WO2002101041A1 - Procede d'amplification de l'acide nucleique et procede de detection du polymorphisme des nucleotides a l'aide d'un analogue de nucleotide - Google Patents

Procede d'amplification de l'acide nucleique et procede de detection du polymorphisme des nucleotides a l'aide d'un analogue de nucleotide Download PDF

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WO2002101041A1
WO2002101041A1 PCT/JP2002/005831 JP0205831W WO02101041A1 WO 2002101041 A1 WO2002101041 A1 WO 2002101041A1 JP 0205831 W JP0205831 W JP 0205831W WO 02101041 A1 WO02101041 A1 WO 02101041A1
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nucleotide
nucleic acid
base
target nucleic
present
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PCT/JP2002/005831
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English (en)
Japanese (ja)
Inventor
Hiroaki Sagawa
Takashi Uemori
Hiroyuki Mukai
Masamitsu Shimada
Junko Yamamoto
Jun Tomono
Eiji Kobayashi
Tatsuji Enoki
Fumitsugu Hino
Kiyozo Asada
Ikunoshin Kato
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Takara Bio Inc.
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Publication of WO2002101041A1 publication Critical patent/WO2002101041A1/fr

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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the present invention relates to a method for detecting a target nucleic acid useful in the clinical field and a method for synthesizing a DNA useful in the field of genetic engineering.
  • the present invention relates to a method for amplifying a ⁇ -type nucleic acid using a nucleotide containing a nucleotide analog, and
  • the present invention relates to a method for detecting base substitution of a target nucleic acid.
  • Nucleotide analogs are used primarily in synthesizing antisense oligonucleotides used in antisense therapy. In other words, when a natural oligonucleotide is used as an antisense molecule, there are problems such as degradation by various nucleases in the living body and poor permeability to cell membranes. Many analogs and analogs have been synthesized and studied.
  • nucleotide analog examples include a case where the nucleotide analog is used in a method for amplifying a target nucleic acid or a method for detecting a target nucleic acid.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription PCR
  • LCR ligase chain reaction
  • a transcription-based amplification system (TAS) method described on pages 245 to 252 is exemplified.
  • TAS transcription-based amplification system
  • the above four methods require repeated high to low temperature reactions to regenerate single-stranded target molecules for the next amplification cycle. Since the reaction is limited by the temperature, the reaction system must be performed in a discontinuous phase or cycle.
  • nucleic acid amplification method examples include, for example, strand displacement amplification (SDA; strand displacement) described in JP-B-7-114178.
  • SDA strand displacement amplification
  • the SDA method is a method for amplifying a target nucleic acid sequence (and its complementary strand) in a sample by displacing a double strand through a DNA polymerase and a restriction endonuclease.
  • four types of primers to be used for amplification are required, and two of them need to be constructed so as to include a recognition site for restriction endonuclease.
  • a modified deoxyribonucleotide triphosphate for example, an oxygen atom of a phosphate group at the ⁇ -position was substituted with a sulfur atom (S) as a substrate for DNA synthesis ( ⁇ — S)
  • S sulfur atom
  • a main object of the present invention is to provide a method for amplifying a target nucleic acid in which a target nucleic acid in a sample is amplified with high sensitivity and specificity by a DNA synthesis reaction using a nucleotide containing a nucleotide analog, and using a nucleotide containing a nucleotide analog. It is an object of the present invention to provide a method for detecting the presence or absence of base substitution based on the presence or absence of a DNA synthesis reaction.
  • the objective of the present invention is to provide a chimeric oligonucleotide primer containing a nucleotide analog and having a ribonucleotide at the 3, 3 or 3 'end, an endoribonuclease, and a DNA polymerase.
  • An excellent amplification reaction system for amplifying the nucleic acid in the region was constructed, and the present invention was completed. That is, the first invention of the present invention relates to a method for amplifying a target nucleic acid using a nucleotide containing a nucleotide analog,
  • nucleic acid to be type III, deoxyribonucleotide triphosphate, a DNA polymerase having strand displacement activity, at least one nucleotide, and RNase H;
  • nucleotide is substantially complementary to the base sequence of the ⁇ -type nucleic acid, and contains at least a deoxyribonucleotide and a ribonucleotide, wherein the ribonucleotide is at the 3 ′ end or 3 ′ of the nucleotide.
  • a nucleotide primer disposed distally, said primer containing one or more nucleotide analogs; and
  • a reaction mixture containing a nucleotide primer having a sequence substantially homologous to the base sequence of the nucleic acid to be type III can be used.
  • LNA [1OcKedNucleicACID] or LNA3 phosphate can be suitably used as a nucleotide analog.
  • the second invention of the present invention uses a nucleotide containing a nucleotide analog.
  • Step of mixing the sample containing the target nucleic acid with the nucleotide The nucleotide is
  • a method for detecting a target nucleic acid using a nucleotide containing a nucleotide analog which comprises the step of detecting the presence or absence of cleavage of the nucleotide by a nuclease.
  • a third invention of the present invention is a method for detecting the presence or absence of base substitution at a specific base on a target nucleic acid using a nucleotide containing a nucleotide analog,
  • Step of mixing the sample containing the target nucleic acid with the nucleotide The nucleotide is
  • the specific In the complex formed from the nucleotide and the target nucleic acid, the specific When there is no mismatch between the base and the base on the nucleotide corresponding to the base, the nucleotide is not cleaved by a nuclease, and the specific base and the base on the nucleotide corresponding to the base are different from each other. If there is a mischid in between, the nucleotide contains a rooster sequence that is cleaved by a nuclease to generate a new 3 ′ end,
  • a method for detecting a target nucleic acid using a nucleotide containing a nucleotide analog which comprises the step of detecting the presence or absence of nucleotide cleavage by a nuclease.
  • LNA loccdNucleicAcid
  • LNA triphosphate can be suitably used as a nucleotide analog.
  • the fourth invention of the present invention relates to a kit for the method of the first to third inventions of the present invention. Detailed description of the invention
  • a nucleotide may be any nucleotide as long as it can be used in the present invention, and may be an oligonucleotide or a polynucleotide. In the present invention, less than 50mer is called an oligonucleotide, and more than 5mer is called a polynucleotide.
  • deoxyribonucleotide refers to a nucleotide in which the sugar moiety is composed of D-2-deoxylipose.
  • adenine or cytosine is used as the base moiety.
  • Guanine and thymine is used as the base moiety.
  • a ribonucleotide refers to a nucleotide in which the sugar moiety is composed of D-ribose, and has adenine, cytosine, guanine, and peracil in the base moiety.
  • a nucleotide analog refers to a deoxyribonucleotide or a substance that can function as a deoxyribonucleotide.
  • a deoxyribonucleotide having a modified base such as 7-deazaguanosine
  • a deoxyinosine nucleotide a modified ribonucleotide in which the oxygen atom of the ⁇ -phosphate group is replaced with a sulfur atom [(a-S) ribonucleotide, (Hi_S) ⁇ and other derivatives
  • LNA locked Nucleic Acid, Proceedings of the USA (Proc. N at 1. Ac a d. S ci. USA), Vol.
  • chimeric oligonucleotide primer refers to a primer containing deoxyliponucleotides and ribonucleotides.
  • the primer may further contain a nucleotide analog.
  • a ribonucleotide is arranged at the 3 ′ end or 3 ′ end side of the primer, and in the method of the present invention, a nucleic acid chain can be extended, cleaved by endonuclease, and a strand displacement reaction. It can be performed.
  • the term "3 terminal" refers to a nucleic acid, for example, a portion of a primer from the center to the 3 terminus.
  • the 5 'end refers to a portion of a nucleic acid from its center to its 5' end.
  • an endonuclease refers to a double-stranded DNA produced by elongating DNA from the chimeric oligonucleotide primer annealed to a type III nucleic acid and acting on the double-stranded DNA to specifically identify a ribonucleotide portion of the primer. If it can be cut off, then.
  • the endonuclease used in the present invention refers to an extended strand that acts on double-stranded DNA generated by performing DNA extension from a chimeric oligonucleotide primer annealed to a type I nucleic acid to cause a strand displacement reaction. What is necessary is just to be able to cut off. That is, it is an enzyme capable of generating a nick in the chimeric oligonucleotide primer of the double-stranded DNA.
  • a ribonuclease can be used, and in particular, an endoribonuclease H (RNase H) which acts on the RNA portion of a double-stranded nucleic acid formed from DNA and RNA can be suitably used.
  • RNase H an endoribonuclease H
  • any of ribonucleases having normal temperature to heat resistance can be suitably used in the present invention.
  • RNase H derived from Escherichia coli (E. coli) can be used in the method of the present invention in a reaction at about 50 ° C. to about 70 ° C.
  • thermostable ribonuclease can also be suitably used.
  • thermostable ribonuclease include, but are not particularly limited to, commercially available Hybridase TM Thermo stable RNase H (manufactured by Epicenter Technologies), thermophilic Bacillus bacterium, Thermus bacterium, and Pyrococcus bacterium.
  • ribonuclease H derived from a genus Thermotoga or a bacterium belonging to the genus Alcaeoglobus can be suitably used.
  • the ribonuclease H preferably has high activity under the same reaction conditions as the DNA polymerase used at the same time, but is not particularly limited.
  • thermostable ribonucleases H include Bacillus' force noredenacus (Bacillus caldotenax), Pyrococcus furiosus
  • the RNaseH is not particularly limited as long as it can be used in the method of the present invention, and may be any of various viruses, phages, prokaryotes, and eukaryotes. Furthermore, it may be either cellular RNase H or viral RNase H.
  • the cellular RNase H is exemplified by Escherichia coli RNase HI
  • the viral RNase H is exemplified by HIV-1.
  • any of Type I, Type II and Type III can be used as RNaseH.
  • RNaseHI derived from Escherichia coli is preferable.
  • DNA polymerase refers to an enzyme that synthesizes a new DNA chain using a DNA chain as a ⁇ -type, and includes a mutant enzyme having the above-mentioned activity in addition to a natural-type DNA polymerase.
  • the enzyme include a DNA polymerase having a strand displacement activity, a DNA polymerase having no 5 ′ ⁇ 3 ′ exonuclease activity, and a DNA polymerase having both a reverse transcriptase activity and a endonuclease activity. Is mentioned.
  • a DNA polymerase having a strand displacement activity of DNA can be used.
  • Those having substantially no 5 ′ ⁇ 3 ′ exonuclease activity can be particularly preferably used.
  • the DNA polymerase used in the present invention is not particularly limited as long as it has the above-mentioned strand displacement activity.
  • Bacillus caldotenax hereinafter, referred to as B. ca
  • Bacillus stearothermophilus Bacillus stearothermophilus
  • B. st Bacillus stearothermophilus
  • E.co1i Escherichia coli
  • DNA polymerase I derived large fragment
  • B. ca is a thermophilic bacterium whose optimal growth temperature is about 70 ° C.
  • Bea DNA polymerase derived from this bacterium has DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase activity (reverse transcription activity), It is known to have 5 ′ ⁇ 3 ′ exonuclease activity and 3 ′ ⁇ 5 ′ exonuclease activity.
  • the above enzyme may be any of those obtained by purifying from its original source or recombinant proteins produced by genetic engineering.
  • the enzyme may be modified by substitution, deletion, addition, insertion or the like by genetic engineering or other methods. Examples of such an enzyme include 5 ′ ⁇ 3 ′ Bca BEST DNA polymerase, a Bea DNA polymerase lacking exonuclease activity
  • the enzyme is Escherichia coli HB 10 l / pU I 205 (F ERM BP-3 720), it can also be prepared by the method described in Japanese Patent No. 2978001.
  • DNA polymerases having an endonuclease activity under specific conditions for example, an RNaseH activity
  • a DNA polymerase can be used in the method of the present invention.
  • the DNA polymerase known to have the above RNaseH activity for example, Tth DNA polymerase derived from Thermus thermophilus and Bca BEST DNA polymerase can be suitably used.
  • the nucleic acid amplification reaction of the present invention can be carried out at room temperature (for example, at 37 ° C.) by using a room temperature DNA polymerase such as Klenow fragment, but an enzyme having heat resistance (endonuclease, DNA polymerase)
  • the reaction can be carried out at a high temperature, for example, at 50 ° C. or more, and further at, for example, 6.0 ° C. or more. In this case, non-specific annealing of primers is suppressed, the specificity of DNA amplification is improved, and the elongation of DNA polymerase is improved by eliminating the secondary structure of type I DNA.
  • NA polymerase (Takara Shuzo) is useful. Also use T5 DNA polymerase and ⁇ 29 DNA polymerase described in Sequenase 1.0 and Citaenase 2.0 (U.S.A. Biochemical), Gene, Vol. 97, pp. 13-19 (1991). Can be. In general, a polymerase having 5 ′ ⁇ 3 ′ exonuclease activity can be used in the method of the present invention if its activity can be inhibited by adding an appropriate inhibitor.
  • the type II or target nucleic acid ie, DNA or RNA
  • the type II or target nucleic acid may be prepared or isolated from any sample that may contain the nucleic acid. Further, the above sample may be directly used for the nucleic acid amplification reaction of the present invention.
  • the sample containing such a nucleic acid is not particularly limited, but includes, for example, whole blood, serum, buffy coat, urine, feces, cerebrospinal fluid, semen, saliva, tissue (eg, cancer tissue, lymph node, etc.)
  • Biological samples such as cell cultures (eg, mammalian cell cultures and bacterial cultures), nucleic acid-containing samples such as viroids, viruses, bacteria, molds, yeasts, plants and animals, and viruses or bacteria.
  • a nucleic acid-containing preparation obtained by treating the sample or the like by a known method.
  • a cell lysate or a sample obtained by fractionating the same, a nucleic acid in the sample, or a specific nucleic acid molecule group, for example, a sample enriched in mRNA, etc. are used in the present invention. Can be used.
  • a nucleic acid such as DNA or RNA obtained by amplifying a nucleic acid contained in the sample by a known method can also be suitably used.
  • the method of the present invention may be carried out using cDNA synthesized by a reverse transcription reaction using the RNA as a type II.
  • the RNA that can be applied to the method of the present invention is not particularly limited as long as a primer used for the reverse transcription reaction can be prepared.
  • a primer used for the reverse transcription reaction can be prepared.
  • mRNA, tRNA , r RNA, etc. or specific RNA molecules.
  • the method for amplifying a nucleic acid using the nucleotide analog of the present invention is not particularly limited, and examples thereof include a nucleic acid amplification method using a chimeric oligonucleotide primer.
  • the ICAN Isotnermal and Chimeric primer-initiated Amplification of Nucleic acids
  • the method of the present invention is characterized in that a nucleotide containing at least a deoxyribonucleotide and a ribonucleotide is used as a chimeric oligonucleotide primer, and the nucleotide contains one or more nucleotide analogs.
  • the chimeric oligonucleotide primer used in the method of the present invention comprises: Has a nucleotide sequence substantially complementary to a part of the nucleotide sequence of the template nucleic acid, and can contribute to DNA chain elongation under the conditions used.
  • This is a chimeric oligonucleotide primer having a ribonucleotide disposed on the terminal side.
  • the primer may contain unmodified ribonucleotides and either Z or a nucleotide analog.
  • the chimeric oligonucleotide primer of the present invention contains a nucleotide analog to the extent that the function of the primer is not lost.
  • the “substantially complementary base sequence” means a base sequence that can be annealed to DNA that becomes type II under the reaction conditions used.
  • the chimeric oligonucleotide primer used in the method of the present invention is not particularly limited, but preferably has a length of about 12 nucleotides to about 100 nucleotides. More preferably, it is a primer having a length of about 15 nucleotides to about 40 nucleotides.
  • the base sequence is preferably a sequence substantially complementary to the type III nucleic acid so as to anneal to the type III nucleic acid under the reaction conditions used.
  • an oligonucleotide having a structure represented by the following general formula can be used as a primer in the method of the present invention.
  • dN deoxyribonucleotide and / or nucleotide analog
  • N unmodified liponucleotide and Z or modified ribonucleotide nucleotides
  • d N a part of d N sites may be substituted by N
  • the chimeric oligonucleotide primer used in the method of the present invention may be used in combination of a plurality of types of nucleotide analogs.
  • the nucleotide analog include, but are not limited to, deoxyinosin nucleotides, deoxyperic nucleotides, and deoxyribonucleotide analogs having a modified base such as 7-desazaguanine, LNA (Locked Nucleic Acid, Proceeding). Zobu The National Academy of Sciences of the USA (Pro Natl. Acad. Sci. USA), Vol. 97, 5563-5638 (2000), WO 99 14226 pamphlet], and nucleotide analogs having a derivative of ribose can be used.
  • the chimeric oligonucleotide used in the present invention may contain a deoxynucleotide, a ribonucleotide, or a nucleotide analog to which various modifications, for example, a labeling compound or the like have been added, as long as the above function is maintained.
  • the chimeric oligonucleotide of the present invention may contain a peptide nucleic acid [PNA, Peptide Nucleic Acid, Nature, Vol. 365, pp. 566-568 (1993)].
  • nucleotide analogues into the primer is also effective from the viewpoint of suppressing the formation of higher-order structure of the primer itself and stabilizing the formation of annealing with type ⁇ .
  • chimeric oligonucleotide primers can be synthesized so as to have an arbitrary nucleic acid sequence by, for example, a phosphoramidite method using a DNA synthesizer type 394 of Applied Biosystems Inc. (ABI). Further, there are other methods, such as the triestenol phosphate method, the H-phosphonate method, and the thiophosphonate method, but any method may be used.
  • the efficiency of the cleavage reaction of endonucleases used in the method of the present invention for example, RNaseH, depends on the base sequence near the 3 ′ end of the primer, and may affect the amplification efficiency of the desired DNA. Therefore, it is natural to design the optimal primer for the RNase H to be used.
  • reaction buffer used in the present invention one containing a buffer component, a magnesium salt or other metal salt, or dNTP is used. It is natural that the type and concentration of the salt are optimized according to the metal requirements of the enzyme used.
  • the buffer component is not particularly limited, for example, bicine, tricine, mouse, tris, phosphate (sodium phosphate, potassium phosphate, etc.) can be suitably used.
  • DNTPs and the like are added to start the nucleic acid amplification reaction of the present invention.
  • the combination of the endonuclease and the DNA polymerase is not particularly limited, and for example, a combination of Pfu-derived, Afu-derived or T1i-derived RNAseH and Bca BEST DNA polymerase is preferred. Good. Furthermore, both the above-mentioned endonuclease and DNA polymerase It is expected that the number of cuts that can be suitably used differs depending on the type. In such a case, the composition of the buffer used and the amount of enzyme added can be adjusted using the improvement in detection sensitivity or the amount of amplification product as an index. In any case, it is natural to optimize the composition of the reaction buffer according to the type of the enzyme to be used.
  • dNTP used in PCR or the like ie, a mixture of dATP, dCTP, dGTP, and dTTP, used as a nucleotide triphosphate serving as a substrate for the extension reaction
  • DUTP may be used as a substrate.
  • the dNTP is an analog of dNTP (deoxyribonucleotide triphosphate) such as 7-deza_dGTP, dITP, and LNA as long as it serves as a substrate for the DNA polymerase used. It may contain an acid or the like.
  • a derivative of dNTP or dNTP analog may be used, and a derivative having a functional group, for example, dUTP having an amino group may be contained.
  • chimeric oligonucleotide primers are used, and the primers can be prepared, for example, using a DNA synthesizer or the like in the same manner as in a usual synthesis method.
  • the above-mentioned chimeric oligonucleotide primer can be used in combination with a usual oligonucleotide primer.
  • a polymer having a series of amplified regions may be produced.
  • Such a polymer has a plurality of amplification regions, all of which are connected in the same direction, and is identified as a ladder-like band in the analysis of amplification products by electrophoresis. It is considered that the production of the polymer is affected by the region to be amplified, the size of the region, the adjacent region, the base sequence of the chimeric oligonucleotide primer used, the reaction conditions, and the like.
  • the production of the polymer can be controlled by using the nucleotide analog.
  • the use of a nucleotide analog reduces the non-specific annealing of the primer, and sets the reaction temperature and stringentity so that the primer specifically anneals to the nucleic acid of type I. Sea levels can be set higher.
  • the method of the present invention can be carried out under high temperature conditions using a thermostable enzyme. Further, from the viewpoint of keeping the reaction efficiency high, it is preferable that the method of the present invention is carried out at an appropriate temperature at which the activity of the enzyme used is sufficiently maintained.
  • a preferred reaction temperature is about 20 ° C. to about 80 ° C., more preferably about 30 ° C. to about 75 ° C., and particularly preferably about 50 ° C. ⁇ 70 ° C.
  • a primer having a longer chain length than when performing the reaction at room temperature.
  • it may be the Tm value reference, or a commercially available primer design software, for example, OL I GO TM P ri me r An alysissoft wa re (Manufactured by Takara Shuzo) may be used.
  • the length of the primer used in the method is not particularly limited.
  • Primers having a length of nucleotides to 100 nucleotides, preferably 14 nucleotides to 50 nucleotides, and more preferably 15 nucleotides to 40 nucleotides can be used.
  • the effect of increasing the reaction temperature in this way is that the secondary structure of type III DNA can be eliminated, and even when type III nucleic acid having a high GC content is used, the desired nucleic acid is amplified.
  • the same effect can be obtained when a long chain region is amplified.
  • the effect is observed in the range from about 60 bp to about 20 kbp, more particularly in the range from about 110 bp to about 4.3 kb, especially in the range from about 130 bp to about 1500 bp.
  • the amplification temperature can be improved by adjusting the reaction temperature according to the GC content of the type III nucleic acid.
  • the amplification reaction of the present invention may be performed at 50 to 55 ° C, depending on the length of the chain to be amplified and the Tm value of the primer. it can.
  • the nucleic acid amplification method of the present invention can be used for various experimental procedures utilizing nucleic acid amplification, for example, detection of nucleic acid, labeling, and determination of a base sequence.
  • the nucleic acid amplification method of the present invention can be used as an in situ nucleic acid amplification method, a nucleic acid amplification method on a solid support such as a DNA chip, or a multiplex nucleic acid amplification method for simultaneously amplifying various types of regions. .
  • Examples of the method of the present invention include a method for amplification and Z or detection of M. tuberculosis.
  • the sample used in the method is not particularly limited, and any sample that may contain M.
  • tuberculosis such as sputum, whole blood, serum, urine, feces, cerebrospinal fluid, semen, saliva, tissue
  • the method of the present invention can be applied to a biopsy sample, a cell culture, or a sample in an environment such as soil, drainage, or air.
  • a sample for which tuberculosis is to be detected can be subjected to a nucleic acid amplification reaction by the above-described method, and then a hybridization between the amplified product and the probe of the present invention can be performed to detect tuberculosis.
  • the Mycobacterium tuberculosis IS 6110 gene is used as a target nucleic acid.
  • the primer for amplifying the nucleic acid by the method of the present invention has the nucleotide sequence shown in SEQ ID NOS: 1 and 2 in the sequence listing.
  • the primer can contain any nucleotide analog.
  • these primers may be labeled with an appropriate substance, for example, a fluorescent substance, a dye, a ligand (eg, biotin, digoxigenin), or colloidal gold.
  • an appropriate substance for example, a fluorescent substance, a dye, a ligand (eg, biotin, digoxigenin), or colloidal gold.
  • a primer labeled with a ligand eg, biotin, digoxigenin
  • colloidal gold e.g., use a primer labeled with a ligand.
  • a highly sensitive and simple quantitative measurement method is provided, such as capturing the amplified target nucleic acid on a carrier.
  • the solid phase include microtiter plates, beads, magnetic beads, membranes, and glass.
  • base substitution means that at a specific site on a nucleic acid, a part of the base is replaced by another base. “Base substitution” causes differences in genetic information between living organisms, and this difference in genetic information is called polymorphism or variation. As used herein, “base substitution” includes base substitution in the above polymorphisms and variations. Further, base substitution artificially introduced into a nucleic acid is also included in the term “base substitution” in the present specification. In the “base substitution” described in the present specification, the number of substituted bases is not particularly limited, and one or more bases may be substituted.
  • the nucleotide used in the detection method of the present invention should detect base substitution on the target nucleic acid.
  • the chain length is not particularly limited as long as it has the above-mentioned properties, and any of an oligonucleotide and a polynucleotide can be used in the present invention.
  • an oligonucleotide of 8 to 50 bases, preferably 10 to 40 bases, particularly preferably 12 to 30 bases is used as the nucleotide of the present invention.
  • the nucleotide used in the detection method of the present invention is usually an oligonucleotide containing deoxyribonucleotide. If necessary, ribonucleotides and nucleotide analogs and derivatives (modifications) can be contained.
  • nucleotide analogs include nucleotide analogs having bases such as inosine and 7-dezaguanine in the base moiety, LNA (Locked Nucleic Acid, Proceedings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), Vol. 97, pp. 5633-5638 (2000), WO 99/14226 pamphlet], and nucleotide analogs having derivative derivatives can be used.
  • examples of the modified nucleotide include a nucleotide in which an oxygen atom bonded to a phosphate group is substituted with a sulfur atom, a nucleotide to which a labeled compound is added, and the like.
  • the nucleotide of the present invention may contain a peptide nucleic acid [PNA, Peptide Nucleic Acid, Nature, Vol. 365, pp. 566-568 (1993)].
  • PNA Peptide Nucleic Acid, Nature, Vol. 365, pp. 566-568 (1993)].
  • the present invention is not particularly limited, but preferably, the nucleotide analogs and derivatives described above are introduced into a site that does not affect the action of the nuclease used.
  • nucleotide analogs and / or modified nucleotides may be contained within a range that retains the function as a nucleotide that can be used in the method for detecting base substitution of the present invention.
  • the nucleotide used in the present invention has the following properties for detecting base substitution at a specific base on a target nucleic acid.
  • A) The 3 'end is modified so that extension from the end by DNA polymerase does not occur.
  • the 5′-side fragment of the nucleotide cleaved by the nuclease can maintain the state of being annealed to the target nucleic acid.
  • a hydroxyl group is present at position 3 of ribose at the 3 ′ end of the 5 ′ side fragment of this nucleotide, and DNA extension from this end by DNA polymerase is possible. That is, when the above-mentioned nucleotide has a nucleotide sequence that is cleaved by a nuclease, it functions as a precursor of a primer.
  • the nucleotide used in the detection method of the present invention is modified so that its 3 ′ end cannot be used for DNA extension reaction by DNA polymerase.
  • the modification means is not particularly limited as long as the above object can be achieved.
  • a 3'-terminal dideoxynucleotide, a nucleotide in which the hydroxyl group at position 3 of ribose is modified, and elongation by DNA polymerase is sterically hindered.
  • adding a nucleotide or the like modified so as to be hindered As the method for modifying the hydroxyl group at the 3-position of the ribose of the above nucleotide, alkynoleic or other known modification methods can be used.
  • a DNA extension reaction can be prevented by aminoalkylation.
  • the nucleotide used in the detection method of the present invention has a base sequence that can anneal to a region where base substitution of a target nucleic acid is to be detected under the conditions used. ing. That is, it is sufficient that the target nucleic acid has a sequence that is substantially complementary to the target nucleic acid, and it has a base sequence that is completely complementary to the target nucleic acid within a range that does not hinder detection of substitution at the target base. You don't have to.
  • nucleotide used in the above-described detection method of the present invention is annealed with a target nucleic acid and incubated in the presence of an appropriate nuclease and DNA polymerase, whether or not the target nucleic acid has a base substitution, that is, Nucleotide cleavage depends on the presence or absence of a mismatch site in the double-stranded nucleic acid formed by annealing with the target nucleic acid.
  • DNA extension occurs with the target nucleic acid as a ⁇ .
  • mismatch that is, the presence or absence of base substitution, can be determined by the presence or absence of DNA extension. it can.
  • both the above nucleotides are prepared so that a mismatch occurs when a base substitution to be detected is present, and on the contrary, the nucleotides are prepared so that a mismatch does not occur when a base substitution exists. It is possible. Furthermore, by preparing and using four nucleotides in which one of the four nucleotides is located at the position corresponding to the target base, and examining which baser has an extension in the primer, the base substitution can be determined. It is also possible to know the presence and the type of the substituting base at the same time. Nucleotides used in the detection method of the present invention are converted to primers capable of DNA extension by cleavage with nucleases as described above.
  • the portion 5'-side from the cleavage site of the nucleotide by the nuclease functions as a primer in DNA extension.
  • the nuclease is not particularly limited as long as it cleaves or does not cleave the nucleotide in response to the presence of a mismatch in a double-stranded nucleic acid formed by annealing a nucleotide and a target nucleic acid.
  • Ribonuclease H restriction enzymes, mismatch-specific nucleases and the like. Contrary to the above two nucleases, an enzyme that recognizes and cuts a mismatch between a target nucleic acid and a nucleotide may be used. MutH and the like can be used as such an enzyme.
  • DNA polymerase used in the step is not particularly limited as long as it can extend the DNA from the 3 'end of the primer depending on the type III DNA sequence. For example, E.
  • thermophilic Bacillus genus DNA polymerase (Bst DNA polymerase, Bea DNA polymerase), Thermus genus DNA polymerase (Taq DNA polymerase and the like, and a mold DNA polymerase derived from a thermophilic archaebacteria (Pfu DNA polymerase and the like).
  • nucleotide of the present invention using ribonuclease H as a nuclease is not particularly limited.
  • an oligonucleotide having a structure represented by the following general formula can be used in the present invention.
  • the site represented by Nb contains a base corresponding to the base to be detected for substitution.
  • Each of the above nucleotides contains nucleotide analogs and derivatives (modified nucleotides) as long as the function is not impaired.
  • the base corresponding to the base to be detected for base substitution is not particularly limited as long as it is located in the portion represented by Nb.
  • the length of the portion represented by (dNc_N ') is 3 bases, and the detection position of the base substitution detection portion represented by Nb is closest to the 3' side.
  • the set nucleotide can be suitably used, and the nucleotide shows good specificity for detecting base substitution.
  • Proper labeling of the nucleotides used in the detection method of the present invention facilitates the detection of the 3′-side fragment separated from the nucleotides by the cleavage by nuclease or the product (extended product) generated by the subsequent DNA extension reaction.
  • the presence of the base substitution can be easily confirmed.
  • the method for labeling a nucleotide for example, radioactive isotopes (3 2 P, etc.), color-containing, fluorescent materials, luminescent materials, various ligands (Piochin, digoxigenin, etc.), enzyme arsenide can be used.
  • the presence of the product derived from the labeled nucleotide can be confirmed by a detection method corresponding to the label.
  • a ligand that cannot be directly detected it may be combined with a ligand-binding substance with a detectable label.
  • a target nucleic acid can be detected with high sensitivity by combining a product derived from a nucleotide labeled with a ligand and an anti-ligand antibody labeled with an enzyme, and amplifying the signal.
  • Examples of the embodiment of fluorescently labeling a nucleotide include, for example, those in which the nucleotide is labeled with both a fluorescent substance and a substance having a function of quenching the fluorescence emitted from the fluorescent substance at appropriate intervals.
  • Such primers do not fluoresce in the intact state, but cleave by nucleases and fluoresce when the distance between the fluorescent and the quencher increases. Since such a nucleotide emits fluorescence simultaneously with the initiation of the DNA extension reaction, the presence or absence of base substitution can be known by directly observing the reaction solution during the reaction.
  • the detection method of the present invention uses the nucleotide of the present invention described in the above (1), and comprises the following steps:
  • nucleotide of the present invention it is determined whether or not the base substitution exists based on the presence or absence of the cleavage by the nuclease.
  • the extension product may be further amplified using a known nucleic acid amplification reaction.
  • a known nucleic acid amplification reaction Such an embodiment is useful from the viewpoint of detecting base substitution with high sensitivity.
  • the nucleic acid amplification reaction described above is not particularly limited, and various nucleic acid amplification methods using a primer having a sequence complementary to the type III nucleic acid can be used.
  • PCR polymerase chain Polymerase chain reaction
  • SDA strand displacement amplification
  • Japanese Patent Publication No. 7-114718 Independent replication method (3 SR; self-sustained sequence replication, NASB A3 ⁇ 4 (nucleic acid sequence based amplification, Patent No. 2650159), TMA method
  • amplification methods such as (transcription-mediated amplification) and the ICAN method (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids, WO 00/56877 or WO 02/16639 pamphlet) can be used.
  • base substitution on a target nucleic acid can be detected by using the nucleotide of the present invention as a primer when synthesizing DNA complementary to type I DNA chain.
  • the detection sensitivity for base substitution on the target nucleic acid can be improved by including a nucleotide analog in the above nucleotide and adjusting the reaction temperature appropriately.
  • the detection method of the present invention it is possible to examine whether the genotype of an individual is homozygous or heterozygous. That is, it is also useful for detecting base substitutions on alleles.
  • the method for detecting a target nucleic acid of the present invention can be carried out by directly amplifying the target nucleic acid from a sample containing the nucleic acid.
  • the chain length of the target nucleic acid to be amplified is not particularly limited, but from the viewpoint of detecting the target nucleic acid with high sensitivity, for example, a region of 200 bp or less, more preferably 150 bp or less is effective. It is.
  • the chimeric oligonucleotide primer of the present invention so as to have the amplified chain length, a target nucleic acid in a sample can be detected with high sensitivity.
  • a trace amount of nucleic acid can be obtained by using a reaction buffer containing a bicine, tricine, a mouse, a phosphate or a tris buffer component, and an annealing solution containing spermidine or propylenediamine.
  • the target nucleic acid can be detected from the sample with higher sensitivity.
  • hybridization with a probe for specifically detecting a target nucleic acid may be combined.
  • the probe is There is no particular limitation as long as the nucleic acid amplified by the nucleic acid amplification method of the present invention can hybridize to the target nucleic acid under ordinary hybridization conditions.
  • the viewpoint of specifically detecting the 1S amplification product for this reason, for example, those hybridizing under strict conditions known to those skilled in the art are preferable.
  • the strict high-priority conditions are described, for example, in 1989, published by Cold Spring Harbor Laboratory, edited by T. Maniatis et al., Molecular Cloning: Laboratory Manual Second Edition (Mo e ecu 1 ar C loning: described in AL aboratory Manual 2nded.)
  • the method for detecting a target nucleic acid of the present invention can be performed as follows.
  • ras Mutant Set c-Ki-ras codon 12 a template DNA and a ras Gene Primer set (ras Gene Primer) corresponding to the above codons in the Takara Bayo Co., Ltd.
  • the amplification product obtained by PCR using Set c_Ki_ras / 12 can be cloned into pT7-Blue vector (Novagen).
  • the recombinant plasmid thus obtained was transformed into type III, and PCR was performed using 1 ⁇ 13 primer # 14 and RV (both manufactured by Takara Bio Inc.).
  • nucleotide sequence of human c—K i—ras exon 1 the nucleotide sequence described in SEQ ID NO: 4 was used as a nucleotide on the side for specifically detecting the template 12G. Can be synthesized.
  • the hydroxyl group at position 3 of the ribose moiety of the nucleotide at the 3 ′ end of the nucleotide can be aminohexylated.
  • any number of nucleotide analogs in the nucleotide can be contained.
  • the nucleotides described above can also be used for nucleic acid amplification by using a primer having the nucleotide sequence of SEQ ID NO: 5 in the sequence listing.
  • each forward nucleoside of 50 pmo 1 A total of 5 X 1 reaction solutions containing the peptide and antisense primer, ⁇ of a 0.25% aqueous propylene diamine solution, and 1 pg of either type ⁇ nucleic acid were prepared, and then Thermal Cycler Personal (Takara Bio Inc.) After heat treatment at 98 ° C for 2 minutes, heat treatment at 53 ° C can be performed to anneal the nucleotide on the primer side and the antisense primer to type III.
  • the kit of the present invention provides a kit used in the nucleic acid amplification method of the present invention described in (1) or the nucleic acid detection method of the present invention.
  • the kit is characterized by including a nucleotide analog.
  • it may contain instructions for the use of DNA polymerase and endonuclease in the strand displacement reaction.
  • a kit containing a DNA polymerase having a strand displacement activity, an endonuclease, and a buffer for a strand displacement reaction is suitably used in the method of the present invention.
  • a commercially available DNA polymerase and / or endonuclease having strand displacement activity may be selected and used according to the instruction.
  • it may contain a reagent for a reverse transcription reaction in the case where the RNA is of type III.
  • the ⁇ instruction sheet '' is a printed material that describes how to use the kit, for example, how to prepare a reagent solution for strand displacement reaction, and recommended reaction conditions.
  • the label attached to the kit and the one described on the package containing the kit are included.
  • the Internet It also includes information disclosed and provided through such electronic media.
  • the kit of the present invention may contain a reaction buffer containing bicine, tricine, mouse, phosphate, or a Tris buffer component, and an annealing solution. Further, DNA polymerase DRNaseH having a strand displacement ability may be contained. Further, it may contain a nucleotide analog. Furthermore, the kit used for the method for detecting a target nucleic acid includes, in addition to the instructions described above, reagents for an amplification reaction, a chimeric oligonucleotide primer suitable for amplifying the target nucleic acid, and the amplified target nucleic acid For example, may include a probe or the like.
  • a known nucleic acid detection method for detecting a target nucleic acid for example, gel electrophoresis (agarose gel, polyacrylamide gel, etc.) or capillary electrophoresis
  • a method of detecting a reaction product of a specific size by a method, a method of detecting by hybridization with a probe, and the like can be used. Further, a detection method using a combination of magnetic beads and the like can also be suitably used.
  • pyrophosphoric acid generated during the step of amplifying the target nucleic acid may be converted into an insoluble substance such as a magnesium salt, and after turbidity, the turbidity may be measured.
  • a fluorescent substance such as ethidium bromide is usually used, but hybridization with a probe may be used.
  • a probe labeled with a radioisotope a probe labeled with a non-radioactive substance such as biotin or a fluorescent substance can be used.
  • a labeled nucleotide in the above-mentioned step (b) it is possible to incorporate a labeled nucleotide into an amplification product to facilitate detection. It is possible to enhance a detection signal using the label.
  • fluorescence polarization method fluorescence resonance energy transfer (FRET), non-fluorescence resonance energy transfer (Non-FRET) and the like can be performed.
  • FRET fluorescence resonance energy transfer
  • Non-FRET non-fluorescence resonance energy transfer
  • a visual detection method using a hybrid dochromatography method can also be suitably used.
  • RNA probes or ribonucleotides and deoxyribonucleic acid labeled with two or more fluorescent substances arranged at a distance so as to be in the quenching state Any of the chimeric oligonucleotide probes composed of nucleotides can be used in the detection method of the present invention.
  • the probe does not fluoresce 1 RNaseH degrades the probe when annealed to amplified DNA from the target nucleic acid that is complementary to it. As a result, the distance between the fluorescent substances on the probe is increased to emit fluorescence, and the presence of the target nucleic acid can be known.
  • a target nucleic acid can be detected only by adding the above probe to the reaction solution.
  • the fluorescent substance used for labeling the probe include 6-FAM (6-carboxyfluorescein) and TAMRA (N, N, N ', N'-tetramethyl-6-carboxyrhodamine), which are FRET label pairs. ) Or No n—FR ET tag pair
  • 6- FAM (6-carboxyfluorescein) and DABBCYL (4- (4'-dimethylaminophenylazo) benzoic acid) power S f Suitable for use.
  • the typical composition of the hybridization solution is as follows: 0.5% SDS, 5X Denhardt's; 0.1% ⁇ serum anolebumin (BSA), 0.1% polyvinyl Pyrrolidone, 0.1% ficoll 400] and
  • the temperature of the hybridization is not particularly limited, for example, the temperature is lower than the Tm value of the probe by 5 ° C. or more.
  • the Tm value of the probe is, for example, the following equation:
  • the Tm value is calculated by the following formula:
  • Ttn (% A + T) X 2 + (% G + C) X 4]
  • the kit is characterized in that it comprises nucleotides containing nucleotide analogues. It may be one containing a set of nucleotides containing each of the four types of bases so that the substituted base can be identified simultaneously with the presence or absence of base substitution. Further, it may contain a nuclease suitable for the nucleotide, a DNA polymerase or its substrate (dNTPs), a buffer suitable for the reaction, etc., or may contain a reagent for detecting a primer extension product. .
  • a kit for detecting base substitution in combination with a nucleic acid amplification method a kit containing a reagent for preparing a reaction solution used in the amplification method is preferable.
  • the substrate may include the nucleotide analog triphosphate.
  • the present invention provides a composition for use in the method of the present invention described above.
  • the composition include those containing the chimeric oligonucleotide primer described in the above (1) or the nucleotide described in the above (2).
  • it may be in the form of a composition for detection containing the above-mentioned probe and a composition for amplification containing the above-mentioned primer.
  • the composition may be in the form of a composition containing magnesium acetate and a composition containing a primer.
  • it may contain a buffer component, dNTPS, and the like.
  • it may contain a modified deoxyribonucleotide or an analog of deoxynucleotide triphosphate.
  • composition of the present invention may contain an internal standard (I C; int rnal contn trol) for confirming a non-amplification situation (false negative) due to decomposition and inactivation of the reagent in the composition of the present invention.
  • the internal standard can be amplified with the primers of the present invention.
  • composition suitable for the detection method of the present invention and containing the various components described above may be mentioned.
  • the composition comprises a suitable type III and chimeric oligonucleotide primer.
  • the amplification reaction can be carried out only by adding.
  • a composition containing one or more chimeric oligonucleotide primers or nucleotides suitable for the amplification of the amplification target is preferable.
  • a primer may be contained as another embodiment.
  • reagents DNA polymerase
  • the kit is suitable for easily and rapidly performing the method for detecting Mycobacterium tuberculosis of the present invention.
  • the kit can provide test results with high reproducibility and reliability, especially when examining the presence of M. tuberculosis in a large number of samples.
  • it may contain an internal standard (IC; internal control) for confirming false negatives and a probe for detecting the internal standard.
  • IC internal control
  • Such an internal standard can be amplified by the primer used in the present invention.
  • the method for amplifying and / or detecting a target nucleic acid using the nucleotide analog of the present invention uses a nucleotide analog, the target nucleic acid is used under stricter reaction conditions than when natural nucleotides are used, for example, under higher temperature conditions. Nucleic acids can be amplified and / or detected. Therefore, the target nucleic acid can be specifically amplified and Z or detected. Sequence listing free text
  • SEQ ID NO: 1 chimeric oligonucleotide primer to amplify the DNA fragment of IS6110 gene derived from Mycobacterium tuberculosis
  • nucleotides 19 to 21 are ribonucleotides-other nucleotides are
  • SEQ ID NO: 2 chimeric oligonucleotide primer to amplify the DNA fragment of IS6110 gene derived from Mycobacterium tuberculosis
  • nucleotides 20 to 22 are ribonucleotides- other nucleotides are
  • SEQ ID NO: 3 Oligonucleotide probe to detect the DNA derived from
  • SEQ ID NO: 4 Chimeric oligonucleotide to detect the nucleotide substitution on human c-Ki-ras gene, ⁇ nucleotides 13 to 15 are
  • ribonucleot ides-other nucleotides are deoxyribonucleotides and the 3'-OH group of the nucleotide at 3 'end is protected with amino hexyl group "
  • SEQ ID NO: 5 Chimeric oligonucleotide primer to amplify the DNA of a portion of human c-Ki-ras gene, ⁇ nucleotides 18 to 20 are
  • ribonucleotides-other nucleotides are deoxyribonucleotides

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Abstract

L'invention concerne un procédé permettant d'amplifier avec une forte sensibilité et de manière spécifique un acide cible dans un échantillon par une réaction de synthèse de l'ADN à l'aide d'un nucléotide contenant un analogue de nucléotide. L'invention traite également d'un procédé permettant de détecter la présence/l'absence de polymorphisme du nucléotide en se fondant sur la présence/absence d'une réaction de synthèse de l'ADN à l'aide d'un nucléotide contenant un analogue de nucléotide.
PCT/JP2002/005831 2001-06-12 2002-06-12 Procede d'amplification de l'acide nucleique et procede de detection du polymorphisme des nucleotides a l'aide d'un analogue de nucleotide WO2002101041A1 (fr)

Applications Claiming Priority (12)

Application Number Priority Date Filing Date Title
JP2001-177381 2001-06-12
JP2001-177737 2001-06-12
JP2001177737 2001-06-12
JP2001177381 2001-06-12
JP2001199552 2001-06-29
JP2001-199552 2001-06-29
JP2001290384 2001-09-25
JP2001-290384 2001-09-25
JP2001-338440 2001-11-02
JP2001338440 2001-11-02
JP2001368929 2001-12-03
JP2001-368929 2001-12-03

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0866071A2 (fr) * 1997-03-20 1998-09-23 F. Hoffmann-La Roche Ag Primaire modifiée
WO1999014226A2 (fr) * 1997-09-12 1999-03-25 Exiqon A/S Analogues d'oligonucleotides
WO2000056877A1 (fr) * 1999-03-19 2000-09-28 Takara Shuzo Co., Ltd. Procede d'amplification d'une sequence d'acide nucleique
WO2000056916A2 (fr) * 1999-03-18 2000-09-28 Exiqon A/S Detection de mutations dans des genes par des amorces de lna specifiques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0866071A2 (fr) * 1997-03-20 1998-09-23 F. Hoffmann-La Roche Ag Primaire modifiée
WO1999014226A2 (fr) * 1997-09-12 1999-03-25 Exiqon A/S Analogues d'oligonucleotides
WO2000056916A2 (fr) * 1999-03-18 2000-09-28 Exiqon A/S Detection de mutations dans des genes par des amorces de lna specifiques
WO2000056877A1 (fr) * 1999-03-19 2000-09-28 Takara Shuzo Co., Ltd. Procede d'amplification d'une sequence d'acide nucleique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIU Q. ET AL.: "Pyrophosphorolysis-activated polymerization (PAP): application to allele-specific amplification", BIOTECHNIQUES, vol. 29, no. 5, November 2000 (2000-11-01), pages 1072 - 1083, XP002201654 *

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