JP2002223761A - Method for detecting target nucleic acid and reagent therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for easily and simply detecting a target nucleic acid. SOLUTION: The subjective method for detecting a target nucleic acid comprises the steps of acting a chimeric nucleic acid probe consisting of DNA and RNA and a double strand-specific exodeoxyribonuclease on a target nucleic acid, hybrid-binding the probe, digesting a DNA part of the chimeric nucleic acid probe by an exodeoxyribonuclease, releasing a residual undigested part from the target nucleic acid, and detecting it, or using the target nucleic acid obtained by the above step as a specimen again, and detecting a residual undigested part after the digestion of the chimeric nucleic acid probe. The subjective reagent for detecting a target nucleic acid contains a chimeric nucleic acid probe consisting of DNA and RNA and a double strand-specific exodeoxyribonuclease.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting a target nucleic acid (DNA or RNA) in a biological or clinical sample with high sensitivity and a reagent therefor.

[0002]

2. Description of the Related Art Conventionally, detection of a target nucleic acid sequence in a sample has been carried out not only in the medical field, such as diagnosis of infectious diseases, identification of microorganisms or diagnosis of hereditary diseases, but also identification of species and evolution of organisms. It is used in various fields such as creation, gene function analysis, and food inspection. At present, a method of exponentially amplifying a nucleic acid containing a target nucleic acid sequence in a sample using two kinds of nucleic acid primers and a heat-resistant DNA polymerase (PCR method, etc.) is mainly used. The amplified nucleic acid is reacted with a complementary labeled nucleic acid probe that recognizes the target nucleic acid sequence, the amplified nucleic acid sequence and the complementary labeled nucleic acid probe are hybridized 1: 1, and based on the label of the bound labeled nucleic acid probe. Methods for detecting a target nucleic acid have been put to practical use.

However, since the target nucleic acid sequence and the labeled nucleic acid probe to be detected are 1: 1, a large number of nucleic acids must be amplified in advance in terms of detection sensitivity. In addition, the low specificity of the probe, to suppress non-specific detection of nucleic acids,
It was necessary to amplify the target nucleic acid and dilute the non-specific detection.

[0004] Without such a nucleic acid amplification step,
As a method for directly detecting a target nucleic acid sequence from a sample nucleic acid, using cleavage of a complementary nucleic acid probe, the target nucleic acid sequence is hybridized with a complementary nucleic acid probe a plurality of times, whereby the cleaved nucleic acid probe is used. Method for detecting and detecting the presence or absence of a target nucleic acid sequence (Patent No. 2856804)
Has been proposed. As a means for specifically realizing this method, a cycling probe technology method (CP
T method) (Patent No. 2839608) and the Invader method (US Pat. No. 5,847,717) have been reported.

[0005] The CPT method is characterized in that a chimeric probe composed of DNA and RNA is used as a complementary nucleic acid probe, and the RNA portion of the probe is cleaved with ribonuclease H. In the CPT method, ribonuclease H is digested only when the RNA portion of the nucleic acid probe forms a double strand with the DNA of the target nucleic acid. When the target nucleic acid is RNA, the target itself bound to the probe is cleaved by ribonuclease H and cannot be used for the subsequent reaction, and thus cannot be directly detected by the CPT method. For this reason, nucleic acids to be subjected to the CPT method are basically DNA, and in the case of detecting RNA, it is necessary to prepare a cDNA which has been reverse-transcribed from RNA in advance.

Further, single nucleotide polymorphisms (Sing
When selectively detecting a nucleic acid sequence that differs by only one base such as le Nucleotide Polymorphism (hereinafter referred to as SNP), it is necessary to shorten the RNA portion of the probe to be prepared. Because, in the CPT method, the probe is designed so that the RNA portion of the probe and the substituted base portion in the SNP (hereinafter, referred to as SNP site) are paired. Since the RNA is digested from an unspecified starting position, if the RNA portion of the probe is long, SNP
DNA: RNA duplexes are formed at sites other than the site,
This is because ribonuclease H digests RNA regardless of SNP.

Further, the invader method uses the target nucleic acid (A)
A nucleic acid probe (B) having a portion complementary to a portion of the target nucleic acid and a nucleic acid probe (C) complementary to another portion of the target nucleic acid and partially overlapping the probe (B) (hereinafter, referred to as “C”). Invader probe) is reacted to form a partially triple-stranded nucleic acid, and only when that part is the enzyme Clearavase
And the nucleic acid probe (B) is cleaved. The nucleic acid probe (B) is cleaved,
The generated nucleic acid sequence is used as an invader probe (D) for the subsequent second step reaction to cleave the labeled probe (E), and detection is performed based on the label of the probe E. The method comprises:
It is suitable for applications such as simultaneous analysis of multiple SNPs, but requires multiple probes (B, C, and E) for one target nucleic acid sequence, and uses a special enzyme, Cleavase, found for the reaction. Since it is used, its versatility is low and it is not a method that can be easily implemented.

[0008]

SUMMARY OF THE INVENTION The present invention has been made in view of such circumstances, and its object is to solve the problem even in the presence of a nucleic acid sequence that differs by only one base such as SNP.
It is an object of the present invention to provide a method and a target nucleic acid detection reagent that can detect a target nucleic acid sequence with high sensitivity, efficiently detect a plurality of target nucleic acid sequences, and can easily set conditions for a series of detection reactions. The object of the present invention is to
It is an object of the present invention to provide a method capable of detecting not only DNA but also RNA, which is an object, without requiring pretreatment.

[0009]

Means for Solving the Problems In view of the above circumstances, the present inventors have made various studies and found that a specific exodeoxyribonuclease (hereinafter referred to as exodeoxyribonuclease) specifically binds to a nucleic acid double strand. React, D
Focusing on digestion of the NA portion from the 3 ′ or 5 ′ side, the enzyme is used to digest the DNA portion of the complementary nucleic acid probe (consisting of DNA and RNA) hybridized to the target nucleic acid sequence. Then, the undigested portion (portion containing RNA) of the digested nucleic acid probe is released to obtain the original target nucleic acid, and the above reaction is repeated a plurality of times to accumulate the undigested portion, and The present inventors have found that a target nucleic acid is detected by detecting, and arrived at the present invention.

[0010] That is, the present invention relates to a target nucleic acid sequence (DNA
Or RNA) is hybridized with a chimeric nucleic acid probe consisting of DNA and RNA (step 1),
Exodeoxyribonuclease is allowed to act to digest the DNA portion in the chimeric nucleic acid probe (Step 2),
Next, a target nucleic acid detection method is characterized in that the undigested portion of the hybridized chimeric nucleic acid probe is released (Step 3), and the released undigested portion is detected (Step 4).

[0011] The present invention is also a method for detecting a target nucleic acid, comprising repeating the following steps 1, 2 and 3 a plurality of times and detecting the undigested portion of the released chimeric nucleic acid probe. Step 1: Target nucleic acid sequence (DNA or RNA) is converted to DNA
Step 2: exodeoxyribonuclease acts to digest the DNA portion in the chimeric nucleic acid probe; Step 3: remove the undigested portion in the hybridized chimeric nucleic acid probe Upon release, the target nucleic acid sequence (DNA or R
NA) is detected.

Further, the present invention is a reagent for detecting a target nucleic acid, comprising a chimeric nucleic acid probe comprising DNA and RNA and an exodeoxyribonuclease.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, exodeoxyribonuclease is DNA: DNA or DN.
A: An enzyme that digests bases in order from the base at the 3 ′ end or 5 ′ end of the DNA portion of the double-stranded nucleic acid composed of RNA. Therefore, for example, when a probe complementary to a single-stranded nucleic acid and a part thereof hybridizes to a portion other than both ends of the single-stranded nucleic acid, only the probe is digested, and the single-stranded nucleic acid portion is not digested. Also, the enzyme is 3 ′ of the target nucleic acid sequence.
Alternatively, it has little endonuclease activity to digest nucleic acid sequences other than the 5 'end. Examples of the enzyme include exonuclease III and lambda exonuclease, and other enzymes having the same activity can be used.

In the present invention, a chimeric nucleic acid probe consisting of DNA and RNA is a DNA portion on the 5 ′ side of the probe when the exodeoxyribonuclease activity acts on the 5 ′ end of the double strand. When the activity acts on the 3 'end, the 3' side is the DNA portion. The length of the DNA portion is about 1 to 20 bases, and an RNA portion having an arbitrary length is present in a portion following the DNA portion. The total length of the chimeric nucleic acid probe is preferably about 10 to 100 bases, and the probe may have a DNA portion and an RNA portion repeatedly. As a specific example, when exonuclease III is used, 5′-RNA (1
Bases) -DNA (1-20 bases) -3 ', as long as it has a sequence, the base sequence upstream of 5'-RNA is DNA or R
Any of NA may be used. When using lambda exonuclease, 5'-DNA (1 to 20 bases)-RNA (1 or more bases)-
As long as it has a 3 ′ sequence, the base sequence downstream of the 3′-RNA may be either DNA or RNA.

In the present invention, a label means any substance that can be used for labeling a nucleic acid containing a radioactive, fluorescent, electrochemical, luminescent or enzymatic label, or other reporter indicator groups. Substances that can be used for labeling nucleic acids include those directly detectable such as dyes and fluorescent dyes, enzymes detectable by providing a substrate such as alkaline phosphatase, and other specific substances such as biotin and digoxigenin. Includes those that have high binding properties and are indirectly detectable.

The detection method of the present invention includes the following steps 1 to 4. FIG. 1 specifically shows the following steps 1 to 3 of the present invention. In FIG. 1, the chimeric nucleic acid probe is DNA-RN
A-DNA, exodeoxyribonuclease refers to exonuclease III that digests double-stranded DNA from the 3 'end. Lambda exonuclease may be used instead of exonuclease III.

Step 1: Target nucleic acid sequence (DNA or RN
A) (in FIG. 1) is hybridized with a chimeric nucleic acid probe consisting of DNA and RNA (in FIG. 1,
). The target nucleic acid to be a sample is purified from the collected sample according to a conventional method, and is preferably dissolved in a TE buffer (pH 7 to 8) or purified water so as to have a concentration of 100 to 500 μg / mL. Sample nucleic acid is 90-95 with buffer and probe
C. for 3 to 10 minutes to denature into single-stranded nucleic acid. At this time, the sample nucleic acid is prepared preferably at 5-150 μg / mL, and the probe is preferably prepared at 10-500 nmol / mL. Hybridization conditions are adjusted to the optimal conditions for the exodeoxyribonuclease to be used and the Tm of the probe to be used. When exonuclease III is used as an example, a 50 mM Tris-HCl buffer (containing 1 mM MgCl2) pH 7.6, and a temperature within 10 ° C. above and below the Tm of the probe.

Step 2: Exodeoxyribonuclease is allowed to act to digest the DNA portion in the chimeric nucleic acid probe (in FIG. 1). Digestion occurs by adding exodeoxyribonuclease to the reaction under hybridization conditions. The amount of enzyme to be added is in the range of 1 to 100 units / μL, and when the difference between the reaction temperature and the optimum temperature of the enzyme is large, the range of 10 to 100 units / μL is desirable.

Step 3: The undigested portion in the hybridized chimeric nucleic acid probe is released (in FIG. 1). The release occurs because the Tm (melting temperature) of the probe is lowered by digestion of the DNA portion of the probe. Therefore, the conditions for hybridization or digestion may be maintained.

Step 4: The released undigested portion is detected. Detection is performed by electrophoresis, nucleic acid staining, or labeled nucleic acid probe. The labeled nucleic acid probe may be immobilized on a latex, polystyrene, cross-linked dextran or glass bead, cellulose, or Teflon or nylon membrane.

In the method of the present invention, the target nucleic acid from which the undigested portion of the chimeric nucleic acid probe is released by performing the above steps 1, 2 and 3 is used a plurality of times as the target nucleic acid sequence of step 1. be able to. By repeatedly performing steps 1, 2 and 3, a large number of undigested portions of the chimeric nucleic acid probe to be detected are accumulated, so that high detection sensitivity can be obtained.

The enzyme used in the process of the present invention is a readily available commercially available exodeoxyribonuclease,
Except for the step of denaturing the target nucleic acid into a single strand and the step of inactivating the enzyme, the detection reaction proceeds at a constant temperature and relatively slow temperature control is required, so that this is a simple and versatile detection method.

The reagent for detecting a target nucleic acid of the present invention contains a chimeric nucleic acid probe and a buffer, exodeoxyribonuclease, and if necessary, control DNA. further,
Includes electrophoresis gel necessary for detection

[0024]

Next, the present invention will be described in detail with reference to examples. The target nucleic acid, nucleic acid probe sequence, enzyme used, reaction conditions, and the like can be appropriately changed as necessary, and the present invention is not limited to these examples.

When performing SNP analysis using the present invention,
SNP site and DN located at 3 'or 5' end of probe
Probe synthesis is performed so that A is complementary. At this time, if the base of the probe corresponding to the SNP site is located within about 5 bases from the digestion start site of exodeoxyribonuclease, SNP-specific detection is possible. This is because, for SNPs other than the target nucleic acid, the exodeoxyribonuclease digestion initiation site of such a probe does not form a double strand, so that digestion by exodeoxyribonuclease does not occur.

For this reason, the length of the DNA portion of the probe is not particularly limited, and the probe can be selected according to the purpose of use. In an actual SNP analysis performed for a plurality of inspection purposes such as a disease-related investigation or a search for a constitutional determinant, it is necessary to simultaneously detect about 100 to about 1000 types of SNPs. On the other hand, it is necessary to design a probe that specifically reacts at almost the same temperature. In such a case, the high degree of freedom in probe selection facilitates probe design.

The SNP means a single nucleotide polymorphism. For example, the SNP means a vitamin D receptor (hereinafter, referred to as a osteoporosis diagnosis).
(VDR) -encoding genes include a site digested with the restriction enzyme BsmI, a gene having gaatgcg, and a gene having no such gene, and are usually called b-type and B-type.

Example 1 In the VDR gene in DNA derived from human blood, the type (b) which is cleaved by the restriction enzyme BsmI present in the intron
SNP analysis with two types (type) and uncleaved type (type B) was performed. (Preparation of chimeric nucleic acid probe) The sequences of probes 1 and 2 used are as follows. Probe 1 "5'-gccca cagac aggc
ctgcg-3 '"probe 2"5'-gggcc acaga cagg
c ctgca-3 ′ ”(The underlined portion represents RNA.) Probe 1 hybridizes with the VDR gene b type (does not hybridize with the B type gene), and probe 2 uses VD
Hybridizes with R gene B type (does not hybridize with b type gene).

(Preparation of Reaction Solutions) The following three types of solutions were prepared. Buffer 500 mM Tris-HCl (pH 7.6) 10 mM MgCl ₂ Probe solution 1 100 pmol / μL (Probe 1) Probe solution 2 100 pmol / μL (Probe 2)

(Preparation of Sample) Nucleic acid extraction kit from human blood (GFX Genomic Blood DNAPu)
Affirmation Kit, AmershamPh
A sample extracted using Armiacia Biotech) is called a PCR-RFLP method, which is a conventional PCR method (sample 1 μL, Tris-HCl buffer 10 mM, primer 1: 5′-gtc agg cga ttc ggt a
gg g-3 ′ 0.5 μM, primer 2: 5′-cca
gcg ga gag gtc aag gg-3 '
0.5 μM, magnesium chloride 1.5 mM, dNT
Ps each 200 μM, heat-resistant DNA polymerase (Toyobo, Taq Polymerase 5 units / μL)
Make 0.2 μL a total volume of 25 μL, 95 ° C. for 5 minutes, [95
C. 30 seconds, 55.degree. C. for 20 seconds, 72.degree. C. for 20 seconds] 40 times, and amplified at 72.degree. C. for 5 minutes).
The sample was treated with 5 μL of a buffer containing units / μL, and the obtained sample was subjected to polyacrylamide gel electrophoresis (PAGE). As a result of the electrophoresis, a band of a restriction enzyme fragment was observed, and it was confirmed that the gene had VDR gene B type.

The gene (VDR) collected from the above human blood
1 μL of human blood-derived DNA having genotype B), 1 μL of the above buffer solution, and 8 μL of a reaction solution A obtained by adding 5 μL of purified water to 1 μL of the probe solution 1 were prepared. Also, 1 μL of the same human blood-derived DNA, 1 μL of the above buffer solution, and 8 μL of a reaction solution B obtained by adding 5 μL of purified water to 1 μL of the probe solution 2 were prepared. After heating the reaction solution A and the reaction solution B at 95 ° C. for 5 minutes, the temperature was lowered to 62 ° C. When the temperature dropped to 62 ° C., 2 μL of exonuclease III (manufactured by Toyobo, 200 units / μL)
Was added to each reaction solution, and the mixture was left at the same temperature for 2 hours. Then, the reaction solution was again heated to 95 ° C. to inactivate the enzyme.
The mixture was heated to 5 ° C., and cooled to 4 ° C. as it was for 5 minutes.

The two reaction solutions after cooling were subjected to electrophoresis on a polyacrylamide gel containing urea so that the nucleic acid did not have a higher-order structure, and visualized with ethidium bromide. The result is shown in FIG. Lane A shows reaction solution A, and lane B shows reaction solution B. From FIG. 2, it is clear that the used target nucleic acid does not react with the probe 1 but reacts with the probe 2. That is, it was confirmed that the VDR genotype existing in the intron portion was B type.

[0033]

According to the method for detecting a target nucleic acid and the reagent for detecting a target nucleic acid of the present invention, not only DNA but also RNA can be used for cDN.
A can be detected directly without creating A. In addition, even in the presence of a target nucleic acid such as an SNP that differs by only one to several bases, a highly specific, simple and versatile detection method is provided.

[0034]

[Sequence List] <110> Nissha Corporation <120> Target nucleic acid detection method and reagents therefor <130> 13-000 <160> 4

<210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> DNA / RNA binding molecule <223> Hybridizes with VDR gene b type (does not hybridize to B type gene) Probe <400> gcccacagac aggcctgcg 19

<210> 2 <211> 20 <213> DNA <213> Artificial Sequence <220> DNA / RNA binding molecule <223> Hybridizes with VDR gene B type (does not hybridize to b type gene) Probe <400> gggccacaga caggcctgca 20

<210> 3 <211> 19 <214> DNA <213> Artificial Sequence <223> Nucleic acid amplification primer by PCR <400> gtcaggcgat tcggtaggg19

<210> 4 <211> 20 <214> DNA <213> Artificial Sequence <223> Nucleic acid amplification primer by PCR <400> ccagcggaag aggtcaagg 20

[Brief description of the drawings]

FIG. 1 is a schematic diagram of the method for detecting a target nucleic acid of the present invention.

FIG. 2 shows the S of the VDR gene BsmI using the method of the present invention.
It is an electrophoretogram which detects NP.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G045 AA24 AA25 AA28 AA35 AA39 BA11 CA25 DA12 DA13 DA14 FB01 FB02 FB03 FB05 FB06 FB07 FB13 FB15 FB20 4B024 AA11 AA20 CA01 CA09 CA11 HA14 HA19 4B063 QA01 QA QA QA QA Q QR32 QR35 QR56 QR66 QR84 QS16 QS25 QS34 QS36 QX02

Claims (12)

[Claims] 1. A target nucleic acid sequence (DNA or RNA) is hybridized with a chimeric nucleic acid probe consisting of DNA and RNA (Step 1), and then exodeoxyribonuclease is allowed to act on the hybridized chimeric nucleic acid probe. A target characterized in that the DNA portion is digested (step 2), and then the undigested portion in the hybridized chimeric nucleic acid probe is released (step 3), and the released undigested portion is detected (step 4). Nucleic acid detection method. 2. A method for detecting a target nucleic acid, comprising repeating the following steps 1, 2 and 3 a plurality of times and detecting the undigested portion of the released chimeric nucleic acid probe. Step 1: Target nucleic acid sequence (DNA or RNA) is converted to DNA
Step 2: exodeoxyribonuclease acts to digest the DNA portion in the chimeric nucleic acid probe; Step 3: remove the undigested portion in the hybridized chimeric nucleic acid probe Upon release, the target nucleic acid sequence (DNA or R
NA) is detected. 3. The exonuclease is specific for a double-stranded nucleic acid and has almost no endonuclease activity.
3. The method for detecting a target nucleic acid according to claim 1, which is an enzyme having III activity or lambda exonuclease activity. 4. The method for detecting a target nucleic acid according to claim 1, wherein the chimeric nucleic acid probe consisting of DNA and RNA is DNA at the 3 ′ side digested by exodeoxyribonuclease. 5. The chimeric nucleic acid probe has a total length of about 10 to 100 bases, and a 3′-side DNA portion digested by exodeoxyribonuclease has a length of about 1 base.
The method for detecting a target nucleic acid according to claim 1 or 2, wherein the number of base nucleic acids is from 20 to 20 bases. 6. The method for detecting a target nucleic acid according to claim 1, wherein the chimeric nucleic acid probe comprising DNA and RNA is a DNA on the 5 ′ side which is digested by exodeoxyribonuclease. 7. The chimeric nucleic acid probe has a total length of about 10 to 100 bases, and a 5 ′ DNA portion digested by exodeoxyribonuclease has a length of about 1 base.
The method for detecting a target nucleic acid according to claim 1 or 2, wherein the number of base nucleic acids is from 20 to 20 bases. 8. The method according to claim 1, wherein the undigested portion of the hybridized chimeric nucleic acid probe is released and detected by electrophoresis, nucleic acid staining or labeled nucleic acid probe.
The method for detecting a target nucleic acid according to the above. 9. The method for detecting a target nucleic acid according to claim 8, wherein the label of the labeled nucleic acid probe is a radioactive, fluorescent, electrochemical, luminescent or enzymatic label, or another reporter indicator. 10. The method according to claim 8, wherein the labeled nucleic acid probe is immobilized on a membrane. 11. The method for detecting a target nucleic acid according to claim 8, wherein the labeled chimeric nucleic acid probe is immobilized on a membrane after or during the exodeoxyribonuclease cleavage reaction. 12. A reagent for detecting a target nucleic acid, comprising a chimeric nucleic acid probe consisting of DNA and RNA and exodeoxyribonuclease.