under the trade name: RF— It was measured with 5300PC (manufactured by Shimadzu Corporation). The results are shown in Figure 3.
  • T—AZACT, T—AZACG, T—CZACI, T_C / A CT or T—C / ACG duplex was used.
  • Complementary T-probes (T probe) containing A allele and C allele (SEQ ID NO: 4) and one extra cytosine residue at the 3 'end of the single nucleotide polymorphism site ) Hybridization with (SEQ ID NO: 2) and G probe (Gprobe) (SEQ ID NO: 3) generates four duplexes containing cytosine bulges as shown in the left panel of FIG. I let you. Forces adjacent to cytosine bulgeka Watson Crick base pair (T—AZACT and T_C / ACG) in two of these four duplexes. (T-AZACG and T-CZA CT, respectively).
  • the relative fluorescence intensity of the A allele duplex at 424 nm determined by the peak height was about 5.5 times the relative fluorescence intensity of the C allele duplex.
  • the AG mismatch base pair in T—AZACG fluoresces the 2,7 diamino-1,8 naphthyridine bound to the adjacent cytosine bulge. It was not extinguished.
  • Absorbance measurements also revealed that 2,7-diamino-1,8-naphthyridine force T — did not bind to cytosine bulge adjacent to the CT mismatch in CZACT!
  • Each duplex comprising a combination of two single nucleotide polymorphism alleles and two probes that are complementary to the alleles and contain a noreji base adjacent to the position corresponding to the single nucleotide polymorphism site.
  • Add 2,7-diamino-1,8-naphthyridine to the rex bind it to the bulge structure, measure fluorescence by excitation at 394 nm, and change the fluorescence intensity between each duplex. It was suggested that the base can be determined.
  • N and N are independently any of T, A, C or G, and these four bases can be considered.
  • an oligonucleotide containing N is referred to as an N allele.
  • N is A
  • N is referred to as an A allele.
  • a leotide is called an N probe; for example, when N is A, it is called an A probe.
  • Each duplex is called an N probe; for example, when N is A, it is called an A probe.
  • Example 3 From Table 1, as in Example 3, the A allele duplex shows strong fluorescence overall. . In contrast, the G allele duplex detected only fluorescence below the background signal, and the differential fluorescence intensity was negative.
  • a single nucleotide polymorphism in a nucleic acid can be detected with low sensitivity and high sensitivity by a simple operation. Therefore, inexpensive, simple and highly sensitive gene diagnosis, gene analysis, and the like are possible.
  • SEQ ID NO: 1 is the sequence of a synthetic DNA (A allele).
  • SEQ ID NO: 2 is the sequence of a synthetic DNA (T probe).
  • SEQ ID NO: 3 is the sequence of a synthetic DNA (G probe).
  • SEQ ID NO: 4 is the sequence of a synthetic DNA (C allele).
  • SEQ ID NO: 5 is the sequence of a synthetic DNA (I probe). Base number: n in 5 is inosine.
  • SEQ ID NO: 6 is the sequence of a synthetic DNA (N allele).
  • N in the base number: 9 is adenine, cytosine, guanine, or thymine.
  • SEQ ID NO: 7 is the sequence of a synthetic DNA (N probe). Base number: 10 n, Ade

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Abstract

L’invention concerne un procédé de détection du polymorphisme nucléotidique caractérisé par le mélange de (A) I) une sonde d’acide nucléique qui peut s’hybrider de manière complémentaire à un acide nucléique devant être évalué présentant au moins un polymorphisme nucléotidique et qui présente une séquence nucléique en addition d’un résidu de cytosine ou de thymine à une position adjacente à l’extrémité terminale en 5’ ou 3’ d’une position s’appariant avec le polymorphisme nucléotidique ou II) une sonde d’acide nucléique s’hybridant de manière complémentaire au brin antisens d’un acide nucléique devant être évalué présentant au moins un polymorphisme nucléotidique et qui présente une séquence nucléique en addition d’un résidu de cytosine ou de thymine à une position adjacente à l’extrémité terminale en 5’ ou 3’ d’une position s’appariant avec le polymorphisme nucléotidique sur le brin antisens, (B) un composé qui peut reconnaître une base de renflement et (C) l’acide nucléique à évaluer, et un procédé de détection d’un signal s’appuyant sur le composé (B) qui reconnaît un renflement de cytosine ou de thymine qui est formé lorsque la sonde d’acide nucléique et l’acide nucléique devant être évalués s’hybride l’un à l’autre, et enfin, l’invention concerne un kit pour ce procédé.
PCT/JP2005/022985 2005-02-01 2005-12-14 Procede de detection d’un polymorphisme nucleotidique WO2006082685A1 (fr)

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WO2008026582A1 (fr) * 2006-09-01 2008-03-06 Osaka University Fragment d'adn utilisé dans la forme attachée à l'extrémité 5' de l'amorce pour une utilisation dans une réaction d'amplification d'un acide nucléique, et son utilisation
WO2009098998A1 (fr) * 2008-02-05 2009-08-13 Olympus Corporation Méthode de détection d'acides nucléiques et kit de détection d'acides nucléiques
JP2011182763A (ja) * 2010-03-11 2011-09-22 Osaka Univ 一塩基多型を検出する方法および試薬キット
WO2013133402A1 (fr) 2012-03-08 2013-09-12 株式会社古河電工アドバンストエンジニアリング Méthode de détection d'un polymorphisme mononucléotidique dans un acide nucléique
JPWO2017138484A1 (ja) * 2016-02-09 2018-09-27 栄研化学株式会社 標的核酸を検出する方法およびそれに用いる核酸プローブ

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JP4873427B2 (ja) * 2006-09-01 2012-02-08 国立大学法人大阪大学 核酸の増幅反応に用いるヘアピンプライマー及びその利用
WO2008026582A1 (fr) * 2006-09-01 2008-03-06 Osaka University Fragment d'adn utilisé dans la forme attachée à l'extrémité 5' de l'amorce pour une utilisation dans une réaction d'amplification d'un acide nucléique, et son utilisation
US8911947B2 (en) 2006-09-01 2014-12-16 Furukawa Electric Advanced Engineering Co., Ltd. DNA fragment used as attached to 5′ end of primer used in nucleic acid amplification reaction and use of DNA fragment
JPWO2008026582A1 (ja) * 2006-09-01 2010-01-21 国立大学法人大阪大学 核酸の増幅反応に用いるプライマーの５’末端に結合して用いるｄｎａ断片及びその利用
EP2058392A4 (fr) * 2006-09-01 2010-09-22 Univ Osaka Fragment d'adn utilisé dans la forme attachée à l'extrémité 5' de l'amorce pour une utilisation dans une réaction d'amplification d'un acide nucléique, et son utilisation
EP2058392A1 (fr) * 2006-09-01 2009-05-13 Osaka University Fragment d'adn utilisé dans la forme attachée à l'extrémité 5' de l'amorce pour une utilisation dans une réaction d'amplification d'un acide nucléique, et son utilisation
US8530165B2 (en) 2008-02-05 2013-09-10 Olympus Corporation Nucleic acid detection method for determining if one or more analyte nucleotides are present in a nucleic acid
WO2009098998A1 (fr) * 2008-02-05 2009-08-13 Olympus Corporation Méthode de détection d'acides nucléiques et kit de détection d'acides nucléiques
JP2011182763A (ja) * 2010-03-11 2011-09-22 Osaka Univ 一塩基多型を検出する方法および試薬キット
JP5401634B1 (ja) * 2012-03-08 2014-01-29 株式会社古河電工アドバンストエンジニアリング 核酸中の一塩基多型の検出方法
WO2013133402A1 (fr) 2012-03-08 2013-09-12 株式会社古河電工アドバンストエンジニアリング Méthode de détection d'un polymorphisme mononucléotidique dans un acide nucléique
US9057105B2 (en) 2012-03-08 2015-06-16 Furukawa Electric Advanced Engineering Co., Ltd. Method for detecting single nucleotide polymorphism in nucleic acid
JPWO2017138484A1 (ja) * 2016-02-09 2018-09-27 栄研化学株式会社 標的核酸を検出する方法およびそれに用いる核酸プローブ
US11434533B2 (en) 2016-02-09 2022-09-06 Eiken Kagaku Kabushiki Kaisha Method for detecting target nucleic acid and nucleic acid probe used therein

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