JP2005151877A - Method for amplifying dna and method for detecting dna - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily detecting whether a DNA in a specimen is a target DNA or not. <P>SOLUTION: The method for amplifying the DNA by amplifying the DNA by a thermal recycling reaction repeating three steps of (a) a step (an annealing step) for recognizing the terminus of a sequence region for amplifying the 3' part of each diverged single-stranded DNA, and hybridizing two kinds of hybridizable oligonucleotides, (b) a step for carrying out an elongation reaction of a primer by a DNA polymerase, and (c) a denaturation step involves using oligonucleotides having polymers having the lower critical solution temperatures and linked to the 5' terminus as the two kinds of the oligonucleotides used in the step (a). The method for detecting the DNA comprises carrying out the amplification method, adding a salt to the product, heating the resultant mixture to a temperature not lower than the lower critical solution temperature of the polymer linked to the 5' terminus of the oligonucleotide and not higher than Tm of the amplified DNA, and measuring the change of turbidity of the solution. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for easily detecting whether a target DNA sequence is present without using an expensive fluorescent dye detection apparatus.

As a technique for amplifying a gene, a polymerase chain reaction method (hereinafter referred to as a PCR method) is generally performed. The PCR method is a method developed by Saiki et al. (Science 230, 1350-1354 (1985)) and detects a specific nucleotide sequence region (such as DNA in a sample). Prepare two oligonucleotides that hybridize by recognizing the 3 'end of the + strand on one end of the specific region of the + strand and the other-strand), and the other on the 3' end of the-strand. The sample DNA that has been made into a single strand by heat denaturation is allowed to function as a primer for a template-dependent nucleotide polymerization reaction, and the generated double-stranded DNA is again separated into single strands to cause a similar reaction again. In this method, the above-mentioned specific region sandwiched between two primers is amplified to a detectable amount by repeating a series of operations.
In a reaction system with appropriately designed primers, it is possible to detect whether or not a very small amount of DNA, for example, one virus, bacteria, or cell is present in a sample by evaluating whether or not DNA has been amplified. Can do.
As a method of confirming the amplification product, there is a PCR-SSCP (PCR-Single Strand Polymorphism) method in which the amplification product is directly electrophoresed using polyacrylamide and a mutation is detected from the difference in the migration degree.
In this method, a change in the three-dimensional structure of the amplification product due to mutation is detected as a difference in electrophoretic mobility. In addition, is it possible to perform PCR by sequencing techniques such as shotgun method, primer walking method, cloning method, etc., or by appropriately cleaving DNA with restriction enzymes and deliberately placing the 3 'end of the PCR primer at the mutated part? PCR-ASP (PCR-Allele Specific Primer) method for discriminating depending on whether or not, and PCR-RFLP (PCR-Restriction Fragment Length Polymorphism) method for discriminating whether or not the mutated portion is amplified by PCR and can be cleaved with a restriction enzyme Further, there is a Taqman probe method in which a mutated portion is further detected with a fluorescently labeled probe. However, any of these methods is a method in which a person skilled in advanced technology can use an expensive device and put a considerable time and money, and only such a method has been developed. It is.

DNA hybridization utilizes the fact that adenine (A) is hydrogen bonded to thymine (T), and cytosine (C) is hydrogen bonded to guanine (G). There are three hydrogen bonds between -C, and in the part where there are many GC sequences, even if the sequence of A or T is slightly shifted, the conventional method may inadvertently hybridize. The accuracy was still insufficient for use in diagnosis.
On the other hand, in the patent application of Maeda et al. (Japanese Patent Laid-Open No. 2001-252098), gene DNA is added to an aqueous solution containing a DNA conjugated substance, which is a complex of single-stranded DNA and a hydrophobic substance, and a metal cation. In addition, a genetic diagnosis method for measuring a change in light scattering intensity or light transmittance of the aqueous solution has been proposed. This is because the DNA is highly water-soluble and forms a colloid. However, when it is double-stranded, adenine has a property of thymine, and cytosine and guanine have hydrogen bonds, resulting in a decrease in hydrophilicity. Only when DNA that forms a complementary strand exists and hybridization occurs, the stability of colloids collapses and aggregation occurs, so that mutation of only one DNA such as single nucleotide polymorphism (SNPS) can be performed. It is an excellent method that can be detected with high accuracy.
However, in order to use this detection method for various genetic tests, a large amount of sample DNA is collected, or the sample DNA is amplified and the obtained DNA is expensive to match the length of the DNA conjugate substance. The sample must be prepared by cutting with a restriction enzyme and removing the other DNA that interferes with the hybridization of the DNA conjugate substance and the DNA. There was a need for further improvements for use in the diagnosis of
JP 2001-252098 A

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a simple DNA testing method, and more specifically, whether or not the sample DNA is the target DNA. It is to provide a simple detection method.

The above object of the present invention is to
(1) (a) Two types of oligonucleotides of sequences designed to recognize and hybridize to the separated single-stranded DNAs by recognizing and hybridizing each 3 'end of the region of the sequence to be amplified , Sometimes referred to as a primer), a step of hybridizing (annealing step),
(B) a step of extending an oligonucleotide with a DNA polymerase in the presence of deoxyribonucleotide triphosphate (dNTP) (polymerase step),
(C) Step of modifying (denaturing step)
When the DNA is amplified by performing a thermal cycle reaction that repeats the above three steps, a polymer having a lower critical solution temperature is linked to the 5 ′ end as two types of oligonucleotides used in the step (a). A method for amplifying DNA, comprising using the oligonucleotide.
(2) After performing the DNA amplification method in the step described in (1) above, a salt is added, and further, the DNA that has been amplified at a temperature above the lower critical solution temperature of the polymer bound to the 5 ′ end of the oligonucleotide A method for detecting DNA, which comprises heating to a temperature below Tm and measuring the change in turbidity of the solution.
Achieved by.

  According to the present invention, it is possible to easily detect whether or not a target gene is present visually without using an expensive fluorescent reagent or a fluorescence detection device.

Hereinafter, the present invention will be specifically described.
First, the principle of the present invention will be described.
The oligonucleotide in which a polymer having a lower critical solution temperature is linked to the 5 ′ end used in the present invention (hereinafter sometimes referred to as an oligonucleotide copolymer) is a transition temperature of the polymer linked to the oligonucleotide. In the following, all components are stretched in a chain, and the structure is easy to hybridize to DNA serving as a template for amplification (sometimes referred to as template DNA or simply a template). When poly (N-isopropylacrylamide) is selected as the polymer having the lower critical solution temperature linked to the oligonucleotide, the lower critical solution temperature of poly (N-isopropylacrylamide) is 32 ° C. The template is annealed at a temperature at which hybridization is easy, for example, around 25 ° C. When the annealing is finished, a polymerase enzyme is used to extend the oligonucleotide along the template DNA. Generally used polymerase enzyme elongates DNA at a temperature of 55 ° C. or higher, so that it becomes higher than the transition temperature of a polymer having a lower critical solution temperature at the 5 ′ end, and this part becomes hydrophobic, making the oligonucleotide hydrophilic. It becomes a micelle structure as a site. Whether the micelle structure is taken or not depends on the structure of the synthesized DNA. That is, what is important is the length of DNA in the synthesized DNA and the content of DNA.
In the amplification method of the present invention, abnormal amplification may occur due to mismatch occurring when annealed at a low temperature. However, when such a micelle structure is adopted, the mismatch portion is removed and only normal amplification proceeds. This primer extends to the annealed part. As a result, DNA in which a polymer having a lower critical solution temperature is linked to the 5 ′ end having a completely complementary strand to the template DNA is synthesized.
Whether this DNA has been successfully amplified depends on whether the DNA having a lower critical solution temperature linked to the 5 'end is agglomerated by increasing the salt concentration to a temperature above the lower critical solution temperature. Can be determined.

Next, the components of the present invention will be described sequentially.
The oligonucleotide copolymer used in the annealing step of the present invention has a polymer having a lower critical solution temperature at the 5 ′ end of the oligonucleotide.
Here, the lower critical solution temperature refers to a critical temperature at which a polymer is rearranged from a state dissolved in water to an insoluble state. Below this temperature, the polymer is in a hydrated state and the molecular chains are spreading, but above this temperature, dehydration occurs and the molecular chains aggregate. This change with temperature is reversible.
The lower critical solution temperature is obtained by using one or more monomers that form a polymer that does not exhibit the lower critical solution temperature together with one or more monomers that can obtain a polymer that exhibits the lower critical solution temperature. Can be changed.
As a monomer that forms a polymer that does not exhibit the lower critical eutectic temperature, for example, when a hydrophobic monomer is used, the lower critical eutectic temperature can be moved to a lower temperature side, and a hydrophilic monomer can be used. The lower critical eutectic temperature can be moved to the higher temperature side.
Examples of the monomer that forms a polymer exhibiting a lower critical eutectic temperature include N-substituted (meth) acrylamide derivatives and vinyl methyl ether, among which N-substituted (meth) acrylamide derivatives are preferable.
The lower critical solution temperature of the polymer obtained by polymerizing these monomers is, for example,
Poly (N-ethylacrylamide): 72 ° C
Poly (N-ethyl-N-methylacrylamide): 56 ° C
Poly (N-pyrrolidinylacrylamide): 56 ° C
Poly (N-cyclopropylacrylamide): 46 ° C
Poly (N-isopropylacrylamide): 32 ° C
Poly (N-diethylacrylamide): 29 ° C
Poly (N-propylacrylamide): 21 ° C
Poly (N-piperidinylacrylamide): 5 ° C
Poly (N-cyclopropylmethacrylamide): 59 ° C
Poly (N-ethylmethallylamide): 50 ° C
Poly (N-isopropylmethacrylamide): 44 ° C
Poly (N-propylacrylamide): 13 ° C
However, poly (N-isopropylacrylamide) (hereinafter sometimes referred to as PNIPAAm) is well known for its temperature response behavior (Schild, HG, Prog. Polym. Sci. 17, 163-249, (1992)) When a complex is formed with a single-stranded DNA, the response temperature range is relatively low, easy to handle and easy to obtain (obtained from Kojin Co., Ltd.). In the invention, PNIPAAm is preferably used as a polymer having a lower critical solution temperature.

In the above polymer, other monomers may be copolymerized as long as the lower critical eutectic temperature is not lost.
When PNIPAAm is used as a polymer having a lower critical solution temperature linked to the 5 ′ end of the oligonucleotide of the oligonucleotide copolymer, when the oligonucleotide is a single-stranded DNA, the oligonucleotide copolymer is: It becomes single-stranded DNA-PNIPAAm. As a method for producing such a single-stranded DNA-PNIPAAm, various methods are applied. Preferably, a terminal-aminated single-stranded DNA and acryloyloxysuccinimide (obtained from Acros) are coupled to each other, Examples thereof include a method obtained by radical-copolymerizing an N-isopropylacrylamide (hereinafter referred to as NIPAAm) monomer after making the terminal amino group acryloylated single-stranded DNA. At this time, any catalyst may be used for the coupling reaction or copolymerization reaction, and the reaction conditions are not particularly limited. For example, terminal aminated single-stranded DNA and acryloyloxysuccinimide are coupled in a reaction system adjusted in pH under Na ₂ CO ₃ -NaHCO ₃ , and the product and NIPAAm are adjusted in pH under Tris-HCl as a buffer. It is also possible to carry out radical polymerization using N, N, N ′, N′-tetramethylethylenediamine as an activator and ammonium persulfate as an initiator. Of course, other catalysts, promoters, and additives may be used.
Specific examples of terminal aminated single-stranded DNA are described in JP-A-59-27900, JP-A-59-93098, JP-A-59-93099, and JP-A-59-93100. A simple synthesis method is described. Further, methods for terminal vinylation and copolymerization thereof with NIPAAm are described in Maeda et al. (Biotechnology and bioengineering, vol. 72, No. 2, 261-268 and polymer journal, vol. 33, No. 10, 830). -833).
Both of the two types of oligonucleotides used in the annealing step of the present invention are preferably oligonucleotide copolymers, but one of them can also be an oligonucleotide copolymer.

The oligonucleotide part of the oligonucleotide copolymer used in the annealing step of the present invention has a base sequence that is substantially complementary to a part of the base sequence of the template DNA, and the (+)-side DNA after denaturation And hybridize to the 3 ′ end of each region of the sequence to be amplified of the (−) side DNA. In addition, after hybridization, the DNA strand is elongated. Here, the “substantially complementary base sequence” means a base sequence that can be annealed to the template DNA without mismatch under the reaction conditions used.
The oligonucleotide portion of the oligonucleotide copolymer is composed of, for example, one selected from deoxyribonucleotides and analogs thereof (also sometimes referred to as deoxyribonucleotides). The deoxyribonucleotides include not only unmodified but also modified ones. The modified deoxyribonucleotide is not particularly limited. For example, (α-S) deoxyribonucleotide in which an oxygen atom bonded to a phosphate group is substituted with a sulfur atom, or the hydroxyl group at the 2-position of ribose is a methoxy group. And deoxyribonucleotides substituted.
As used herein, deoxyribonucleotide refers to a nucleotide whose sugar moiety is composed of D-2-deoxyribose. For example, the base moiety includes adenine (A), cytosine (C), guanine (G), thymine ( Those having T). Furthermore, deoxyribonucleotide analogs such as deoxyribonucleotides and deoxyinosine nucleotides having modified bases such as 7-deazaguanosine are also included in the above deoxyribonucleotides.

  Making the oligonucleotide part of the oligonucleotide copolymer into a nucleotide analog is effective also from the viewpoint of suppressing higher-order structure formation and stabilizing the formation of annealing with the template. These oligonucleotide moieties can be synthesized, for example, by the phosphoramidite method using a DNA synthesizer type 394 from Applied Biosystems Inc. (ABI, Applied Biosystems Inc.). Moreover, although the phosphoric acid triester method, the H-phosphonate method, the thiophosphonate method, etc. are used as another method, what was synthesize | combined by what kind of method may be used.

Next, the polymerase process of the present invention will be described. This step elongates the DNA to be detected.
The polymerase step is performed by extending a primer (oligonucleotide) hybridized to DNA with DNA polymerase in the presence of deoxyribonucleotide triphosphate (dNTP).
Deoxyribonucleotide triphosphates (dNTPs) extend oligonucleotides, but as these deoxyribonucleotide triphosphates (dNTPs), dNTPs used in PCR methods, that is, mixtures of dATP, dCTP, dGTP, and dTTP are preferably used. it can.
These deoxyribonucleotide triphosphates (dNTPs) are analogs of deoxyribonucleotide triphosphates (dNTPs), for example, nucleotide triphosphates such as 7-deaza-dGTP and dITP, as long as they are substrates for the DNA polymerase used. Is also included. Deoxyribonucleotide triphosphate (dNTP) and deoxyribonucleotide triphosphate (dNTP) analog derivatives are also included, and these derivatives include derivatives having functional groups, such as amino derivatives.
Deoxyribonucleotide triphosphate (dNTP) can be prepared, for example, using a DNA synthesizer or the like in the same manner as a normal synthesis method.

  The amount of deoxyribonucleotide triphosphate (dNTP) and oligonucleotide copolymer used in the present invention may be adjusted according to the base sequence of the DNA to be detected, the target amplified fragment length, and the reaction system composition used, For example, the amount of amplification product can be adjusted as a guide, but is not particularly limited.

DNA polymerase refers to an enzyme that synthesizes a new DNA strand using a DNA strand as a template. In addition to natural DNA polymerase, a mutant enzyme having the above activity is also included, but 5 ′ → 3 ′ exo A DNA polymerase that does not have nuclease activity is preferred.
The DNA polymerase used in the present invention is not particularly limited, and examples thereof include Bacillus caldotenax (hereinafter referred to as B.ca) and Bacillus stearothermophilus (hereinafter referred to as B.st). A mutant lacking the 5 ′ → 3 ′ exonuclease activity of a thermophilic Bacillus genus DNA polymerase such as E. coli (hereinafter referred to as E. coli) or a large fragment (Klenow) of DNA polymerase I Fragment) and the like. In the present invention, as the DNA polymerase, any of room temperature to heat resistant can be suitably used. B. ca is a thermophilic bacterium having an optimum growth temperature of about 70 ° C. The ca DNA polymerase is known to have DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase activity (reverse transcription activity), 5 ′ → 3 ′ exonuclease activity, and 3 ′ → 5 ′ exonuclease activity. The enzyme may be either purified and obtained from its original origin or a recombinant protein produced by genetic engineering. In addition, 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 ′ exonuclease. B. deficient in activity B. a ca DNA polymerase. and ca BEST DNA polymerase (Takara Shuzo).
Some DNA polymerases are known to have endonuclease activity, for example, RNase H activity, under specific conditions. Such a DNA polymerase can be used in the present invention. That is, an embodiment in which the DNA polymerase is used under such conditions that RNase H activity is expressed, for example, in the presence of Mn ²⁺ can be mentioned. In this embodiment, the present invention can be carried out without adding the RNase H. That is, the above B. in buffer containing Mn ²⁺ ca DNA polymerase can exhibit RNase H activity. Note that the above-described aspect is the same as that of The present invention is not limited to ca DNA polymerase, and known DNA polymerases known to have RNase H activity, for example, Tth DNA polymerase derived from Thermos thermophilus can also be used in the present invention.

  In the method of the present invention, when the activity of the enzyme used may be lowered during the reaction, the enzyme can be further added during the reaction. The enzyme to be added may be the same as the enzyme contained in the reaction solution at the start of the reaction, or may be a different type of enzyme that exhibits the same action. That is, if the effect of improving the detection sensitivity or increasing the amount of amplified product can be obtained by adding during the reaction, the kind of enzyme to be added and the nature of the enzyme are not limited.

According to the DNA amplification method of the present invention, not only double-stranded DNA but also single-stranded DNA can be amplified, and by using cDNA obtained by reverse transcription reaction using RNA as a template, Nucleotide sequences can also be amplified.
The DNA or RNA used in the practice of the present invention can be prepared or isolated from any sample that may contain the DNA or RNA.
In the DNA amplification reaction and detection method of the present invention, the sample may be used directly.
Samples containing DNA or RNA are not particularly limited. Examples of these samples include 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), DNA-containing samples such as viroids, viruses, bacteria, molds, yeasts, plants and animals, such as viruses or bacteria Samples that may be contaminated or infected with various microorganisms (food, biological preparations, etc.), or samples that may contain organisms such as soil and wastewater. Moreover, the preparation containing DNA and RNA obtained by processing the said sample etc. by a well-known method may be sufficient.
As the preparation, for example, a cell disrupted product, a sample obtained by fractionating the same, DNA in the sample, or a specific DNA molecule group, for example, a sample enriched with mRNA can be used. Furthermore, DNA or RNA amplified by a known method can be suitably used.
There is no particular limitation on the method for preparing the preparation from the above sample, and for example, methods such as dissolution treatment with a surfactant, ultrasonic treatment, shaking and stirring using glass beads, and the use of a French press are used for the preparation. Can do. In some instances (eg, when an endogenous nuclease is present), it may be advantageous to further manipulate and purify the DNA. In these examples, purification is performed by known methods such as phenol extraction, chromatography, ion exchange, gel electrophoresis or density gradient centrifugation.

The RNA used to form the DNA having the sequence derived from RNA is not particularly limited as long as the primer used for the reverse transcription reaction can be prepared, and can be the total RNA in the sample. An RNA molecule group such as mRNA, tRNA, rRNA, or a specific RNA molecular species can be mentioned.
The primer used for the reverse transcription reaction is not particularly limited as long as it anneals to the template RNA under the reaction conditions used. The primer may be a primer having a base sequence complementary to a specific template RNA, an oligo dT (deoxythymine) primer, or a primer having a random sequence (random primer). The length of the primer for reverse transcription is preferably 6 nucleotides or more, more preferably 9 nucleotides or more from the viewpoint of performing specific annealing, and preferably 100 nucleotides or less from the viewpoint of oligonucleotide synthesis. More preferably 30 nucleotides or less.
The enzyme used in the reverse transcription reaction is not particularly limited as long as it has cDNA synthesis activity using RNA as a template. For example, avian myeloblastosis virus-derived reverse transcriptase (AMV RTase), Moloney murine Examples include reverse transcriptases of various origins such as leukemia virus-derived reverse transcriptase (MMLV RTase) and rous-associated virus 2 reverse transcriptase (RAV-2 RTase). In addition, a DNA polymerase having reverse transcription activity can also be used. Enzymes having reverse transcription activity at high temperatures are also suitable. For example, DNA polymerases derived from thermus bacteria (Tth DNA polymerase, etc.), DNA polymerases derived from thermophilic Bacillus bacteria, etc. can be used. Although these are not particularly limited, for example, a thermophilic Bacillus bacterium-derived DNA polymerase is preferable. st-derived DNA polymerase (B.st DNA polymerase); ca DNA polymerase is preferred. For example, B.I. The ca DNA polymerase does not require manganese ions for the reverse transcription reaction, and can synthesize cDNA while suppressing secondary structure formation of the template RNA under high temperature conditions.
As the enzyme having the above-mentioned reverse transcriptase activity, any of a natural body and a mutant can be used as long as the enzyme has the activity.

As another aspect of the present invention, DNA or RNA containing the base sequence to be amplified may be replicated in advance, and then used as the template DNA of the method of the present invention or after forming the template DNA. Good.
The method for replicating DNA is not particularly limited, but after transforming a suitable host with a vector into which a DNA fragment containing the nucleotide sequence to be amplified is inserted, the obtained transformant is cultured, A method of extracting and using a vector into which a DNA fragment containing a base sequence to be amplified is inserted is exemplified. The vector is not particularly limited as long as it is stably replicated in the host. For example, any of pUC system, pBluescript system, pGEM system, cosmid system, and phage system can be suitably used. The host is not particularly limited as long as it can hold the vector to be used, and examples thereof include E. coli that can be easily cultured. Furthermore, as another embodiment of the above-mentioned replication method, a large number of RNAs having the nucleotide sequence are prepared by RNA polymerase using a DNA fragment containing the nucleotide sequence to be amplified as a template, and then converted into a large number of cDNAs by reverse transcription reaction. A method is mentioned.
RNA replication can be performed using a DNA fragment containing a base sequence to be amplified. The DNA fragment is not particularly limited as long as it has an RNA polymerase promoter sequence, and may be inserted into a vector having an RNA polymerase promoter sequence, or an adapter having an RNA polymerase promoter sequence at the end or The cassette may be ligated, or may be synthesized enzymatically using a primer having an RNA polymerase promoter sequence and an appropriate template. That is, the DNA fragment containing the base sequence to be amplified can be replicated and amplified in the form of RNA using the RNA polymerase promoter sequence arranged as described above. The vector is not particularly limited as long as it has an RNA polymerase promoter sequence. For example, any of pUC system, pBluescript system, pGEM system, cosmid system, and phage system can be suitably used. In addition, as the vector, any of those circularly processed or linearly processed can be preferably used. Furthermore, the RNA polymerase used in the above-described replication and amplification methods is not particularly limited, and for example, SP6 RNA polymerase, T7 RNA polymerase, T3 RNA polymerase, or the like can be preferably used.

Both the double-stranded DNA such as genomic DNA and PCR fragment isolated by the above method and the single-stranded DNA such as cDNA prepared by reverse transcription reaction from total RNA or mRNA are all of the DNA of the present invention. It can be suitably used as DNA as a template for the amplification method.
For the purpose of amplification of DNA having a sequence derived from RNA, the RNA strand of RNA-cDNA double-stranded DNA obtained by reverse transcription reaction using RNA as a template is decomposed with RNase H or the like to produce a single strand. An amplification reaction is carried out as cDNA. By adding an RNA strand degrading enzyme such as RNase H to the amplification reaction solution of the present invention, the amplification reaction of the present invention can be started without degrading the RNA strand in advance. . Furthermore, by using a DNA polymerase having reverse transcriptase activity and strand displacement activity in the DNA amplification method of the present invention, reverse transcription reaction using RNA as a template, and DNA using the cDNA generated by the reaction as a template The amplification reaction can be performed with one type of DNA polymerase.
The length of the DNA used as the template is such that the target sequence is completely contained in the fragment, or sufficient part of the target sequence is present in the fragment, thereby providing sufficient binding of the primer sequence. It should be long enough.
In the method of the present invention, although not particularly limited, when the template DNA is a double-stranded DNA, the oligonucleotide can be bound to the template DNA strand by denaturing them into a single strand. Can be made. Holding at a temperature at which the double-stranded DNA is denatured, for example, about 95 ° C. is a preferred denaturing method. Other methods include increasing the pH, but in order to bind the oligonucleotide to the target, it is necessary to decrease the pH during the amplification reaction. After the step of denaturing the double strand as described above into a single strand DNA, or the step of preparing cDNA (single strand DNA) by reverse transcription when the template is RNA, the DNA is changed by changing the temperature. Amplify. For example, this temperature change is performed by annealing at 25 ° C., amplifying DNA at 55 ° C., and denaturing at 95 ° C., whereby DNA is continuously amplified.

The DNA amplification reaction of the present invention can be carried out at room temperature (for example, 37 ° C.) by using a room temperature DNA polymerase such as Klenow fragment, but an enzyme having heat resistance (endonuclease, DNA polymerase) is used. Then, it can be carried out at a high temperature, for example, 50 ° C. or higher, and further, for example, 60 ° C. or higher.
Further, the amplification reaction may be carried out in a manner that the reverse transcription reaction and the DNA amplification are continuously performed. The RNA reaction is performed by combining the above reaction with a reverse transcriptase or using a DNA polymerase having reverse transcription activity. Can be amplified.
The DNA polymerase used contributes to the synthesis of the extended strand downstream from the 3 ′ end of the oligonucleotide portion and, importantly, it does not exhibit 5 ′ → 3 ′ exonuclease activity that can degrade the substituted strand. It is. Klenow fragment, which is an exonuclease-deficient mutant of such a DNA polymerase, for example, E. coli-derived DNA polymerase I; a similar fragment derived from st DNA polymerase (manufactured by New England Biolabs); B derived from ca. ca BEST DNA polymerase (Takara Shuzo) is useful. T5 DNA polymerase and φ29 DNA polymerase described in Sequene 1.0 and Sequene 2.0 (US Biochemical), Gene, Vol. 97, pages 13 to 19 (1991) can also be used. Usually, even a DNA polymerase having 5 ′ → 3 ′ exonuclease activity can be used in the DNA synthesis method of the present invention if the activity can be inhibited by addition of an appropriate inhibitor.
In the present invention, amplification may be performed automatically using a reaction device such as a thermal cycler, or amplification may be performed by sequentially placing in a heat block adjusted to each temperature of the annealing step, polymerase step, and denaturing step. It doesn't matter.
The polymerase process of the present invention is preferably performed at an appropriate temperature at which the activity of the enzyme used is sufficiently maintained. Therefore, depending on the enzyme used, the 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. to about 70 ° C. is there.
In the present invention, the amplification efficiency can be improved by adjusting the reaction temperature according to the GC content of the template DNA. For example, when the template DNA has a low GC content, the polymerase step can be performed at 50 to 55 ° C., depending on the chain length to be extended and the Tm value of the oligonucleotide.
Also, a DNA polymerase having reverse transcriptase activity, such as B.I. When ca BEST DNA polymerase is used, amplification of RNA-derived DNA including a step of preparing cDNA from RNA (reverse transcription reaction) can be carried out conveniently with only one type of enzyme. Further, the step of preparing cDNA from RNA can be carried out independently, and the product (cDNA) can be used as template DNA in the method of the present invention.

The DNA amplification method of the present invention can be used for various experimental operations utilizing DNA amplification, such as DNA detection, labeling, and base sequence determination.
The DNA amplification method of the present invention can be used as an in situ DNA amplification method, a DNA amplification method on a solid phase carrier such as a DNA chip, or a multiplex DNA amplification method for simultaneously amplifying multiple types of regions. .
Tm in the present invention refers to the temperature at which double strands of DNA are thermally denatured to become single strands, and is calculated by the following formula according to “Current Protocols in Molecular Biology”.
Tm (℃) = 81.5 + 16.6 * log [S] +0.41 * (% GC)-(500 / n)
[S]: Molar concentration of salt
(% GC): GC content in oligo DNA (%)
n: Length of oligo DNA (bp)
However, [S] means [(sodium ion concentration) + (potassium ion concentration) + (Tris ion concentration) × 0.67], and the magnesium ion concentration is not included in the calculation of the concentration.

According to the DNA detection method of the present invention, the difference in the base sequence on the target DNA can be discriminated.
In this embodiment, for example, the base and the base in the intermediate part of the primer form a hydrogen bond so that the intermediate part of the primer is located at a specific base to be discriminated from the target base sequence. Primers are designed. When an amplification reaction is carried out using such a primer, amplification does not occur if there is a mismatch between the base sequence of the middle part of the primer and the base sequence of DAN used as a template. Generation is not seen.
According to the method of the present invention, it is possible to obtain information about a specific base on a gene such as a point mutation or single nucleotide substitution (SNP).
The detection of the target DNA using the DNA detection method of the present invention can be carried out directly using a sample containing DNA or by amplifying the target DNA. In this case, the length of the target DNA to be amplified is not particularly limited, but from the viewpoint of detecting the target DNA with high sensitivity, for example, a region of 200 bp or less, more preferably 150 bp or less is effective.
Furthermore, in the detection method of the present invention, a reaction buffer containing a bicine, tricine, hepes, phosphate or tris buffer component, and an annealing solution containing spermidine or propylenediamine are used to further increase the amount of DNA sample. Target DNA can be detected with high sensitivity. In this case, the DNA polymerase to be used is not particularly limited.
When genetic diagnosis is performed using the DNA detection method of the present invention, the content of single-stranded DNA in the DNA amplification solution obtained by the DNA amplification method of the present invention is such that a stable micelle structure can be formed. It is not limited. If the content is too low, not only is it difficult to confirm the change in light dispersion intensity due to the addition of a complementary gene, but it is also not possible to form micelles. In addition, when the content of single-stranded DNA is too high, DNA having a polymer (referred to as a DNA copolymer) is unlikely to become micelles, and even if micelles are formed, they are often unstable. Therefore, about 0.1 mol% or more and 0.5 mol% or less is preferable. However, since the content of single-stranded DNA is desirably changed according to the length of the single-stranded DNA chain and the structure of the hydrophobic substance, it is not limited to the above range.

In gene diagnosis using the DNA detection method of the present invention, a salt, for example, a metal cation such as magnesium or sodium is allowed to coexist in the amplified DNA solution.
In ionic surfactant micelles and polymer micelles, it is known that as the counter ion concentration in the solution increases, the critical micelle concentration (CMC) decreases and the number of associations increases. That is, when the counter ion increases, micelles can be formed even with a small amount of surfactant, and the particle size of micelles increases.
When genetic diagnosis is performed using the DNA detection method of the present invention, micelles are formed even when the concentration of the formed single-stranded DNA (single-stranded DNA copolymer substance) is small, and the micelle particles It is preferable to allow a metal cation to coexist for reasons such as an increase in diameter and ease of light scattering measurement.
The metal cation used here is not particularly limited, but is preferably a magnesium ion or a sodium ion. The magnesium ions can be used MgCl ₂ or the like, its concentration is preferably from 20 to 100 mm, more preferably 30 to 50 mm. Moreover, NaCl etc. can be used as a sodium ion, The density | concentration is 0.1-2M, More preferably, it is 0.5-1.5M.
In the method for detecting DNA of the present invention, a phase transition temperature of a polymer having a lower critical solution temperature after adding 30 mM of Mg ²⁺ ions in the range of the content of single-stranded DNA of 0.1 to 0.5 mol%. The above is preferable (32 ° C. or more for PNIPAM). However, if the temperature is higher than the Tm of the amplified DNA, the hybridization between the DNAs will be loosened. For example, the temperature is preferably 32 to 40 ° C.
In the method for detecting DNA of the present invention, other DNA is contained in the amplified solution of the amplified DNA as long as it does not interfere with the formation of a double helix between the single-stranded DNA and the complementary strand or the measurement of light scattering intensity. These substances may be added.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
Example 1
Pseudomonas aeruginosa was smeared on an agar medium and cultured at 37 ° C. overnight.
DNA of Pseudomonas aeruginosa was extracted according to the following procedure using ICAN GN Sample Preparation kit (manufactured by Takara Bio Inc.).
(1) Add 200 μL of Lysis Solution to a 1.5 mL microtube.
(2) One of the colonies of Pseudomonas aeruginosa cultured was struck with a toothpick, placed in the microtube of (1), and well dispersed by vortexing.
(3) This was placed in a 37 ° C. heat block and kept warm for 30 minutes.
(4) Further, heat treatment was performed for 5 minutes in a 95 ° C. heat block to separate the double-stranded DNA into two single-stranded DNAs.
(5) This was set in a centrifuge cooled to 5 ° C. and centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected.
Since Pseudomonas aeruginosa is a ceramidase-producing bacterium, Pseudomonas aeruginosa was detected by detecting this ceramidase-producing gene, and the sequence of this part was investigated using the genbank database.

1 ggatcctctt cggcatctgg atgatggcgg tgcagtacat cgactatccg gcggacaacc
61 acaagctcgg ctggaacgag atgctcgcct ggctgcgcag caagcgctgg gcgtgcatgg
121 gtttcggcgg ggtgacctac ctggcgctgc tgatcccgct ggtcaacctg gtcatgatgc
181 ccgccgccgt cgccggcgcc accctgttct gggtccgcga ggaaggcgag aaggcgctgg
241 tgaaataagc atgcgcccgg tgtccgctgt cggcattgtc aggcgggcgt cagcgccctg
301 acagccggcg tcgggcacac tacgaatgcc ctcgggagcg tcggcgcagg ccgcagccga
361 gacctgcgcc agcccttgca gaccggctcg acgctgccga accgactggc cgccggtgcc
421 tccccacgca ggcggccttt ttttacccgc ctgccttgcc gtccatgcag gatggccggc
481 gctcaccccc ttcctgtccg ctacgccccc ctcgactcca ccaccctggc actacagtgg
541 caaccaggcg aagcaggctc cggcccgctt tcgatgacca ccgtccccag cccgcccagc
601 ggcggaaaac aagaagaggg tcgccatgtc acgttccgca ttcaccgcgc tcttgctgtc
661 ctgcgtcctg ctggcgctct ccatgcctgc cagggccgac gacctgccct accgcttcgg
721 cctgggcaag gcggacatca ccggcgaagc cgccgaagtc ggcatgatgg gttactcctc
781 cctcgaacag aagaccgccg gcatccacat gcgccagtgg gcgcgtgcct tcgtgatcga
841 ggaagcggcc agcggacgtc gcctggtcta cgtcaacacc gacctgggga tgaccttcca
901 ggccgtgcac ctgaaggtcc tggcccggct caaggcgaag taccccggtg tctacgacga
961 gaacaacgtg atgctcgccg ccacccacac ccactccggt ccgggcggct tctcccacta
1021 cgcgatgtac aacctgtcgg tgctcggctt ccaggaaaag accttcaacg ccatcgtcga
1081 cggcatcgtc cgctccatcg agcgggccca ggccaggttg cagcccggcc gcctgttcta
1141 cggcagcggc gagctgcgca acgccagccg caaccgttcg ctgctgtcgc acctgaagaa
1201 tccggacatc gccggctacg aggatggcat cgacccgcag atgagcgtgc tcagcttcgt
1261 cgacgccaac ggcgagctgg ccggcgcgat cagttggttc ccggtgcaca gcacctcgat
1321 gaccaacgcc aatcacctga tctccccgga caacaagggc tacgcctcct atcactggga
1381 gcacgacgtc agccgcaaga gcggtttcgt cgccgccttc gcccagacca atgccggcaa
1441 cctgtcgccc aacctgaacc tgaagcccgg ctccggtccc ttcgacaacg agttcgacaa
1501 cacccgcgag atcggtctgc gccaattcgc caaggcctac gagatcgccg gccaggccca
1561 ggaggaagtg ctcggcgaac tggattcgcg cttccgtttc gtcgacttca cccgcctgcc
1621 gatccgcccg gagttcaccg acggccagcc gcgccagttg tgcaccgcgg ccatcggcac
1681 cagcctggcc gccggtagca ccgaagacgg tccaggcccg ctggggctgg aggaaggcaa
1741 caatccgttc ctctcggccc ttggcgggtt gctcaccggc gtgccgccgc aggaactggt
1801 gcaatgccag gcggaaaaga ccatcctcgc cgacaccggc aacaagaaac cctacccctg
1861 gacgccgacg gtgctgccga tccagatgtt ccgcatcggc cagttggaac tgctcggcgc
1921 ccccgccgag ttcaccgtga tggccggggt gcggatccgc cgcgcggtgc aggcggccag
1981 cgaagcggcc ggtatccgcc atgtggtctt caatggctac gcgaatgcct atgccagcta
2041 cgtcaccacc cgcgaggaat acgccgccca ggaatacgaa ggcggctcga ccctctacgg
2101 cccctggacc caggccgcct accagcagtt gttcgtcgac atggcggtgg cgctgcgcga
2161 acgcctgccg gtggaaacct cggcgatagc gccggacctg tcctgctgcc agatgaactt
2221 ccagaccgga gtagtggccg atgatcccta tatcggcaag tccttcggcg acgtgttgca
2281 acaacccagg gaaagttatc gcatcggcga caaggtgacc gtcgctttcg tgaccggaca
2341 tccgaagaat gacttgcgca ccgagaagac tttcctggaa gtggtgaata tcggcaagga
2401 tggcaaacag acgcccgtga ccgttgccac cgataatgac tgggataccc aataccgctg
2461 ggagagagtg ggtatatctg cctcgaaagc gactatcagc tggtccattc caccagggac
2521 cgagcccggc cattactaca tcaggcacta tggcaacgcg aagaacttct ggacccagaa
2581 gatcagcgaa atcggcggct cgacccgctc cttcgaggtg ctcggcacca ctccctagcg
2641 ggctccagcc aaggtttcga gattcgccag ccaactttat gacgcatgaa agtcgtcaaa
2701 taaaatgtga tttaaaacac atgaacaagt gaccttttca ttca

In this sequence, 456 bp from 1045 to 1500 is amplified and used as a primer.
foward primer: 5'-CGGCTTCCAGG-3 '
reverse primer: 5'-TTGTCGAACTC-3 '
Was set.
The absorbance (260 nm) of the oligonucleotide can be calculated from the following formula.
E ₂₆₀ = A * 15300 + C * 7400 + G * 11800 + T * 9300
In the formula, A represents the number of adenosine in the oligonucleotide, C represents the number of cytidine in the oligonucleotide, G represents the number of guanidine in the oligonucleotide, and T represents the number of thymidine in the oligonucleotide.
From the above formula, the absorbance (EF) of the forward primer is EF = 1 * 15300 + 4 * 7400 + 4 * 11800 + 2 * 9300 = 110700
Absorbance (ER) of reverse primer is ER = 2 * 15300 + 3 * 7400 + 2 * 11800 + 4 * 9300 = 113600
The forward primer, reverse primer, forward primer, and reverse primer, which were aminohexylated at the 5 ′ end, were ordered from an oligo DNA synthesizer (Sigma Genomics) and obtained.

The forward primer 0.6 μmol in which 5 ′ end was aminohexylated was dissolved in 150 μL of carbonate buffer (Na ₂ CO ₃ (10 mM) -NaHCO ₃ (90 mM)) to prepare a primer solution.
8.5 μmol of acryloyloxysuccinimide (manufactured by Acros) was dissolved in 460 μL of dimethyl sulfoxide, added to the primer solution, and allowed to react at room temperature for 24 hours. This solution was added to 10 mL of isopropanol and stirred well, and then centrifuged to remove the supernatant and introduce a vinyl group at the 5 ′ end.
In the same manner, the 5 ′ end of the reverse primer in which the 5 ′ end was aminohexylated was vinylated.
Each of the obtained terminal vinylated forward primer and terminal vinylated reverse primer was radically copolymerized with ammonium persulfate as an initiator in the presence of N-isopropylacrylamide (NIPAAm) and tetramethylethylenediamine, and forward primer-PNIPAAm complex and A reverse primer-PNIPAAm complex was synthesized. After dialysis of the obtained reaction product, gel filtration was performed with a column packed with SHEPHADEXG100 to remove unreacted products.
The obtained polymer was freeze-dried and then dissolved in 25 mL of purified water. The absorbance at 260 nm was as follows.
Obtained from forward primer 2.16 (hereinafter referred to as primer A solution)
Obtained from reverse primer 2.29 (hereinafter referred to as primer B solution)
A kit “Pyrobest DNA Polymerase” of Takara Bio's polymerase, PCR reaction buffer and dNTP mixed solution was purchased.

100 μL of supernatant obtained by extracting Pseudomonas aeruginosa DNA in a 1.5 ml PCR tube
10 x PyrobestBuffer 100μL
dNTP Mixture 80μL
Primer A solution 253μL
Primer B solution 245μL
Sterile distilled water 222μL
And a thermal cycle of 98 ° C. for 10 seconds, 25 ° C. for 2 minutes and 68 ° C. for 1 minute was repeated 40 times.
The obtained reaction solution was heated to 40 ° C., which is 32 ° C. or higher, the lower critical solution temperature of PNIPAM. The reaction solution was transparent, but when a 330 mM magnesium chloride aqueous solution and 100 μL were added, the reaction solution became cloudy, and the transmittance became 5% or less after 10 minutes.
Similarly, it can be said that the white turbidity has detected that the PCR reaction has progressed since salt was added to the reaction solution not subjected to the thermal cycle and the white turbidity did not occur even when heated to 40 ° C.

Claims (2)

(A) A step of hybridizing each of the separated single-stranded DNAs with two kinds of oligonucleotides having a sequence designed to recognize and hybridize each 3 ′ end of the region to be amplified (Annealing process),
(B) a step of extending an oligonucleotide with a DNA polymerase in the presence of deoxyribonucleotide triphosphate (dNTP) (polymerase step),
(C) Step of modifying (denaturing step)
When the DNA is amplified by performing a thermal cycle reaction that repeats the above three steps, a polymer having a lower critical solution temperature is linked to the 5 ′ end as two types of oligonucleotides used in the step (a). A method for amplifying DNA, comprising using the oligonucleotide.   After performing the DNA amplification method in the step according to claim 1, a salt is added, and a temperature not lower than the lower critical solution temperature of the polymer bonded to the 5 ′ end of the oligonucleotide and not higher than Tm of the amplified DNA And detecting a change in the turbidity of the solution.