JP2005000162A - Pcr amplification method for nucleic acid, pcr primer set, pcr amplification product, and method for detecting the nucleic acid using the amplification method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for amplifying a nucleic acid, capable of preferentially amplifying a nucleic acid chain having a desired nucleic acid sequence from a nucleic acid molecule used as a template, by utilizing a PCR amplification method, simpler than ever, and having high repeatability. <P>SOLUTION: This method for amplifying the nucleic acid comprises conducting PCR amplification reaction from the nucleic acid molecule used as the template by using two primers having melting temperature (Tm) values different from each other, wherein base sequences of the two primers are each designed so that the Tm values of the two primers which are used in the PCR amplification reaction and positioned on either sides of a base sequence region to be amplified are different from each other under a PCR amplification reaction condition. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for preferentially amplifying a nucleic acid chain having a desired nucleic acid sequence using a PCR amplification method from a nucleic acid molecule as a template. The present invention also relates to a PCR primer set used in such a nucleic acid PCR amplification method, a PCR amplification product obtained using the method, and a nucleic acid detection method using the amplification method.

  In recent years, with the dramatic improvement of gene sequence analysis technology, the decoding of many gene base sequences has progressed mainly in the human genome, and the functions of proteins and nucleic acid molecules encoded by the decoded genes in vivo have been elucidated. Has been. As the functions of the proteins and nucleic acid molecules encoded by genes continue to be elucidated, the expression status of these genes, mutations in the nucleotide sequence, and variations in the amino acid sequence of the encoded protein are associated with various diseases and constitutions. It has also been elucidated. Against the background of accumulation of such genetic information, the need for a technique for detecting a nucleic acid molecule having a specific base sequence contained in a specimen sample with high accuracy is rapidly increasing. In accordance with the demand, various related technologies applicable to the detection technology have been developed so far.

  Among many nucleic acid detection techniques, the most widely used is a nucleic acid molecule detection technique using a hybridization method. In this detection method using a hybridization method, a nucleic acid probe corresponding to a complementary strand to the base sequence of a nucleic acid molecule to be detected is prepared in advance, and the nucleic acid probe is immobilized on a solid phase or in a liquid phase as it is. Performing a hybridization reaction between the nucleic acid molecule to be detected and the nucleic acid probe contained in the specimen, detecting the obtained hybrid by an appropriate method, and verifying the presence or absence of the nucleic acid molecule having the base sequence to be detected; Also attempts to quantitatively detect it.

  Detecting a nucleic acid molecule having a specific base sequence contained in a sample is not limited to application to diagnosis of various genetic diseases using a marker gene or the like, but also in cancer, infectious diseases, etc. Correlations between specific nucleic acid molecules contained therein and their cases have been revealed one after another, and a wide range of applications in the diagnosis of various diseases is expected.

  In the detection method of nucleic acid molecules based on this hybridization method, a technique that has been especially expanded in recent years is a detection technique using a DNA chip or a DNA microarray in which a nucleic acid probe is immobilized on a solid phase. . In this method using a DNA chip or a DNA microarray, a synthetic complementary strand (DNA probe) to the base sequence of a nucleic acid molecule to be detected is fixed on a solid phase surface such as a glass substrate and labeled with a fluorescent substance or the like in advance. Measure the fluorescence intensity derived from the nucleic acid molecule that has been subjected to a hybridization reaction on the solid phase with the nucleic acid molecule in the applied specimen, and formed a hybrid on the substrate, for example, the nucleic acid molecule previously fluorescently labeled, This is a technique for examining the presence or absence of a nucleic acid molecule having a target base sequence in a sample, and further the amount contained in the sample. As a result of various technological developments as a technique for producing DNA chips, various nucleic acid probes (DNA probes) can be arranged at high density, and can be applied to high sensitivity and simultaneous detection of multiple items. It is highly expected as a possible breakthrough technology.

  Although a DNA chip can be detected with high sensitivity, generally the absolute amount of nucleic acid to be detected contained in a sample is extremely small, and it is necessary to label with a fluorescent substance or the like by an appropriate method. In the detection of nucleic acid sequences using the DNA, the specimen processing technique is very important along with the DNA chip production technique.

  The most widely used among various nucleic acid amplification techniques is a method called polymerase chain reaction (PCR). This PCR method is a method of amplifying a specific sequence contained in a sample at an extremely high amplification rate. The PCR method is described in US Pat. Nos. 4,683,195, 4,683,202, 4,965,188 and the like.

When preparing a sample for hybridization on a solid phase such as a DNA chip, set the amplification region to include the probe region immobilized on the solid phase, and design primers on both ends of the amplification region . Then, a PCR reaction is performed using both designed primers, and a large amount of double-stranded nucleic acid containing the desired probe region is synthesized. In addition, when it is desired to label the sample with some labeling substance, a PCR reaction substrate (that is, a nucleic acid monomer) is added with a labeled monomer bound to the labeling substance at a certain ratio, or a PCR reaction is performed. Is labeled with some labeling substance and PCR reaction is performed to obtain a labeled double-stranded nucleic acid.
US Pat. No. 4,683,195 US Pat. No. 4,683,202 US Pat. No. 4,965,188

  When detecting a nucleic acid molecule having a specific base sequence using a hybridization reaction, the higher the amount of a single-stranded nucleic acid molecule that forms a hybrid with a probe, the higher the accuracy of detection. It becomes possible. Usually, the content of the nucleic acid molecule to be detected contained in the primary sample is often not sufficient, and the nucleic acid molecule to be detected is contained in advance by an amplification reaction using the nucleic acid molecule to be detected contained in the primary sample as a template. A method of detecting a nucleic acid molecule after preparing a specimen sample containing an amplification product quantitatively amplified according to the amount is used. In the sample preparation process, it is desirable to synthesize a large number of DNA strands on the complementary strand side of the probe. That is, among the double strands contained in the specimen sample, the presence of more DNA strands (target strands) containing a complementary base sequence that hybridizes with the probe than the DNA strands containing the same base sequence as the probe, It is desirable for the subsequent hybridization reaction to proceed efficiently. This is not only because the higher the concentration of the target strand that undergoes the hybridization reaction with the probe is more sensitive, but the DNA strand that contains the same base sequence as the coexisting probe is However, by reducing the concentration of DNA strands containing the same base sequence as that of the probe, there is an effect of further increasing the efficiency of the hybridization reaction by the probe. For these two reasons, the same base as the probe is used. It is desired that the content ratio of the DNA strand (target strand) containing a complementary base sequence is higher than that of the DNA strand containing the sequence.

  Various devices have been devised in order to amplify one DNA strand more efficiently. As a representative method, in the PCR amplification reaction, a method of performing PCR amplification reaction (asymmetric PCR) by preparing a higher concentration of primers used for elongation synthesis of a DNA strand to be preferentially synthesized, or once There is a method in which a double-stranded DNA containing a target strand is synthesized by a normal PCR amplification reaction, and then only a primer used for target strand extension synthesis is added and the amplification reaction is carried out again (two-step PCR).

  However, these conventional methods often fail to achieve preferential amplification of the target strand of interest due to various factors. Specifically, with respect to the asymmetric PCR method, the yield of the PCR amplification product may not be sufficiently obtained, and even when the yield is sufficiently obtained, the yield of the PCR amplification product does not become asymmetric. As a result, the PCR amplification product may be almost double-stranded DNA. In addition, the two-step PCR method has a problem in terms of reproducibility because the amplification yield in the whole process is likely to vary in addition to the complexity of performing the PCR reaction in two steps. Furthermore, in the second-stage PCR reaction, only one strand is added as a primer. However, in some cases, the second-stage PCR reaction may not proceed at all. Therefore, it is desired to develop a nucleic acid amplification method that can preferentially amplify and synthesize the target single-stranded DNA (target strand) with a simpler and higher reproducibility.

  The present invention solves the above-mentioned problems, and an object of the present invention is to preferentially amplify a nucleic acid chain having a desired nucleic acid sequence using a PCR amplification method rather than a nucleic acid molecule as a template. Another object of the present invention is to provide a nucleic acid amplification method that is simpler and has high reproducibility.

  The present inventor uses a hybridization reaction to detect a nucleic acid molecule having a specific base sequence by a simpler and more reproducible means that can be applied to the preparation of a specimen sample. In order to develop a method capable of preferential amplification of a nucleic acid chain having a desired nucleic acid sequence by using a PCR amplification method from the nucleic acid molecules to be studied, research has been advanced. At that time, the base sequences of the two primers were designed so that the melting temperature (Tm) values of the two primers sandwiching the base sequence region to be amplified used in the PCR amplification reaction differ under the PCR reaction conditions. Furthermore, it was found that the yield of single-stranded DNA extended from each primer can be made different with high reproducibility by performing PCR amplification reaction using two primers having different Tm values. That is, it has been found that by applying the above-described method, a single-stranded DNA extended from one primer can be preferentially amplified and synthesized. In particular, when the annealing temperature in the PCR amplification reaction is set between the Tm values of two primers designed to be different from each other, by performing the PCR amplification reaction under such a temperature condition, an extended chain from a primer having a high Tm value is obtained. The present inventor confirmed that amplification was performed with significantly higher efficiency compared to an extended strand from a primer having a low Tm value, and the present invention was completed.

That is, the nucleic acid amplification method according to the present invention includes:
Using a primer set composed of at least two primers from a nucleic acid molecule contained in a sample, an amplification product having at least one specific base sequence in the nucleic acid molecule is produced by a PCR amplification method. A method,
A primer having a melting temperature (Tm) value different from that of other primers under the conditions of the PCR amplification reaction is used as at least one primer among a plurality of kinds of primers constituting the primer set, The nucleic acid amplification method.

  According to the nucleic acid amplification method of the present invention, PCR is performed by appropriately setting the Tm values of plural kinds of primers used in the PCR amplification reaction, the chemical modification groups bound to the primers, and the annealing temperature during the amplification reaction. Among the two extended strand DNAs having complementary base sequences that can be synthesized as extended strands from each primer in the amplification product, the desired primer extended strand has priority over the complementary strand. Can be synthesized. In addition, the preferential amplification of a desired primer extension chain achieved by the nucleic acid amplification method according to the present invention is performed by detecting a complementary base sequence that hybridizes with a probe DNA in detection of a nucleic acid molecule using a DNA chip or the like. The present invention can be suitably applied to the preparation of a specimen sample in which efficient synthesis of the contained single-stranded DNA is desired.

  Next, a mode for carrying out the nucleic acid amplification method according to the present invention, a primer set exclusively used for the nucleic acid amplification method according to the present invention, and a PCR obtained by application of the nucleic acid amplification method according to the present invention The invention of the proliferation product and nucleic acid detection method will be described.

First, in the nucleic acid amplification method according to the present invention described above,
Among a plurality of types of primer extension strands that are amplified in the PCR amplification reaction and extended from the plurality of types of primers,
As a primer constituting a primer extension strand to be preferentially amplified, a primer having a higher Tm value than at least one other primer constituting the primer set can be used. In addition, when the sample is a specimen in which plural types of nucleic acid molecules are mixed,
Selecting one or more nucleic acid molecules to be amplified from a plurality of types of nucleic acid molecules mixed in the specimen;
Selecting one or more base sequence regions to be amplified from the entire base sequences of the selected one or more nucleic acid molecules to be amplified;
Designing a forward primer and a reverse primer for PCR amplification with respect to the selected base sequence region to form a primer set composed of the at least two primers,
Want to preferentially amplify one or more of the plurality of types of primer extension strands that are extended from the plurality of types of primers that are amplified in a PCR amplification reaction using at least two primers constituting the primer set. Preferably, the method further comprises a step of selecting a primer extension strand.

For example, in a PCR amplification reaction, when amplifying an amplification product having a specific base sequence in a nucleic acid molecule from a nucleic acid molecule contained in a sample, two primers are used,
Of the two primer extension strands extended from the two primers,
A primer having a higher Tm value than other primers can be used as a primer constituting a primer extension strand to be preferentially amplified.

Alternatively, in the PCR amplification reaction, the upper limit is set to the highest Tm value and the lowest Tm value among the Tm values of each primer indicated by the plurality of types of primers constituting the primer set under the conditions of the PCR amplification reaction. Within the temperature range that is the lower limit,
An annealing temperature in an annealing process for binding a primer to a template may be set. In that case, when amplifying an amplification product having a specific base sequence in the nucleic acid molecule from the nucleic acid molecule contained in the sample in the PCR amplification reaction, two primers are used under the conditions of the PCR amplification reaction. It is preferable to set the annealing temperature in the annealing step for bonding the primer to the template within a temperature range in which the Tm values indicated by the two primers are the upper limit and the lower limit, respectively.

  More specifically, the temperature difference between the Tm values indicated by the two primers is more preferably selected in the range of 10 ° C. to 20 ° C., and the temperature difference between the Tm values indicated by the two primers is determined. It is also preferable to select in the range of 5 ° C to 10 ° C. Furthermore, the temperature difference between the Tm values indicated by the two primers can be selected in the range of 1 ° C to 5 ° C.

In addition, as means for using at least one primer among a plurality of types of primers constituting the primer set as a primer having a Tm value different from other primers under the conditions of the PCR amplification reaction,
By adjusting the GC% in the base sequence of the primer, the means for adjusting the Tm value indicated by the primer of the base sequence,
By selecting the base sequence of the primer, a difference in Tm value from other primers can be provided. Alternatively, as means for using at least one of the plurality of primers constituting the primer set as a primer having a Tm value different from that of other primers under the conditions of the PCR amplification reaction,
By adjusting the base chain length of the base sequence of the primer, using means for adjusting the Tm value indicated by the primer of the base sequence,
By selecting the base sequence of the primer, a difference in Tm value from other primers can be provided.

  In addition, the primer Tm value can be adjusted by applying some chemical modification to the primer. This is based on a phenomenon that a modified chemical modification group or the like affects the duplex stability of the primer and its complementary strand, and as a result, changes the Tm value of the primer. That is, when the introduced chemical modifying group increases the stability of the double strand, the Tm value increases. Conversely, when the stability is decreased, the Tm value can be decreased.

  Adjustment of the Tm value with a chemical modifying group can have a significant effect on double-stranded stability, particularly when the chemical modification has properties as an intercalator or as a groove binder. Therefore, it is possible to adjust the Tm value of the primer by introducing a chemical modifying group that functions as an intercalator or a groove binder.

On the other hand, a step of comparing the Tm values of the primers under the conditions of the PCR amplification reaction with respect to at least two primers among the plurality of types of primers constituting the primer set,
As a Tm value of the primer to be compared, a value calculated based on the base sequence of the primer is used,
As a method for calculating the Tm value of the primer, the closest base pair method can be used. Alternatively, the method further comprises a step of comparing the Tm values of the primers under the conditions of the PCR amplification reaction with respect to at least two of the plurality of primers constituting the primer set,
As a Tm value of the primer to be compared, a value calculated based on the base sequence of the primer is used,
The Wallace method can also be used as a method for calculating the Tm value of the primer. Furthermore, among a plurality of types of primers constituting the primer set,
A step of comparing Tm values of the primers with each other under the conditions of the PCR amplification reaction for at least two primers,
As a Tm value of the primer to be compared, a value calculated based on the base sequence of the primer is used,
The GC% method can also be used as a method for calculating the Tm value of the primer.

  In the nucleic acid amplification method according to the present invention, the amplification product obtained by PCR amplification can include an amplification product labeled with a labeling substance.

  At that time, the amplification product labeled with the labeling substance contains a labeled deoxynucleotide in a deoxynucleotide used as a substrate in a PCR amplification reaction, and a label derived from the labeled deoxynucleotide is added to an extended chain by the PCR amplification reaction. It may be an amplification product. Similarly to PCR using the chemically modified primer, a pre-labeled primer is contained in a plurality of types of primers constituting the primer set, and the labeled primer is extended by a PCR amplification reaction. The amplification product may be a chain. In that case, in the pre-labeled primer contained, it is more preferable that the label previously applied to the primer is a 5 ′ end label. In this case, it is desirable to adjust the primer sequence or the base length in consideration of the influence of the Tm value due to the labeling substance as in the case of using the chemical modification group.

  In addition, in the amplification product labeled with the labeling substance, the labeling substance is preferably a fluorescent substance. Alternatively, in the amplification product labeled with the labeling substance, the labeling substance may be a radioisotope.

As an application of the nucleic acid amplification method according to the present invention described above,
The sample is a sample targeted for detection of a nucleic acid molecule using a hybridization reaction,
In the detection of nucleic acid molecules using the hybridization reaction, at least one nucleic acid molecule to be detected is at least one or more produced by a PCR amplification method using a primer set composed of the at least two primers. Amplification product having a specific base sequence of
A sample containing the amplification product prepared by amplification can be used as a specimen in detection of nucleic acid molecules using the hybridization reaction. In the detection of nucleic acid molecules using the hybridization reaction, it is preferable to use DNA molecules immobilized or adsorbed on the solid surface as the probe for hybridization reaction. For example, in the DNA molecule fixed or adsorbed on the solid phase surface, the solid phase is more preferably in the form of a DNA chip or a DNA microarray. Further, in the DNA molecule immobilized or adsorbed on the solid surface, the solid phase may be a bead.

The present invention further provides an invention of a primer set for PCR exclusively used for the above-described nucleic acid amplification method according to the present invention, that is, the primer set according to the present invention comprises:
From a nucleic acid molecule contained in a sample, a primer set composed of at least two primers used for producing an amplification product having at least one specific base sequence in the nucleic acid molecule by a PCR amplification method There,
Using the nucleic acid amplification method according to the present invention having any one of the forms described above, the amplification product is a primer set composed of a plurality of types of primers suitable for the step of producing the amplification product by a PCR amplification method. It is a primer set.

At the same time, the present invention provides an invention of a PCR amplification product obtained by using the above-described nucleic acid amplification method according to the present invention, that is, the PCR amplification product according to the present invention comprises:
An amplification product having at least one specific base sequence in a nucleic acid molecule produced by a PCR amplification method using a primer set composed of at least two primers from a nucleic acid molecule contained in a sample Because
The amplification product is a PCR amplification product prepared by the nucleic acid amplification method according to the present invention having any one of the forms described above.

The invention of the nucleic acid detection method using the nucleic acid amplification method according to the present invention also provides the present invention, that is, the nucleic acid detection method according to the present invention comprises:
A method for detecting a nucleic acid molecule using a hybridization reaction,
PCR amplification method for amplification product having at least one specific base sequence in nucleic acid molecule using primer set comprising at least two primers from sample containing nucleic acid molecule to be detected Manufactured by
A sample containing the amplification product prepared by amplification is used as a specimen in the detection of a nucleic acid molecule using the hybridization reaction. In producing the amplification product by the PCR amplification method, the invention has any one of the forms described above. A nucleic acid detection method using such a nucleic acid amplification method.

Hereinafter, the nucleic acid amplification method according to the present invention will be described in more detail.

  In the nucleic acid amplification method according to the present invention, a reaction used for PCR amplification reaction when amplifying and synthesizing a nucleic acid chain corresponding to a desired nucleotide sequence region selected from the entire nucleotide sequence of a nucleic acid molecule as a template by PCR amplification reaction. The melting temperature (Tm) value of each primer in the liquid composition and the annealing temperature in the annealing process for bonding the template and each primer in the PCR amplification reaction are appropriately set. With this setting, it is possible to efficiently amplify the extended strand of a specific primer by providing a difference in the yield of single-stranded DNA molecules extended from primers having different Tm values. Therefore, the base sequence of each primer is selected so that the Tm value of the primer used for extension of the single-stranded DNA molecule to be preferentially amplified is higher than the Tm values of the other primers. In many cases, the base sequence is designed so that the Tm value of the primer used for extending a single-stranded DNA molecule to be amplified is higher than the annealing temperature. If the base sequence is set so that the Tm value of the primer used for the extension of the single-stranded DNA molecule on the strand side is lower than the annealing temperature, only the desired single-stranded DNA strand is preferentially and simultaneously efficient. Can be amplified.

  The technical principle used for the nucleic acid amplification method according to the present invention is schematically shown in FIG. In the annealing process, the primer 2 having a higher Tm value forms a stronger bond to the template, so that the yield of the primer 2 extended chain extended from the primer 2 having a higher Tm value is higher. Become. On the other hand, in the annealing process, the primer 1 having a lower Tm value can only form a weaker bond with the template, so that the yield of the extended strand of the primer 1 extended from the primer 1 having a lower Tm value is lower. It will be a thing. In the PCR amplification reaction, by repeating the temperature cycle, the DNA strand from the primer is extended using the single-stranded DNA molecule synthesized up to the previous stage as a template, and the amplification proceeds repeatedly. Thus, as a result of equalizing the addition amounts of the plurality of primers, the yield of the primer 1 extended strand extended from the primer 1 having a lower Tm value is also maintained within a certain range. As a result, in each stage, in the extension of the DNA strand from the primer 2 having a higher Tm value, the content of the primer 1 extension strand that is extended from the primer 1 having a lower Tm value as the template is extremely reduced, While avoiding the situation where the progress of amplification substantially stops, it is possible to increase the yield of the primer 2 extended strand extended from the target primer 2 having a higher Tm value.

  The PCR amplification product amplified and synthesized by the nucleic acid amplification method according to the present invention can incorporate a labeling substance such as a fluorescent substance in the process of extending a DNA chain from such a primer. As a method for incorporating this labeling substance, a method in which a label is added to a primer to be used in advance, or a DNA strand that is elongated by using a labeled nucleotide as a substrate in the elongation of a DNA strand by a polymerase enzyme. A technique for introducing a label into the part can be used.

  These labeled single-stranded DNA molecules can easily be quantitatively analyzed by using this label when the resulting hybrid is used as a specimen sample in the detection of nucleic acid molecules using a hybridization reaction with a DNA probe. It is possible to detect. For example, in the preparation of a specimen sample to be hybridized to a DNA chip or the like prepared for nucleic acid detection, the target single-stranded DNA molecule is preferentially and quantitatively amplified, and a predetermined fluorescent substance or the like is labeled. The nucleic acid amplification method according to the present invention can be suitably applied to a sample preparation step involving nucleic acid amplification for incorporating a substance.

In the nucleic acid amplification method according to the present invention, first, a primer that sandwiches a base sequence region to be amplified of a target nucleic acid molecule is designed. In primer design, two primers (one primer set) for forward primer and reverse primer sandwiching a base sequence region to be amplified are designed so that Tm values in PCR reaction conditions are different. At that time, as the Tm value of the primer under the PCR reaction conditions, the Tm value of the primer used for the extension of the DNA strand to be preferentially amplified is
By designing two primers so as to be higher than the Tm value of the primer used for the extension of the other DNA strand, it is possible to amplify many desired DNA strands that are extended from the primer having a higher Tm value. it can.

  As a method of designing the primer so that the Tm value of the primer under the PCR reaction conditions becomes a desired temperature, the length of the base sequence of the primer is changed within a range that does not significantly impair the sequence specificity as a primer, or In general, the base sequence of the primer is selected so as to change the GC% (the guanine and adenine composition ratio in the nucleic acid sequence) in the primer base sequence. In addition, the designed primer Tm value is calculated by changing the temperature under the actual PCR reaction conditions and measuring the ratio of the primer binding to the template, for example, the absorption intensity derived from the fluorescent label attached to the primer. It is also possible to calculate from the temperature dependence. In addition, as a technique for estimating the Tm value of a primer with high accuracy based on the base sequence of the primer, generally known methods such as the nearest neighbor pairing method, the Wallace method, and the GC% method are used. It is also possible to obtain a calculated value.

For the closest base pair method,
Breslauer KJ, Frank R., Blocker H., Markey LA (1986) Predicting DNA duplex stability from the base sequence-Proc. Natl. Acad. Sci. USA 83, 3746-3750,
Freier SM, Kierzek R., Jaeger JA, Sugimoto N., Caruthers MH, Nielson T., Turner DH (1986) Improved free-energy parameters for predictions of RNA duplex stabilit.- Proc. Natl. Acad. Sci. USA 83, 9373-9377,
See Schildkraut C., Lifson S. (1965) Dependence of the melting temperature of DNA on salt concentration-Biopolymers 3, 195-208, etc.
Regarding the Wallace method,
Wallace, RB; Shaffer, J .; Murphy RF; Bonner, J .; Hirose, T .; Itakura, K .; (1979) Nucelic Acid Res.
Regarding the GC% method,
Dependence of the Melting Temperature of DNA on Salt Concentration, Schildkraut C., Lifson S. (1965) BIOPOLYMERS 3.195-208,
See, for example, Optimization of the annealing temperature for DNA amplification in vitro, W. Rychlik, Nucl. Acids Res. (1990) 18 (21) 6409-6412.

  In addition, as a method of designing a primer so that the primer Tm value under PCR reaction conditions is a desired temperature, the primer is chemically modified and the primer Tm value is adjusted by the action of the chemical modification group. Can do. The chemically modified group attached to the primer affects the duplex stability of the primer. For example, in the case of a chemically modified group that has the effect of enhancing the stability of the duplex, the Tm value is increased and, on the contrary, the stability is increased. In the case of a chemical modifying group that has the effect of reducing the Tm value, the Tm value decreases.

  The chemical modification group can be used with no particular restrictions on both the type and the bonding mode, but among them, substances having properties such as intercalators or groove binders that interact strongly with the double-stranded structure of nucleic acids, This is particularly effective for adjusting the Tm value. As an intercalator, ethidium bromide, 9-aminoacridine, acridine orange, proflavine, and other polycyclic aromatic molecules such as anthracene are generally used. Alternatively, examples of groove binders include Hoechst 33258, Hoechst 33342, Pentamidine, Netropsin, and DAPI, which are commercially available. Aromatic compounds such as ethidium bromide and anthracene are commonly used. In addition, pyrylium dyes disclosed in Japanese Patent Application Laid-Open No. 7-233065 are often used as substances that interact strongly with double-stranded nucleic acids. Some groove binders have been reported to interact more frequently in areas where there are many ATs, such as DAPI. On the other hand, many intercalators or groove binders interact strongly with the double-stranded structure of nucleic acid, non-specifically to the base sequence. At that time, the structure and thermal stability of the resulting complex vary depending on the base sequence of the double-stranded structure portion of the nucleic acid to be interacted with and the type of intercalator or groove binder.

  The intercalator enters between stacks of base pairs of double-stranded nucleic acid, and the groove binder enters the groove of the double-stranded helical structure, both of which strongly affect the stability of the double-stranded structure. Some increase the stability of the double-stranded structure and others decrease it.

  As a method for introducing these chemically modified groups into the primer, for example, an amino group or biotin is modified in advance at the 5 ′ end of the primer, and various functional groups such as amino group and hydroxy group are used, for example. Although there is a method of bonding a desired chemical modification group via the bivalent reagent, various bonding modes can be used without particular limitation.

  In a basic PCR amplification reaction, a primer set to be used is composed of a set of a forward primer and a reverse primer that sandwich a base sequence region to be amplified. It is not limited to this. In the case of amplifying one base sequence region with one or more sets of primers, in the case of amplifying a plurality of amplification regions with one set of primers, or in complex PCR amplification combining these, The nucleic acid amplification method can be applied. A PCR amplification reaction using a plurality of primer sets selects one or more nucleic acid molecules to be amplified from one or more nucleic acid molecules, and one or more regions to be amplified from the base sequence of the selected nucleic acid molecule After designing a primer for each selected region and further selecting one or more DNA strands to be preferentially amplified by a PCR reaction reaction, the nucleic acid amplification method of the present invention is performed. Thus, the nucleic acid amplification method of the present invention can be applied by designing the primers to be used and setting the conditions for the PCR amplification reaction.

  The PCR amplification reaction using the designed primer is possible under any reaction conditions, but in order to more effectively implement the nucleic acid amplification method according to the present invention, the annealing temperature in the PCR amplification reaction is set to a set of forwards. A PCR amplification reaction may be performed by setting between different Tm values of two primers, a primer and a reverse primer. Depending on the PCR amplification reaction, the annealing temperature and the DNA chain elongation reaction temperature by the polymerase enzyme may be the same temperature, and it may not always be necessary to carry out the annealing reaction separately. In that case, the annealing treatment is performed simultaneously. Preferential nucleic acid amplification by the method of the present invention is possible by setting the Tm value of each primer so that the enzyme reaction temperature is between the Tm values of the two primers.

  The relationship between the Tm value of the two primers and the annealing temperature in the PCR amplification reaction is preferably as described above. For example, even if the annealing temperature is lower than the Tm value of the two primers, the PCR amplification reaction is possible. is there. However, the substantial difference between the Tm values of the two primers relative to the annealing temperature is reduced, and the effect of the present invention is reduced. On the other hand, when the annealing temperature is higher than the Tm values of the two primers, there is a concern that the efficiency of the PCR amplification reaction itself is lowered. Therefore, in the amplification method of the present invention, it is most desirable to set the annealing temperature of the PCR amplification reaction between the Tm values of the two primers, and when it cannot be set, within 5 ° C. from the Tm value of one primer. It is desirable to set the annealing temperature within the range.

  If there is an appropriate difference between the Tm values of the two primers in the PCR amplification reaction conditions, the nucleic acid amplification method of the present invention can be carried out, but the temperature difference between the Tm values is in the range of 1 to 5 ° C. Desirable if present. Furthermore, it is more desirable if the temperature difference between the Tm values is in the range of 5 to 10 ° C. More desirably, the temperature difference between the Tm values is in the range of 10 to 20 ° C.

  More specifically, when there is a large difference in Tm values between two primers, it is desirable from the viewpoint of “amplifying a primer extension strand to be preferentially amplified more than a complementary strand”, which is an object of the present invention. However, an excessively large difference is not so preferable in terms of the efficiency of the PCR amplification reaction. Therefore, when asymmetry of the amplification product is strongly required rather than the amplification rate, a difference in Tm value of 10 to 20 ° C. or 5 to 10 ° C. is set between the two primers. In the case where the amplification factor is required to be stronger than the control, it is more preferable to set the difference in the Tm value relatively small.

  The nucleic acid amplification method according to the present invention can be applied without particular limitation to various sample preparations that require PCR amplification. In particular, as an embodiment in which the effect is expected, a specific nucleic acid sequence utilizing a hybridization reaction is used. It is applicable to the detection of nucleic acid molecules having Among them, as a nucleic acid detection method, an application method widely used in recent years includes a detection method using a hybridization reaction with a DNA probe immobilized on a solid phase. As the solid phase, glass, plastic, metal and the like are often used, and the nucleic acid amplification method of the present invention can be used without any particular limitation on the kind of the solid phase. Among them, a nucleic acid molecule detection method using a DNA chip or a DNA microarray in which a large number of DNA probes are fixed in a flat plate shape is a nucleic acid in which the nucleic acid amplification method of the present invention can be most suitably used for the preparation process of the sample. This is one of the detection methods. In addition to the plate-like DNA chip, a nucleic acid detection method using beads to which a DNA probe is immobilized has been frequently used in recent years. The nucleic acid amplification method of the present invention is a DNA probe immobilized on the bead surface. It can also be applied to the preparation of a specimen sample in a nucleic acid detection method using

  The DNA probe fixed on the solid phase is a single-stranded DNA fragment, and when the single-stranded DNA fragment has a relatively long chain length of about 60 mer, the single-stranded DNA probe has a high Tm value and is relatively strong. Hybridization reaction is possible. Therefore, even if the PCR amplification product to be detected forms a double strand with its complementary strand, heat treatment is performed to dissociate the double strand and efficiently form a hybrid with the DNA strand to be detected. can do. However, when the strand length of the single-stranded DNA probe on the solid phase is relatively short, such as about 20 mer, the Tm value of the single-stranded DNA probe is low, and the double strand of the PCR amplification product is dissociated. Competing with the process of reconfiguring, it becomes impossible to efficiently form a hybrid. When using a DNA probe as short as about 20 mer, the nucleic acid amplification method according to the present invention is particularly effective because it can preferentially amplify the target strand, and is preferentially amplified and synthesized. By using a specimen sample containing a target strand, highly sensitive detection is possible.

  When carrying out this nucleic acid amplification method in the preparation process of a sample sample containing a target strand that forms a hybrid with an immobilized DNA probe such as a DNA chip, the target strand, which is a primer extension strand to be amplified in some form, is somehow formed. Signs are often applied. The nucleic acid amplification method according to the present invention can also be applied to a nucleic acid amplification method involving labeling, and in that case, it can be carried out without being limited to the labeling method. As a method for performing labeling, the most commonly performed method is to perform some kind of labeling on a part of the primer in advance and perform a PCR amplification reaction with the labeled primer in the same manner as the primer subjected to the chemical modification described above. It is one of. In particular, a method in which a fluorescent substance or the like is labeled in advance on the 5 ′ end of the primer and the like and a PCR amplification reaction is performed using this labeled primer is representative. However, in that case, it is necessary to design the base sequence of the primer in consideration of the influence of the Tm value due to the binding of the labeling substance, as in the case of the primer using the chemical modification group. In addition to this method, as a substrate for the PCR amplification reaction, deoxynucleotides incorporated into the extended DNA strand are labeled with a fluorescent substance or the like, and this substrate is added to the PCR reaction solution to label the PCR amplification product. There is also a method of making it widely used. The nucleic acid amplification method according to the present invention can be applied to any of these labeling methods without particular limitation.

  As a labeling substance to be used, labeling with a fluorescent substance is common because of its high sensitivity and ease of handling. However, labeling with a radioactive isotope or a PCR amplification product labeled with biotin is performed by using labeled avidin as biotin. It is also possible to label indirectly by binding to.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the examples described below are examples of the best mode according to the present invention, but the technical scope of the present invention is not limited to these examples.

<Example 1> pUC118 EcoRI / BAP PCR
(1) Primer design Four primers having the following sequences were designed from the base sequence of a commercially available vector pARA118 EcoRI / BAP (full length 3162 bp) manufactured by Takara. In addition, pUC118 EcoRI / BAP base sequence information is provided by Takara and is also available from public databases.

Table 1 shows the melting temperature Tm values obtained by calculation based on the base sequences of these four kinds of primers. The calculation method of the Tm value used is the above-described method. In the calculation, the conditions for the PCR reaction are as follows: Na ⁺ concentration in reaction solution 50 mM, Mg ²⁺ concentration 1.5 mM, and each primer concentration 0 .5M.

  Among the primer sets composed of these four kinds of primers, the base sequence chain length of the double-stranded PCR amplification product obtained by the combination of the following forward primer and reverse primer is as shown in Table 2. Become.

(2) Synthesis of primers Four types of primers designed in Example 1 (1) were synthesized. For synthesis of each primer, a DNA chain having the respective base sequence was synthesized by a DNA synthesizer according to a conventional method, and further, an amino group was modified on the 5 ′ end side of the DNA chain via a hexamethylene linker. The obtained 5′-terminal amino-modified DNA (primer) was purified with a cartridge, and with regard to F1, F2, and F3, it was treated with an NHS type Cy3 labeling reagent (Amersham Pharmacia) that binds to the amino group, and with respect to R1, This was treated with an NHS type Cy5 labeling reagent (Amersham Pharmacia), and a fluorescent dye label was introduced at the 5 ′ end. The obtained labeled primers were each purified by HPLC to obtain four types of primers: Cy3 labeled F1, Cy3 labeled F2, Cy3 labeled F3, and Cy5 labeled R1. Each obtained labeled primer was diluted with TE buffer to a concentration of 10M.

(3) PCR amplification reaction Using 4 types of labeled primers synthesized in (2) of Example 1, Takara vector pUC118 EcoRI / BAP as template gene DNA, and QIAGEN PCR kit HotStarTaq Master Mix A PCR amplification reaction was performed.

  As a combination of the forward primer and the reverse primer, the PCR reaction conditions for the three combinations described in Table 2 in (1) are as follows. went.

  About the prepared reaction liquid, PCR amplification reaction was performed according to the temperature cycle protocol of the following Table 4 using the commercially available thermal cycler.

  After the amplification reaction was completed, the PCR amplification product was purified using a purification column (QIAGEN QIAquick PCR Purification Kit). After purification, the volume of the PCR amplification product solution was adjusted to 50 l. A portion of the resulting purified PCR amplification product solution was taken and subjected to electrophoresis according to a conventional method, and it was confirmed that a target band corresponding to 760 bp had appeared. FIG. 2 shows an electrophoresis image of the PCR amplification product obtained when the forward primer and reverse primer combinations [1], [2], and [3] are used.

(4) Absorption measurement The absorption of the solution of the four types of labeled primers synthesized in Example 1 (2) and the PCR amplification product solution prepared in (3) were measured. Each labeled primer solution is prepared at a concentration of 0.5M, and the absorption intensity derived from the labeled fluorescent substance at the following absorption wavelength is measured according to the labeled fluorescent substance (Cy3 or Cy5) of each labeled primer. did.

  For the PCR amplification product solution, the absorption intensity was measured at both absorption wavelengths of the labeled fluorescent material derived from the labeled forward primer and reverse primer.

  Subsequently, the incorporation yield of each labeled primer by the PCR amplification reaction was calculated based on the absorption intensity measured in the PCR amplification product solution. This incorporation yield indicates how many percent of the primers added to the reaction solution in the PCR amplification reaction caused an extension reaction to become a primer extension chain of the PCR amplification product.

  In the PCR amplification product [2], the Tm values of Cy3-labeled F2 and Cy5-labeled R1 are almost equal, and as a result, there is no significant difference in the uptake yield between the two, and the corresponding duplexes are synthesized evenly. I understood. On the other hand, in the PCR amplification product [1], since the Tm value of Cy3-labeled F1 is lower than the Tm value of Cy5-labeled R1, the extended strand from Cy5-labeled R1 is preferentially synthesized, and each extension is accompanied by the extension. The uptake yield of Cy5 labeled R1 was significantly higher than the uptake yield of Cy3 labeled F1 in the chain. On the other hand, in the PCR amplification product [3], since the Tm value of Cy3 labeled F3 is higher than the Tm value of Cy5 labeled R1, the extended chain from Cy3 labeled F3 is preferentially synthesized. The uptake yield of Cy3 labeled F3 was also higher than the uptake yield of Cy5 labeled R1 in the chain.

<Example 2> Hybridization reaction in a DNA chip (1) Preparation of a DNA microchip (1) -1 Cleaning of a glass substrate A synthetic quartz glass substrate (size (WLT): 25 mm 75 mm 1 mm, made by Iiyama Special Glass Co., Ltd.) It was placed in an alkali-resistant rack and immersed in a cleaning solution for ultrasonic cleaning adjusted to a predetermined concentration. After soaking in the cleaning solution overnight, ultrasonic cleaning was performed for 20 minutes. Subsequently, the glass substrate was taken out and lightly rinsed with pure water, followed by ultrasonic cleaning for 20 minutes in ultrapure water. Next, the glass substrate was immersed in a 1N sodium hydroxide aqueous solution heated to 80 ° C. for 10 minutes. Again, pure water cleaning and ultrapure water cleaning were performed to prepare a cleaned quartz glass substrate for a DNA chip.

(1) -2 Surface Treatment A silane coupling agent KBM-603 (manufactured by Shin-Etsu Silicone) was dissolved in pure water to a concentration of 1% and stirred at room temperature for 2 hours. Subsequently, the washed quartz glass substrate was immersed in this aqueous silane coupling agent solution and left at room temperature for 20 minutes. The glass substrate was pulled up, the surface was lightly washed with pure water, and then dried by blowing nitrogen gas on both sides of the glass substrate. Next, the glass substrate dried with nitrogen blow was baked in an oven heated to 120 ° C. for 1 hour to complete the coupling agent treatment. By this coupling agent treatment, an amino group derived from a silane coupling agent was introduced on the surface of the glass substrate.

  On the other hand, N-maleimidocaproyloxy succinimide (N- (6-Maleimidocaproxy) succinimido); hereinafter abbreviated as EMCS) manufactured by Dojindo Laboratories Ltd. was finally added in a 1: 1 mixed solvent of dimethyl sulfoxide and ethanol. An EMCS solution dissolved so as to have a concentration of 0.3 mg / ml was prepared. After completion of baking, the glass substrate treated with the coupling agent was allowed to cool and immersed in the prepared EMCS solution at room temperature for 2 hours. During this immersion treatment, the amino group introduced on the surface of the glass substrate treated with the coupling agent and the succinimide group of EMCS reacted to introduce a maleimide group derived from EMCS on the surface of the glass substrate. The glass substrate pulled up from the EMCS solution was washed with the above-mentioned mixed solvent of dimethyl sulfoxide and ethanol, further washed with ethanol, and then dried in a nitrogen gas atmosphere.

(1) -3 Synthesis of Probe DNA Probe P1 having the following base sequence was synthesized. This base sequence is a base sequence that is 100% complementary to the partial base sequence of the extended strand on the reverse side of the PCR amplification products [1], [2], and [3] in Example 1. .
Probe P1: 5 'ccttaacgtgagttttcg 3'
The probe DNA was subjected to thiolation at the 5 ′ end in accordance with a conventional method in order to introduce a maleimide group on the surface and covalently bond it to the glass substrate. Thereafter, in order to avoid side reactions during DNA synthesis, the protected protecting groups were deprotected, and further subjected to HPLC purification and desalting treatment.

  The obtained probe DNA was dissolved in pure water and dispensed to a final concentration (at the time of ink dissolution) of 10 M, and then freeze-dried to remove moisture.

(1) -4 Probe DNA ejection by BJ printer and binding to substrate surface Glycerin 7.5 wt%, Thiodiglycol 7.5 wt%, Urea 7.5 wt%, Acetylenol EH (manufactured by Kawaken Fine Chemical Co., Ltd.) 1.0 wt % Aqueous solution was prepared. Subsequently, the dispensed probe DNA was dissolved in the above mixed solvent so as to have a specified concentration (10M). The obtained probe DNA solution was filled in an ink tank for a bubble jet printer (trade name: BJF-850 manufactured by Canon Inc.) and mounted on a print head.

  The bubble jet printer is modified so that ink jet printing onto a flat plate is possible. The modified bubble jet printer is capable of spotting about 5 pl of DNA solution droplets at a pitch of about 120 m by inputting a print pattern according to a predetermined file creation method.

  Subsequently, using this modified bubble jet printer, the probe DNA solution was spotted on the surface of the glass substrate. A printing pattern was prepared in advance so that 16 spots were ejected per DNA chip, and ink jet printing was performed. After confirming that the target solution has been spotted with the DNA pattern with a magnifying glass or the like, it is allowed to stand in a humidified chamber for 30 minutes, and the maleimide group on the surface of the glass substrate and the sulfanyl group on the probe DNA 5 ′ end -SH).

(1) -5 Washing After the reaction for 30 minutes in the humidified chamber, unreacted probe DNA remaining on the glass substrate surface was washed away with 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl. A DAN microarray-type DNA chip was obtained in which predetermined single-stranded probe DNAs were immobilized on 16 spots per each DNA chip on the glass substrate surface.

(2) Hybridization reaction Using the DNA chip prepared in (1) of Example 2 and the PCR amplification product prepared in Example 1 as a sample, a detection reaction for the target nucleic acid molecule was performed.

(2) -1 Blocking of DNA microarray BSA (bovine serum albumin Fraction V: manufactured by Sigma) was dissolved in 100 mM NaCl / 10 mM Phosphate Buffer so as to be 1 wt%, and this solution was dissolved in (1) of Example 2 The prepared DNA microarray (DNA chip) was immersed for 2 hours at room temperature to block the glass substrate surface. After the blocking, washing with 2SSC solution (NaCl 300 mM, sodium citrate (trisodium citrate dihydrate, C ₆ H ₅ Na ₃ .2H ₂ O) 30 mM, pH 7.0) containing 0.1 wt% SDS (sodium dodecyl sulfate) After performing, it rinsed with the pure water. Thereafter, the DNA microarray was drained with a spin dry apparatus.

(2) -2 Preparation of Hybridization Solution In order to make the amount of target nucleic acid (number of moles) in each PCR amplification product solution the same, three kinds of absorption strengths by Cy5 labeled on the reverse strand are the same. The concentration of the PCR amplification product was adjusted. Subsequently, three types of hybridization solutions were prepared using equal amounts of PCR amplification product solutions so that the final concentrations were as follows.

<Hybridization solution>
6SSPE / 10% Formamide / PCR amplification product solution (6 × SSPE: NaCl 900 mM, NaH ₂ PO ₄ .H ₂ O 60 mM, EDTA 6 mM, pH 7.4)
(2) -3 Hybridization The drained DNA chip was set in a hybridization apparatus (Genomic Solutions Inc. Hybridization Station), and a hybridization reaction was performed using the hybridization solution having the above composition under the following procedures and conditions.

<Hybridization conditions and procedures>

(3) Fluorescence measurement After the completion of the hybridization reaction, a fluorescence measurement derived from the hybrid was performed on the spin-dried DNA chip using a fluorescence detection apparatus for DNA microarray (Anexon, Genepix 4000B). The fluorescence derived from the hybrid is derived from the fluorescent label on the reverse strand of the PCR amplification products [1], [2] and [3] in Example 1 that form a hybrid with the probe DNA. It is.

  On the DNA chip, the fluorescence intensity observed in the portion without the spot of the probe DNA was used as a background value, and the value obtained by subtracting the background value from the apparent fluorescence intensity from each spot was taken as the actual measurement value of fluorescence intensity. Table 9 below shows the average value of actually measured values obtained by performing the measurement twice.

  From this result, the PCR amplification product [1] in which the Reverse strand is preferentially synthesized as compared with the Forward strand compared to the specimen sample by the PCR amplification product [2] in which the Forward strand and the Reverse strand are synthesized equally. ], It can be seen that reverse strand hybridization occurs more efficiently on the DNA chip. Therefore, by applying the nucleic acid amplification method according to the present invention, it is possible to preferentially synthesize a desired PCR-amplified extended chain, and a specimen sample preparation process for detection of nucleic acid molecules using a DNA chip Furthermore, it was found that the nucleic acid amplification method according to the present invention can be suitably applied.

Example 3 PCR and Hybridization Using Anthracene Modified Primer (1) Synthesis of Anthracene Modified Primer Primer F2 of pUC118 designed in Example 1 was synthesized. Primers were synthesized by synthesizing a DNA chain having the same base sequence with an automatic DNA synthesizer according to a conventional method, and further modifying the thiol group via a linker composed of a methylene chain at the 5 ′ end of the DNA chain. The obtained 5′-terminal thiol-modified DNA was purified by gel filtration and ethanol precipitation after removing the protective group bonded to each functional group.

  Subsequently, 30 nmol of the obtained thiol-modified DNA was dissolved in 100 microliters of a mixed solvent (water 50%, ethanol 25%, dimethyl sulfoxide 25%), and further 1 mg of EMCS was added and reacted at room temperature for 3 hours. The resulting mixture was purified by gel filtration and ethanol precipitation. By this reaction, a modified DNA in which EMCS was bound to the 5 'end of the DNA and a succinimide group was bound to the end was synthesized.

  Subsequently, after dissolving the modified DNA in 100 microliters of a mixed solvent of water and dimethylformamide (1: 1), 10 mg of 2-aminoanthracene was added and stirred to dissolve as much as possible. Furthermore, the reaction was carried out at 45 ° C. for about 5 hours while stirring was continued. By this reaction, a succinimide group at the DNA end and an amino group bonded to anthracene reacted to obtain an anthracene-modified primer (hereinafter abbreviated as F2AN) in which anthracene was bonded to the 5 'end.

  The precipitated components were removed by filtration and then purified according to a conventional method. After purification, the concentration of the nucleic acid component was measured and diluted to a concentration of 10M with TE buffer.

(2) PCR amplification reaction Each primer having the F2 and R1 sequences designed in Example 1 was synthesized according to a conventional method. In the synthesis, the terminal is not particularly modified. The synthesized primer was diluted with TE buffer to a concentration of 10M.
Subsequently, PCR was performed using three types of primers, F2AN, F2, and R1. The primer combinations were those shown in Table 10.

  The detailed reaction composition of PCR was as shown in Table 11 below. The PCR product obtained was labeled by carrying out PCR by mixing Cy3dUTP as a labeling substrate.

  About the prepared reaction liquid, PCR amplification reaction was performed according to the temperature cycle protocol of the following Table 12 using the commercially available thermal cycler.

  After the amplification reaction was completed, the PCR amplification product was purified using a purification column (QIAGEN QIAquick PCR Purification Kit). After purification, the volume of the PCR amplification product solution was adjusted to 50 l. A portion of the resulting purified PCR amplification product solution was taken and subjected to electrophoresis according to a conventional method, and it was confirmed that a target band corresponding to about 760 bp had appeared.

(3) Hybridization reaction The two kinds of PCR products obtained were each hybridized to the DNA chip prepared in Example 2 (1). The composition of the hybridization solution, the hybridization procedure, and the chip scan were performed in the same manner as in Example 2. The results of calculating the average of the two scans in the same manner as in Example 2 are shown in Table 13.

  From this result, it can be seen that the primer F2AN in which an anthracene as an intercalator is bound to a primer has an increased Tm value due to the effect of anthracene, and a PCR extension reaction was performed preferentially over R1. As a result, the fluorescence brightness was higher when F2AN ([4]) was used than with unmodified F2 ([5]).

Example 4 PCR and Hybridization Using Adamantane Modified Primer (1) Synthesis of Adamantane Modified Primer Primer R1 of pUC118 designed in Example 1 was synthesized. Primers were synthesized by synthesizing a DNA chain having the same base sequence with an automatic DNA synthesizer according to a conventional method, and further modifying the thiol group via a linker composed of a methylene chain at the 5 ′ end of the DNA chain. The obtained 5′-terminal thiol-modified DNA was purified by gel filtration and ethanol precipitation after removing the protective group bonded to each functional group.

  Subsequently, 30 nmol of the obtained thiol-modified DNA was dissolved in 100 microliters of a mixed solvent (water 50%, ethanol 25%, dimethyl sulfoxide 25%), and further 1 mg of EMCS was added and reacted at room temperature for 3 hours. The resulting mixture was purified by gel filtration and ethanol precipitation. By this reaction, a modified DNA in which EMCS was bound to the 5 'end of the DNA and a succinimide group was bound to the end was synthesized.

  Subsequently, after the modified DNA was dissolved in 100 microliters of water, 10 mg of amantadine hydrochloride was added and stirred to dissolve as much as possible. Furthermore, the reaction was carried out at 45 ° C. for about 5 hours while stirring was continued. By this reaction, a succinimide group at the DNA end and an amino group bound to adamantane reacted to obtain an adamantane-modified primer (hereinafter abbreviated as R1AD) having adamantane bound to the 5 'end.

  The precipitated components were removed by filtration and then purified according to a conventional method. After purification, the concentration of the nucleic acid component was measured and diluted to a concentration of 10M with TE buffer.

(2) PCR amplification reaction Each primer having the F2 and R1 sequences designed in Example 1 was synthesized according to a conventional method. In the synthesis, the terminal is not particularly modified. The synthesized primer was diluted with TE buffer to a concentration of 10M.

  Subsequently, PCR was performed using three types of primers F2, R1, and R1AD. The primer combinations were those shown in Table 10.

  The detailed reaction composition of PCR was as shown in Table 15 below. The PCR product obtained was labeled by carrying out PCR by mixing Cy3dUTP as a labeling substrate.

  About the prepared reaction liquid, PCR amplification reaction was performed according to the temperature cycle protocol of the following Table 16 using the commercially available thermal cycler.

  After the amplification reaction was completed, the PCR amplification product was purified using a purification column (QIAGEN QIAquick PCR Purification Kit). After purification, the volume of the PCR amplification product solution was adjusted to 50 l. A portion of the resulting purified PCR amplification product solution was taken and subjected to electrophoresis according to a conventional method, and it was confirmed that a target band corresponding to about 760 bp had appeared.

(3) Hybridization reaction The two kinds of PCR products obtained were each hybridized to the DNA chip prepared in Example 2 (1). The composition of the hybridization solution, the hybridization procedure, and the chip scan were performed in the same manner as in Example 2. The results of calculating the average of the two scans in the same manner as in Example 2 are shown in Table 17.

  From this result, primer R1AD, which is chemically modified with adamantane on the primer, decreases the duplex stability of the primer due to the effect of adamantane and lowers the Tm value, and the extension strand from F2 is given priority, and the PCR extension reaction takes place. You can see that it was done. As a result, the fluorescence brightness was higher when R1AD ([6]) was used than with unmodified R1 ([7]).

  The present invention can be applied to the preparation of a specimen in which the content ratio of a desired single-stranded nucleic acid is increased using a PCR amplification method, and in particular, the effect of increasing the content ratio of the desired single-stranded nucleic acid is utilized. Thus, it can be suitably applied to detection of a nucleic acid molecule having a specific nucleic acid sequence by a hybridization reaction.

It is a figure which shows typically the technical principle of the basic invention in the nucleic acid amplification method concerning this invention. 2 is a diagram showing an electrophoresis image of a purified PCR amplification product solution obtained by the PCR amplification reaction in Example 1. FIG.

Claims (32)

Using a primer set composed of at least two primers from a nucleic acid molecule contained in a sample, an amplification product having at least one specific base sequence in the nucleic acid molecule is produced by a PCR amplification method. A method,
A primer having a melting temperature (Tm) value different from that of other primers under the conditions of the PCR amplification reaction is used as at least one primer among a plurality of kinds of primers constituting the primer set, A nucleic acid amplification method. At least one constituting the primer set as a primer constituting a primer extension strand to be preferentially amplified among a plurality of types of primer extension strands amplified from the plurality of types of primers amplified in a PCR amplification reaction. The method according to claim 1, characterized in that a primer having a higher Tm value than one other primer is used. The sample is a specimen in which plural types of nucleic acid molecules are mixed,
Selecting one or more nucleic acid molecules to be amplified from a plurality of types of nucleic acid molecules mixed in the specimen;
A step of selecting one or more base sequence regions to be amplified from all the base sequences of the selected one or more nucleic acid molecules to be amplified, and forward amplification for PCR amplification with respect to the selected base sequence regions Designing a primer and a reverse primer to form a primer set composed of the at least two primers;
Want to preferentially amplify one or more of the plurality of types of primer extension strands that are extended from the plurality of types of primers that are amplified in a PCR amplification reaction using at least two primers constituting the primer set. The method of claim 2, further comprising selecting a primer extension strand. In the PCR amplification reaction, when amplifying an amplification product having a specific base sequence in the nucleic acid molecule from the nucleic acid molecule contained in the sample, two primers are used,
Of the two types of primer extension strands extended from the two primers, a primer having a higher Tm value than other primers is used as a primer constituting a primer extension strand to be preferentially amplified. The method of claim 2. In the PCR amplification reaction, the upper limit and the lower limit of the highest Tm value and the lowest Tm value among the Tm values of each primer indicated by a plurality of types of primers constituting the primer set under the conditions of the PCR amplification reaction. 5. The method according to claim 1, wherein an annealing temperature in an annealing step in which a primer is bonded to a template is set within a temperature range. In the PCR amplification reaction, when amplifying an amplification product having a specific base sequence in the nucleic acid molecule from the nucleic acid molecule contained in the sample, two primers are used,
An annealing temperature in the annealing step for binding the primer to the template is set within a temperature range in which the Tm values indicated by the two primers are the upper and lower limits under the conditions of the PCR amplification reaction, respectively. The method according to claim 5. The method according to claim 4, wherein the temperature difference between the Tm values indicated by the two primers is selected in the range of 10 ° C to 20 ° C. The method according to claim 4, wherein the temperature difference between the Tm values indicated by the two primers is selected in the range of 5 ° C to 10 ° C. The method according to claim 4, wherein the temperature difference between the Tm values indicated by the two primers is selected in the range of 1 ° C to 5 ° C. As a means for using at least one of the plurality of primers constituting the primer set as a primer having a Tm value different from that of other primers under the conditions of the PCR amplification reaction, in the nucleotide sequence of the primer By adjusting the GC%, a means for adjusting the Tm value indicated by the primer of the base sequence is used, and by selecting the base sequence of the primer, a difference in Tm value from other primers is provided. The method according to any one of claims 1 to 9. As a means for using at least one of the plurality of primers constituting the primer set as a primer having a Tm value different from that of other primers under the conditions of the PCR amplification reaction, the nucleotide sequence of the primer By adjusting the base chain length, using means for adjusting the Tm value indicated by the primer of the base sequence, and selecting the base sequence possessed by the primer, a difference in Tm value from other primers is provided. The method according to any one of claims 1 to 9. Chemical modification of the primer as a means for using at least one of the plurality of primers constituting the primer set as a primer having a Tm value different from other primers under the conditions of the PCR amplification reaction The method according to claim 1, wherein the method is performed. The method according to claim 12, wherein the chemical modification is a chemical substance having a property of increasing the double-stranded stability of the nucleic acid. The method according to claim 12, wherein the chemical modification is a chemical substance having a property of reducing the double-stranded stability of the nucleic acid. 15. The method according to claim 13, wherein the chemical substance has a property as an intercalator of nucleic acid. The method according to claim 13 or 14, wherein the chemical substance has a property as a groove binder of nucleic acid. A step of comparing the Tm values of the primers with each other under the conditions of the PCR amplification reaction for at least two of the plurality of primers constituting the primer set;
As a Tm value of the primer to be compared, a value calculated based on the base sequence of the primer is used,
10. The method according to any one of claims 1 to 9, wherein a closest base pair method is used as a method for calculating the Tm value of the primer. A step of comparing the Tm values of the primers with each other under the conditions of the PCR amplification reaction for at least two of the plurality of primers constituting the primer set;
As a Tm value of the primer to be compared, a value calculated based on the base sequence of the primer is used,
The method according to claim 1, wherein a Wallace method is used as a method for calculating the Tm value of the primer. A step of comparing the Tm values of the primers with each other under the conditions of the PCR amplification reaction for at least two of the plurality of primers constituting the primer set;
As a Tm value of the primer to be compared, a value calculated based on the base sequence of the primer is used,
The method according to claim 1, wherein a GC% method is used as a method for calculating the Tm value of the primer. The method according to any one of claims 1 to 9, wherein the amplification product obtained by PCR amplification includes an amplification product labeled with a labeling substance. The amplification product labeled with the labeling substance contains a labeled deoxynucleotide in a deoxynucleotide used as a substrate in a PCR amplification reaction, and an amplification product having a label derived from the labeled deoxynucleotide in an extended chain by the PCR amplification reaction The method of claim 20, wherein: The amplification product labeled with the labeling substance contains a pre-labeled primer in a plurality of types of primers constituting the primer set, and is made an extended strand of the labeled primer by a PCR amplification reaction. 21. The method of claim 20, wherein the method is an amplification product. 23. The method according to claim 22, wherein in the pre-labeled primer contained, the label previously applied to the primer is a 5 'end label. The method according to any one of claims 20 to 23, wherein in the amplification product labeled with the labeling substance, the labeling substance is a fluorescent substance. 24. The method according to any one of claims 20 to 23, wherein in the amplification product labeled with the labeling substance, the labeling substance is a radioisotope. The sample is a sample targeted for detection of a nucleic acid molecule using a hybridization reaction,
In the detection of nucleic acid molecules using the hybridization reaction, at least one nucleic acid molecule to be detected is at least one or more produced by a PCR amplification method using a primer set composed of the at least two primers. Amplification product having a specific base sequence of
The method according to any one of claims 1 to 25, wherein a sample containing the amplification product prepared by amplification is used as a specimen in detection of a nucleic acid molecule using the hybridization reaction. 27. The method according to claim 26, wherein in the detection of nucleic acid molecules using the hybridization reaction, a DNA molecule immobilized or adsorbed on a solid phase surface is used as the hybridization reaction probe. 28. The method according to claim 27, wherein in the DNA molecule immobilized or adsorbed on the solid surface, the solid phase is in the form of a DNA chip or a DNA microarray. 28. The method according to claim 27, wherein in the DNA molecule immobilized or adsorbed on the solid surface, the solid phase is a bead. From a nucleic acid molecule contained in a sample, a primer set composed of at least two primers used for producing an amplification product having at least one specific base sequence in the nucleic acid molecule by a PCR amplification method There,
A primer set comprising a plurality of types of primers suitable for the step of producing the amplification product by a PCR amplification method using the nucleic acid amplification method according to any one of claims 1 to 29. Primer set. An amplification product having at least one specific base sequence in a nucleic acid molecule produced by a PCR amplification method using a primer set composed of at least two primers from a nucleic acid molecule contained in a sample Because
A PCR amplification product, wherein the amplification product is prepared by the nucleic acid amplification method according to any one of claims 1 to 29. A method for detecting a nucleic acid molecule using a hybridization reaction,
PCR amplification method for amplification product having at least one specific base sequence in nucleic acid molecule using primer set comprising at least two primers from sample containing nucleic acid molecule to be detected Manufactured by
Using a sample containing the amplification product prepared by amplification as a specimen in detection of nucleic acid molecules using the hybridization reaction,
A nucleic acid detection method using the nucleic acid amplification method according to any one of claims 1 to 24 when the amplification product is produced by a PCR amplification method.