JPH0775560B2 - Method for detecting target nucleic acid in sample - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for detecting a base sequence of a specific gene without using a so-called hybridization method.
More specifically, a pair of primers having a functional group complementary to the nucleic acid sequence to be detected is used to perform an extension reaction of the primer complementary to the nucleic acid sequence to form extension products of the primers. The present invention relates to a method for detecting a nucleic acid sequence having a double-stranded portion after immobilizing it on a solid phase carrier.

2. Description of the Related Art With the rapid progress of molecular biology of genes, it has become extremely important to detect the nucleotide sequence of a specific gene. For example, it is of great significance to detect genes in prenatal diagnosis of genetic diseases, molecular diagnosis of cancer, or detection of pathogens such as viruses.

A method generally called hybridization is used to detect such a gene (BDHame
s and SJ Higgins: Nucleic acid hybidization, a pra
ctical approach. IRL Press, 1985). In this method, a single chain or a double chain having a base sequence complementary to a target sequence is labeled with a radioactive or non-radioactive labeling substance, and then it is bound using its complementarity with the target sequence, that is, a hybrid. This is a method of detecting a target sequence by allowing soybean. In this case, generally, a dot hybridization method [DNA, 4 , 327-331, (198
5)] or Southern hybridization method [Molec
ular Cloning, p382, Cold Spring Harbor (1982)] and so on. However, even though these methods are cumbersome, time-consuming, and mechanized, it is still impossible to analyze a large number of samples as a routine work. In order to solve these problems, a hybridization method in which a probe is immobilized on a carrier has been devised (for example, TR Gingeras et al .: Nucleic Acids Res. 1
5 5373-5390], there is a limit to such hybridization between liquid phase and solid phase, and it cannot be a method that can be actually applied in terms of sensitivity and the like. In order to overcome these drawbacks of liquid-solid hybridization, sandwich type liquid-liquid hybridization has been devised [eg Ann-Christine Syvaenen et al., N.
Nucleic Acids Res. 14 , 5037-5048 (1986), JP-A-62-
229068]. However, these methods have also not been satisfactory in terms of high background and sensitivity resulting from the large excess of probe usage.

Further, a method for amplifying a specific nucleic acid sequence has been developed in order to improve the sensitivity (Japanese Patent Laid-Open No. 62-281), but this method also requires an operation of hybridization, which is complicated. Is not to be reduced.

[Outline of Invention]

The purpose of the present invention is to solve the above problems and to provide a method for easily and sensitively detecting a specific base sequence, and a pair of primers having a functional group complementary to a nucleic acid sequence to be detected. Using, the nucleic acid sequence having a double-stranded portion formed between the extension products of the primer obtained by performing the extension reaction of the primer complementary to the nucleic acid sequence, after being immobilized on a solid phase carrier, It seeks to solve this problem by providing a method of detection.

Therefore, the method for detecting at least one target nucleic acid in a sample according to the present invention is characterized by carrying out the following steps (a) to (i).

(A) To prepare a sample to detect the presence or absence of at least one target nucleic acid (hereinafter referred to as nucleic acid (I)).

(B) When the nucleic acid (I) is double-stranded, it is complementary to each strand, and when the nucleic acid (I) is single-stranded, the nucleic acid (I) and the nucleic acid complementary to the nucleic acid (I) A pair of single-stranded nucleic acids that are complementary to each strand and that are shorter than nucleic acid (I) but long enough to specifically hybridize with nucleic acid (I); Nucleic acid into which a functional group consisting of a detectable label has been introduced, and a functional group consisting of a site capable of binding to a solid phase carrier has been introduced into the other (hereinafter referred to as nucleic acid (I))
To prepare.

(C) If the nucleic acid (I) is a single-stranded nucleic acid, the sample of the step (a) is left as it is, and if it is a double-stranded nucleic acid, it is single-stranded and then subjected to the next step.

(D) Whether the single-stranded nucleic acid obtained in the previous step is hybridized with the nucleic acid (I), and in the presence of a unit nucleic acid, the single strand is used as a template to extend the chain length to form a double-stranded nucleic acid If necessary, the double-stranded nucleic acid is made into a single strand, and then the synthetic nucleic acid strands are hybridized to each other to obtain a double-stranded nucleic acid having both functional groups.

(E) Making the double-stranded nucleic acid obtained in step (d) single-stranded.

(F) Using the single-stranded nucleic acid obtained in the step (e), the step (d) is performed, or the steps (d) and (e) are sequentially repeated step by step, and finally the step To obtain a double-stranded nucleic acid having both functional groups consisting of a detectable label and a site capable of binding to a solid phase carrier, which is completed at the stage corresponding to (d).

(G) The double-stranded nucleic acid having both functional groups, which is the product of the step (d) or (f), is solid-phased in the sample using a functional group composed of a site capable of binding to the solid-phase carrier. To be combined with a carrier.

(H) Washing the solid phase carrier obtained in step (g) to remove the nucleic acid not bound to the solid phase carrier.

(I) The solid-phase carrier obtained in step (h) is subjected to a detection operation using a functional group consisting of the label, and the presence or absence of nucleic acid bound to the solid-phase carrier corresponds to the nucleic acid to be detected. Detect as something to do.

As described above, the method for detecting a nucleic acid sequence according to the present invention obtains a copy (nucleic acid sequence) to be detected labeled with two types of labels using a polymerase or the like, and Is used for immobilizing a solid phase carrier and the other is used as a label for detection to detect a gene of interest, and does not require a complicated operation called so-called hybridization, and is currently used in other fields such as the field of antigen-antibody reaction. The device used in 1. can be easily applied to this method. As a result, multiple samples can be analyzed at once.

Since nucleic acid hybridization and gel nucleic acid separation by gel electrophoresis are not required, the sample containing the nucleic acid may be in a roughly purified state, and the sample can be easily prepared and can be used by using an apparatus.

Further, in the present invention, the extension reaction by the primer is performed without performing the hybridization, so that the analysis time can be significantly shortened. Further, as the labeling substance prepared in advance in this measurement method, labeled primer or labeled mononucleotide can be chemically synthesized in large quantities, and a natural DNA fragment can be synthesized with an enzyme or the like as in the conventional hybridization method. There is no need to use and label.

Furthermore, in each sample to be detected,
Even when the base sequence of the target nucleic acid (I) is slightly different (more than one base), by appropriately adjusting the reaction conditions such as polymerase, the case where the primer is completely complementary to the target nucleic acid and the case where it is not Can be distinguished. That is, point mutations can be easily detected without relying on the hybridization method or the like.

Then, by using a plurality of primers each labeled with a labeling substance having a different functional group, an extension reaction for one or more kinds of target nucleic acid (I) can be performed at the same time, and the functional groups of the respective labeling substances are used. By performing the detection operation, the presence or absence of many target nucleic acids can be examined at the same time.

DETAILED DESCRIPTION OF THE INVENTION Detection Principle The method for detecting at least one target nucleic acid according to the present invention comprises the steps (a) to (i) described above.
(I) A target nucleic acid (referred to as nucleic acid (I)) in the sample is prepared with a nucleic acid complementary thereto (this nucleic acid is referred to as a synthetic nucleic acid), and detection is carried out; (ii) Synthesis in the sample Immobilizing nucleic acid on a solid phase carrier and performing detection using a label on synthetic nucleic acid, (iii) In order to perform (ii) above, nucleic acid (I) (each strand in the case of double-stranded) , A single strand, which is complementary to the original strand and a nucleic acid strand complementary thereto and is shorter than the nucleic acid (I) but long enough to specifically hybridize with the nucleic acid (I). For single-stranded nucleic acid,
By carrying out an amplification reaction using a nucleic acid (II) having a detectable label on one side and a site capable of binding to a solid phase carrier on the other side, a site capable of binding a synthetic nucleic acid to the solid phase carrier is formed. A functional group and a functional group consisting of a label are obtained, and the functional group is introduced so that only the synthetic nucleic acid has both functional groups, but the coexisting nucleic acid does not have both functional groups. The basic principle is to make the detection on the solid phase carrier selective.

As described above, the synthetic nucleic acid obtained by copying the base sequence of the target nucleic acid has only the both functional groups in the sample, that is, the coexisting non-target nucleic acid in the sample does not have these functional groups at all. Since it has no or only one (for example, the binding site to the solid phase carrier), if the solid phase carrier is washed after contacting the sample with the solid phase carrier, the binding site to the carrier is The coexisting nucleic acid not having it is washed away, while the coexisting nucleic acid having such a site is bound to the solid-phase carrier, but since it does not have a label, it can be detected even if the detection operation is performed on the nucleic acid-bound solid-phase carrier. Without detection, only the target nucleic acid is detected, and the above selective detection becomes possible. If the target nucleic acid does not exist in the sample, no synthetic nucleic acid is produced, and there is no labeled nucleic acid that binds to the solid phase carrier, so that the detection result shows that the target nucleic acid is absent in the sample.

The introduction of both the functional groups which should realize such selective detection is carried out by the primer having the above-mentioned both functional groups (the above-mentioned nucleic acid (I
I)) is used, for example, if the target nucleic acid is DNA, it is made into a single strand and then the DNA polymerase is used. Then, gene amplification is performed to obtain the synthetic nucleic acid chain having both functional groups. To give a specific example of the thus-obtained synthetic nucleic acid chain having both functional groups, when the target nucleic acid (I) is a double-stranded DNA, a primer having a binding site to the solid phase carrier ( Nucleic acid (II)), and dATP, dTTP, dGT as unit nucleic acids
DNA in the presence of at least one of P, dCTP, etc. (details below)
The polymerase is used to extend the chain length of the primer to form a double strand of the original DNA strand and the synthetic nucleic acid strand, and the double strand D
A primer (nucleic acid (II)) having a detectable label for the other NA is hybridized, and an extension reaction is performed in the same manner as above to form a double-stranded structure.
A DNA-derived strand is removed to release a synthetic strand, and both synthetic strands are hybridized to form a double strand having both functional groups supplied from a primer, or a target nucleic acid (I)
When is a single-stranded DNA, a site (or a detectable label) capable of binding to the solid phase carrier as described above.
With a primer (nucleic acid (II)) that has a nucleic acid (II) and is subjected to an extension reaction to form a double strand of an original DNA chain and a synthetic nucleic acid chain. A primer (nucleic acid (II)) having a binding site) is hybridized, and a similar extension reaction is performed to form a double-stranded chain having both functional groups. For example, a double strand consisting of a synthetic chain is amplified by adding and / or extending the chain length or forming a synthetic chain.

It should be noted that one of the synthetic nucleic acids also has a binding site to the solid phase carrier, but in this case, the "binding site to the solid phase carrier" is not necessarily a site that can be directly linked to the solid phase carrier. May be. That is, a substance capable of intervening between the site on the side of the synthetic nucleic acid and the binding site on the side of the solid phase carrier to enable or promote the binding of the both (the solid phase carrier and The capture reagent may be bound in advance to the binding site with the solid phase carrier on the synthetic nucleic acid side, or the binding site on the solid phase carrier side. May be combined with.

Further, in the primer extension reaction by a polymerase, it is possible to simultaneously detect a plurality of target nucleic acids (I) in the same sample by using different primers having different functional groups (labels).

Actual Detection a. Nucleic acid The nucleic acid to be detected in the present invention includes a base sequence to be detected and may be DNA or RNA. Such nucleic acids can be prepared from all living organisms such as E. coli, viruses and higher animals and plants. When the above nucleic acid is used in the present detection method, the nucleic acid may or may not be purified.

b. Primer and its extension reaction (i) Primer (nucleic acid (II)) When the above-mentioned nucleic acid (I) to be detected is a double strand, the primer in the present invention is a nucleic acid (I) for each strand. Is a single strand, the strand and each strand of the nucleic acid complementary thereto (DN
In the case of A, the double-stranded nucleic acid sequence is made single-stranded by means such as denaturation, and in the case of RNA, it is made into DNA by reverse transcriptase or the like as described above. ) Is a set of single-stranded nucleic acids that specifically forms a complementary strand with a mononucleotide sequentially added to its 3'end (a hydroxyl group at the 3'end is essential). One of the double-stranded nucleic acids has a detectable label, and the other has a site capable of binding to a solid phase carrier. Generally, a primer refers to an oligodeoxyribonucleotide, but may be prepared from a long DNA fragment obtained from nature. It should be of sufficient length to specifically hybridize with the nucleic acid of interest (nucleic acid (I)).

When a point mutation is to be detected, a primer and a point mutation that are sufficiently complementary and long enough to specifically hybridize with the point-mutated target nucleic acid Two primers are used, which are of sufficient length and are completely complementary to specifically hybridize with the nucleic acid which is not present. All of these primers are oligodeoxyribonucleotides.

The site of the primer that can bind to the labeling substance or the solid phase carrier may be any position as long as it does not interfere with the extension reaction of the primer, but is preferably the 5'end.

As the labeling substance, either a non-radioactive substance or a radioactive substance may be used.

Examples of the non-radioactive labeling substance include, in addition to the biotin shown in the experimental examples described below, a 2,4-dinitrophenyl group, fluorescein and its derivative [fluorescein isothiocyanate (FITC)], rhodamine and its derivative [eg, tetramethyl Rhodamine Isothiocinate (TRITC), Texas Red, etc.], 4-Fluoro-7-
There are fluorescent substances or chemiluminescent substances such as nitrobenzofuran (NBDF) and dansyl, and labeling is performed by known means (see JP-A-59-93098 and JP-A-59-93099). be able to.

When labeling with a radioactive substance, for example, ¹³¹ I,
^The labeling substance can be introduced by a known means using a radioactive isotope such as ¹³³ I, ¹⁴ C, ³ H, ³⁵ S, ³² P.

On the other hand, the site capable of binding to the solid phase carrier may be any site as long as it can selectively react with the carrier. As an example, the non-radioactive labeling substance can be used as it is, and in that case, the labeling substance to be used for detection is
They cannot be the same. As a preferred specific example, a fluorescent substance such as biotin or fluorescein or a hapten such as a 2,4-dinitrophenyl group can be introduced into a primer in advance. The solid-phase carrier as used herein is defined below.

When the primer is an oligodeoxyribonucleotide, the labeling of the primer with these is carried out chemically after or simultaneously with the chemical synthesis of the oligodeoxyribonucleotide (JP-A-59-93098 and JP-A-59-93099). ).

It can also be chemically labeled when the primer is a natural fragment [LE Orgel et al., Nucleic Acids R
es. 14 , 5591-5603, (1986)].

ii) Extension reaction of primer There are four types of extension reactions using the above-mentioned primers (primers different from each other, one having a site capable of binding to a solid phase carrier and the other having a labeling substance introduced). It can be carried out by incorporating at least one of deoxyribonucleotide triphosphates deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate and thymidine triphosphate as a substrate into a primer. E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, or reverse transcriptase can be used for this extension reaction. In particular, a thermostable enzyme capable of elongation reaction at high temperature can be used to enhance the specificity of target sequence recognition by a primer [FF Chehab et al .: Nature 329 , 293-294 (198
7)].

Further, when more sensitive detection is required, particularly when the amount of the base sequence to be detected is small, a nucleic acid sequence amplification method can be used [JP-A-62-281]. That is,
If the labeled primer and labeled mononucleotide triphosphate described above are used, a target base sequence having two kinds of functional groups (a site capable of binding to a solid phase carrier or a labeling substance used for detection) can be easily amplified. Obtainable.

In order for the primer extension reaction to properly start at the target position, the degree of complementarity between the primer and the template (that is, the target nucleic acid (I)), the length of the primer,
Factors such as reaction temperature must be considered. It goes without saying that the reaction generally has to be performed at a lower temperature when the length of the primer is short or the degree of complementarity is low.

Moreover, in order to carry out the primer extension reaction at a more correct target position, after making the double-stranded nucleic acid obtained after the first primer (nucleic acid (II)) extension reaction into a single strand, , Complementary to the base portion of the target nucleic acid (I) 5 ′ to the base portion that hybridizes with the nucleic acid (II), and shorter than the base portion, but sufficient to specifically hybridize with the base portion. Single-stranded nucleic acid of length (nucleic acid (II
I)) can be used to carry out a new extension reaction (Japanese Patent Application No. 63-149157).

Furthermore, when using this method to detect a sudden change in a point, the extension conditions of the primer must be considered in addition to the above. For example, DMSO may be added to the reaction solution in order to make a difference in the stability of the double strand (whether it is completely complementary or not) formed between the primer and the target nucleic acid (I), or a competitive primer ( When investigating a point mutation in the target nucleic acid (I), a primer that is completely complementary to a normal base sequence and a primer that is completely complementary to a base sequence that has a point mutation are mixed and the primer When the base sequence in the target nucleic acid (I) is normal, the latter primer is a competitive primer, and conversely, a point mutation occurs in the base sequence in the target nucleic acid (I). In this case, the former serves as a competitive primer.) And the extension reaction must be performed.

c. Solid-phase carrier The solid-phase carrier in the present invention may be any solid carrier that is inert to the solvent and all reagents used in the reaction and that can be separated from the solution by some method. As such, for example, microtiter wells, polystyrene balls, agarose beads, polyacrylic beads, etc. can be used.

The solid phase carrier has introduced therein a capture reagent for capturing a double-stranded structure (in which a site capable of binding to the solid phase carrier and a labeling substance have been introduced) previously generated by the primer extension reaction. By doing so, immobilization can be performed easily and selectively.

Such a capturing reagent may be one that selectively reacts with a site capable of binding to the solid phase carrier existing in the above double-stranded structure, and preferably capable of reacting under mild conditions. Things are good. The binding mode between the two may be covalent bond or non-covalent bond as long as a specific bond is generated. Preferably, a binding method that can retain the activity of the capture reagent to the maximum is preferable. Specific examples of these capture reagents include streptavidin and antibodies.

For example, in order to capture the biotin-labeled copy, a carrier bound with streptavidin is used, and fluorescein or 2,4-
For the labeled product of the dinitrophenyl group, one in which each antibody is bound to a carrier can be used.

d. Detection method The solid phase carrier prepared according to the method described above and the nucleic acid having both functional groups prepared in the above steps (a) to (f) are mixed to bind both, and then bound to the carrier. Unpurified nucleic acids other than the target nucleotide sequence and impurities other than the nucleic acid contained in the sample are washed with an appropriate solvent. The term “suitable solvent” as used herein means that all reagents such as nucleic acids and labeling substances must be kept stable, as well as solid phase carriers and synthetic nucleic acids, solid phase carriers and capture reagents, and capture reagents. The conditions under which the bond between the synthetic nucleic acid or the label and the synthetic nucleic acid is broken should not be established. Further, when both functional groups in the nucleic acid sequence having a double-stranded portion are present on separate strands of the complementary strand, the conditions should not be such that the complementary strand is dissociated.

The washing method varies depending on the property of the carrier for immobilization, and may be a method generally used in the field of antigen-antibody reaction.

By such a washing operation, only the copy (synthetic nucleic acid) of the target nucleic acid to be detected (nucleic acid (I)) can be selectively immobilized on the carrier.

Next, the synthetic nucleic acid having the labeling substance immobilized on the carrier is
It can be detected while being immobilized on the carrier or in the form of being liberated in the solution. For detection in a free form, for example, there is a method of breaking the bond between the labeling substance and the capture reagent, or breaking the bond between the labeling substance and the synthetic nucleic acid [Herman, TM Et al. Anal.Bioche
mistry 156 , 48-55 (1986)]. Also, when both functional groups are present on separate strands of the complementary strand of the synthetic nucleic acid, the double strands are separated by heat denaturation or other known methods, and the strand containing the detection labeling substance is released into the solution. Can be made.

In addition, when the amplification reaction is carried out using primers labeled with two different labels, if there is a restriction enzyme site in the amplified sequence, fix it on a carrier and then use the restriction enzyme for detection. The fragment containing the label can be released from the solid support.

On the other hand, when there is no restriction enzyme cleavage site in the amplified sequence, the fragment containing the detection labeling substance cannot be released from the immobilization carrier, and this method is used to cut a specific restriction enzyme cleavage site in the amplified sequence. You can find out if exists.

For the actual detection of the labeling substance, a general method may be used depending on the labeling substance used. For example, if the labeling substance is a radioisotope, the activity may be measured as it is, and if it is biotin, for example, it is reacted with a substrate using an avidin- or streptavidin-enzyme conjugate,
The detectable component can be obtained by chromatic or fluorescent means. In addition, for example, if the labeling substance is fluorescent, the intensity can be directly measured using a fluorometer.

Now, the following points are important for measuring an actual sample in the method described above.

The first is a non-specific extension reaction in the primer extension reaction. This is a primer that binds to a site other than the intended nucleotide sequence and causes an extension reaction.
In order to prevent such a non-specific extension reaction, as is generally considered, the GC content and length of the primer must be thoroughly examined for each detection target. The non-specific extension reaction can be sufficiently eliminated by raising the reaction temperature, and it can be improved by using heat-resistant DNA polymerase (NEB).

Secondly, the unreacted label binds to the capture reagent immobilized on the carrier and interferes with the original reaction with the labeled copy (synthetic nucleic acid) to be detected. In order to solve this problem, firstly, it is necessary to increase the capturing ability of the capturing reagent immobilized on the carrier. The second is to remove the unreacted labeled substance outside the system. This is due to the difference in the properties of the labeled primer and the replicate (synthetic nucleic acid) to be detected, such as a simple gel method. Can be separated with [Ma
niatis et al .: Molecular Cloning p. 466 (1982)]. However, these methods are not necessarily preferable methods in view of mechanization. From that point,
It is important to select reaction conditions that minimize the amount of unreacted label remaining. For example, using a kind of primer introduced with a site capable of binding to a solid phase carrier,
When an extension reaction occurs using a unit nucleic acid (mononucleotide triphosphate) as a substrate, the amount of the primer must be such that it does not exceed the carrier-capturing ability.

In addition, even if the efficiency of producing a copy is reduced to some extent, do not use an excessive amount of primer. However, the reaction condition that reduces the amount of unreacted labeled substance remaining is not necessary when the capturing ability of the carrier into which the capturing reagent is introduced is sufficient.

[Experimental example]

Example 1 This example illustrates a method for detecting the E. coli β-galactosidase gene.

A part of β-galactosidase gene is deleted in Escherichia coli JM103 (Pharmacia), and if the extension reaction is carried out using a primer corresponding to that part, a deletion strain (here JM103) and a wild strain (here It is possible to distinguish from HB101 (BRL), so an experiment was carried out to distinguish between a wild strain and a deletion strain using the method described below.

The gene of Escherichia coli is obtained by the method of Raymond L. [Recombinant DN
A Techniques.p.45-46, Addison-Wesley Publishing C
ompany (1983)] and extracted from JM103 and HB101.
The structure of the primer is biotin (Bio) introduced at the 5'end as shown below. An aminated oligonucleotide was synthesized using an automatic DNA synthesizer NS-1 (Shimadzu), and succinimide ester of piotin was synthesized. Was used for biotinization (JP-A-59-93098 and JP-A-59-93099).

(Bio) -GGGTTTTCCCAGTCACGACGTTGTA Escherichia coli DNA digested with the restriction enzyme EcoRI was used as a polymerase reaction solution [50 μl in total. 10% DMSO, 0.05 μg primer, 67
_{mM Tris-HCl pH8.8,6.7mM MgCl 2,} 6.6mM ammonium sulfate, 10 mM beta-mercaptoethanol, 6.7Myu
M EDTA, 20 μM dATP, 20 μM dGTP, 20 μM TT
P, 1 μM [α- ³² P] dCTP (NEG-013H) was added, heated at 95 ° C for 7 minutes, and then kept at room temperature for 5 minutes to be heat-resistant.
DNA polytalase (0.5 U, New England Biolab) was added and reacted at 50 ° C. for 10 minutes.

Streptavidin-agarose (BR
L) is a washing solution (0.01M phosphate buffer, pH7.2, 0.15M
NaCl) and washed twice. Next, the extension reaction mixture (50μ) and 0.02M phosphate buffer pH7.2, 0.3M NaCl.
(50μ) mixed and pretreated Streptavidin-
It was added to agarose and left at room temperature for 30 minutes. After the reaction, the plate was washed 5 times with a washing solution (200 μ), and the radioactivity immobilized on streptavidin-agarose was measured. as a result,
HB10 is wild type for the β-galactosidase gene
It was possible to distinguish 1 from the deletion type JM103.

Example 2 This example illustrates a method for discriminating between the normal β-globin gene and the β-globin sickle cell anemia allele.

Both the normal and sickle cell anemia β-globin genes had pBR322 with the BamHI fragment inserted into plasmid pBR322.
-HβPst and pBR322-HβS were used [RB Walla
ce et al .: DNA 3 , 7-15, (1984)]. The primers are of the structure shown below, each complementary to a different strand,
One is polynucleotide kinase and [γ- ³² P] ATP
The 5'end was labeled with and the other was synthesized and biotinylated as in Example 1. ³² P- ^{5 '} TTCTGACACAACTGTGTTCACTAGC ^3' -Primer A (Bio) -5 ^' ACCACCAACTTCATCCACGTTCACC ^3' -Primer B Plasmid digested with restriction enzyme EcoRI (pBR322-HβPst
Alternatively, pBR322-HβS) 10 ng is used as a heat-resistant DNA polymerase reaction solution [50 μm. 10% DMSO, 0.3 μg Primer A, 0.3 μg Primer B, 67 mM Tris-HCl pH 8.8, 6. according to New England Biolabs protocol.
7 mM MgCl ₂ , 6.6 mM ammonium sulfate, 10 mM β-mercaptoethanol, 6.7 μM EDTA, 33 μM dATP, 33
μM dGTP, 33 μM dCTP, 33 μM dTTP].
The mixture was heated at 95 ° C for 7 minutes, returned to room temperature and annealed for 5 minutes. Next, add 0.5 U of heat-resistant DNA polymerase (New England Biolabs) at 55 ° C.
The first extension reaction was performed for 5 minutes. Thereafter, the cycle of denaturation at 91 ° C for 1 minute + annealing at room temperature for 5 minutes + extension reaction at 55 ° C for 5 minutes was repeated 20 times. Next, streptavidin-agarose (50 μm) prepared in the same manner as in Example 1 was added to the above reaction mixture (25 μm) and water (75 μm).
) And 0.02M phosphate buffer pH 7.2, 0.3M NaCl
(100 μ) was added, and the mixture was reacted at room temperature for 30 minutes with slow mixing. It was washed 5 times with a washing solution (200 μ). Next, streptavidin-agarose was divided into two tubes in equal amounts. On the other hand, the reaction solution of the restriction enzyme DdeI [10
mM Tris-HCl (pH 7.5), 7 mM MgCl ₂ , 150 mM NaCl, 7
mM mercaptoethanol, 100 μg / ml, calf serum albumin] 100 μ was added, and the restriction enzyme DdeI (Toyobo, 10 U /
μ) 5μ, and slowly mix at 37 ℃.
Reacted for hours. After the reaction, it was washed 5 times with a washing solution (200 μ). Plasmids pBR322-HβPst and pBR322-H
For each βS, the radioactivity remaining in the carrier before and after DdeI digestion was measured to examine the decrease in radioactivity due to DdeI digestion. As a result, the residual radioactivity due to DdeI digestion was 40% in the case of pBR322-HβPst and pBR322-H.
In the case of βS, it was 92%, and both could be distinguished.

Example 3 This example shows a method for simultaneously detecting two types of genes in one sample.

The human β-clobin gene and human papillomavirus 16 (HPV-16) were selected as the target genes. As a primer for amplifying the β-globin gene, β-
Bio-NH-CAACTTCATCCACGTTCAAC (Bio-PG1), each of which is complementary to the duplex of the globin gene and labeled with biotin
And fluorescently labeled with EITC (eosin isothiocyanate)
EITC-NH-ACACAACTGTGTTCACTAGC (EITC-PG2) was used. In addition, Bio-NH-TGAGCAATTAAAT labeled with biotin was used as a primer for amplifying the E6 gene of HPV-16.
FITC-NH-T fluorescently labeled with GACAGC (Bio-PVO1) and FITC
GTGCTTTGTACGCACAAC (FITC-PVO2) was used. As a sample to be detected, a normal human placenta gene and Caski cells (ATCC: CRL1550) were used. The former has the human globin gene, but does not have the papillomavirus gene. The latter is naturally derived from human cells and has the human β-globin gene, but it has been clarified that the human papillomavirus 16 gene is amplified and present {The EMBO Journal 6 , 139-144.
(1987)}.

Reaction 1: Human placenta gene (1 μg) and primer {Bi
o-PVO1 (300ng), FITC-PVO2 (300ng), Bio-PG1 (3
00ng), EITC-PG2 (300ng) in reaction solution {67mM Tris ・ HCl
_{pH8.8,6.7mM MgCl 2, 16.6mM (NH4)} 2SO 4 · 10mM mercaptoethanol, 6.7mM EDTA, 200μM dATP, 200μM dGT
P, 200 μM dCTP, 200 μM TTP, 10% DMSO} (total volume:
49 μl) and denatured at 95 ° C. for 5 minutes. After annealing at 55 ° C for 1 minute, Taq polymerase (NEB, 20 / µl; 1µ
1) was added and extension reaction was carried out at 70 ° C. for 2 minutes. Next 92 ° C
It was denatured for 1 minute at room temperature and annealed at 55 ° C for 1 minute. This denaturation, annealing and extension reaction was repeated 25 times.

Reaction 2: Using DNA (1 μg) obtained from Caki cells, the same extension reaction was carried out with the same primers as in Reaction 1.

Preparation of Streptavidin Agarose: Streptavidin agarose (BRL) is a washing solution (10m
M Tris ・ Hcl pH7.5,1mM EDTA pH8.0,0.1M NaHclO ₄ 2
After washing twice, the salmon gene (1 μg) was added to this for pretreatment. 20 μl of reaction solution 1 or reaction solution 2 was added to pretreated streptavidin agarose (50 μl) and left at room temperature for 15 minutes. This is the previous washing solution (500 μl)
It was washed twice with 1 M NaHClO ₄ (500 μl) and then twice. Next, wash twice with the previous washing solution (500 μl),
0 mM Tris ・ HCl pH7.5,1 mM EDTA pH8.0,50 mM NaCl (500
μl) and washed 3 times. To this, 100 mM NaOH (50 μl) was added to denature the DNA to obtain a supernatant, and 10 mM Tris pH7.
Fluorescence was measured by adding 5,1 mM EDTA pH8.0, 50 mM NaCl solution (450 μl). FITC fluorescence was measured at an excitation wavelength of 489 nm and an emission wavelength of 520 nm, and EITC was measured at an excitation wavelength of 520 nm and an emission wavelength of 540 nm. The respective fluorescence intensities are shown below as relative intensities.

From this result, it was possible to determine that only the β-globin gene was present in normal human placental DNA, and both β-globin gene and human papilloma gene were present in Caski cells.

Claims (1)

[Claims] 1. A method for detecting at least one target nucleic acid in a sample, which comprises carrying out the following steps (a) to (i). (A) To prepare a sample to detect the presence or absence of at least one target nucleic acid (hereinafter referred to as nucleic acid (I)). (B) When the nucleic acid (I) is double-stranded, it is complementary to each strand, and when the nucleic acid (I) is single-stranded, the nucleic acid (I) and the nucleic acid complementary to the nucleic acid (I) A pair of single-stranded nucleic acids that are complementary to each strand and that are shorter than nucleic acid (I) but long enough to specifically hybridize with nucleic acid (I); Nucleic acid having a functional group consisting of a detectable label and a functional group consisting of a site capable of binding to a solid phase carrier (hereinafter referred to as nucleic acid (II))
To prepare. (C) If the nucleic acid (I) is a single-stranded nucleic acid, the sample of the step (a) is left as it is, and if it is a double-stranded nucleic acid, it is single-stranded and then subjected to the next step. (D) Whether the single-stranded nucleic acid obtained in the previous step is hybridized with nucleic acid (II) and the length of the single strand is extended in the presence of a unit nucleic acid to form a double-stranded nucleic acid. If necessary, the double-stranded nucleic acid is made into a single strand, and then the synthetic nucleic acid strands are hybridized to each other to obtain a double-stranded nucleic acid having both functional groups. (E) Making the double-stranded nucleic acid obtained in step (d) single-stranded. (F) Using the single-stranded nucleic acid obtained in the step (e), the step (d) is performed, or the steps (d) and (e) are sequentially repeated step by step, and finally the step To obtain a double-stranded nucleic acid having both functional groups consisting of a detectable label and a site capable of binding to a solid phase carrier, which is completed at the stage corresponding to (d). (G) The double-stranded nucleic acid having both functional groups, which is the product of the step (d) or (f), is solid-phased in the sample using a functional group composed of a site capable of binding to the solid-phase carrier. To be combined with a carrier. (H) Washing the solid phase carrier obtained in step (g) to remove the nucleic acid not bound to the solid phase carrier. (I) The solid-phase carrier obtained in step (h) is subjected to a detection operation using a functional group consisting of the label, and the presence or absence of nucleic acid bound to the solid-phase carrier corresponds to the nucleic acid to be detected. Detect as something to do.