JP5197661B2 - Probe carrier for nucleic acid detection - Google Patents

Description

The present invention relates to a detecting method using a probe carrier for nucleic acid detection probe set composed is fixed a plurality of probes for detecting a nucleic acid to be detected and the same.

  As represented by the Human Genome Project, genes of various organisms have been elucidated, and the relationship between genes of life activity mechanisms, diseases, constitutions, and the like has been investigated one after another. It has been found that knowing the presence or absence of a gene and its abundance (expression level) makes it possible to select, for example, more detailed features such as illness, typing, or effective treatment methods.

  Many methods have been devised since the past to determine the presence or abundance of a specific gene contained in a specimen, but among these methods, detection is possible because it has a wide range of applications and can be applied regardless of the detection target. A method is widely used in which a characteristic partial sequence of a target gene or nucleic acid is selected and the presence or abundance of the partial sequence is examined. Specifically, a nucleic acid sequence (probe) corresponding to the complementary strand of the selected partial sequence is prepared, and the presence of the nucleic acid sequence in the sample is detected by detecting the hybridization between the sample and the probe by some means. It is a method to check.

  A method for detecting a specific nucleic acid using hybridization can be used regardless of a solid phase or a liquid phase. For example, when performed in the liquid phase, prepare a probe solution to which a labeling substance that changes in spectral characteristics during double strand formation is bound, add it to the specimen, and measure the change in the spectral characteristics. One example is a method for examining the presence or absence of a specific nucleic acid therein.

  When hybridization is performed on a solid phase, the probe is fixed or adsorbed on the solid phase, a sample labeled with some detectable labeling substance is added to the solid phase, and the label from the solid phase is added. A typical method is detection by measuring a signal of a substance. Among them, a chip in which a probe is fixed on a flat substrate such as glass or metal, or a bead fixed on the surface of a minute particle is a typical form of solid-phase hybridization. The reason why hybridization on a solid phase is preferred is that B / F separation is easy, the detection area can be physically miniaturized and high sensitivity can be expected, and multiple types of probes are physically isolated. This is because simultaneous detection of multiple items is possible and the handling and application can be facilitated because of the solid phase.

Among the advantages of solid-phase hybridization, the feature that multiple items can be detected simultaneously enables various detection methods. For example, when detecting a family gene having the same sequence partially, one probe is set in a region having a common sequence between family genes, and conversely, in a unique region having a sequence different between family genes. If one probe is set, it becomes possible to compare gene expression levels among a plurality of specimens and accurately identify which type of family gene is expressed. Further, when distinguishing infectious disease-causing fungi, generally, a sequence portion encoding 16S ribosomal RNA is detected from the genes of the fungus, and the bacterial species is often discriminated based on the detected sequence. In this case, however, it is possible to discriminate the target level by selecting a specific sequence or a common sequence according to the bacterial species, strain, and genus levels. For example, if a probe is set from a region that is common within the same bacterial species and different from the other bacterial species, and at the same time, a probe is set from a different region depending on the strain within the same bacterial species, the bacterial species is identified by these multiple probes. The stock can be discriminated at the same time.

  An example of a DNA array in which a plurality of probes are set for the same nucleic acid to be detected is GeneChip of Affymetrix, USA. In the detection method using DNA arrays manufactured by Affymetrix, labeled nucleic acid is allowed to act on oligo DNA synthesized on a flat substrate, and its hybridization is measured by fluorescence detection, so that it is included in the sample. The presence or amount of a specific nucleic acid to be detected is detected (see Patent Document 1). Then, about 10 to 20 types of probes are set for the same nucleic acid to be detected, the signals obtained from these probes are comprehensively determined, and the expression level of the gene is measured.

  On the other hand, regardless of the liquid phase or the solid phase, various improvements have been made in the method of detecting a nucleic acid to be detected by hybridization using a probe nucleic acid, and the sensitivity has greatly increased compared to the previous method. However, in order to detect a nucleic acid to be detected by direct hybridization, a very large amount of sample is required and often unrealistic, and the sample amount obtained in general is insufficient in sensitivity. . In general, it is necessary to label the nucleic acid to be detected with a fluorescent substance or the like. In many cases, the nucleic acid to be detected is amplified and labeled by a PCR method or RNA polymerase reaction, and then hybridized with the prepared probe nucleic acid. Has been done.

  These amplified nucleic acids to be detected and probe nucleic acids form a hybrid. Since probes are generally artificially chemically synthesized nucleic acids, their base length is often about 15 mer to 80 mer. In addition, the nucleic acid to be detected amplified by the PCR method or the like is generally 100 mer or more. Therefore, the hybrid formed by the probe and the nucleic acid to be detected has a structure in which the probe binding region has a double-stranded structure and a single-stranded portion of the nucleic acid to be detected protrudes on at least one side thereof.

US Pat. No. 6,410,229

  In the case of family gene detection and bacterial species discrimination as exemplified above, it is effective to set multiple different probes that detect multiple different regions of the target single-stranded nucleic acid, but only for example Tm value or GC% If a plurality of different probes having the same level of binding force are set with a focus on the above, there may be a large variation in the stability of the hybrid formed in the fixed region of each probe. In spite of the amount of the specimen, there is a problem that the fluorescence luminance value varies greatly between the probe fixing regions. In the measurement of DNA microarrays, a scanner that can measure the fluorescence brightness precisely is used for the measurement, but most of them use a method that scans the entire surface of the microarray at once, and adjusts the detection sensitivity by changing the probe. In general it is not possible.

  Accordingly, even in a probe set that can detect different regions in the same nucleic acid to be detected, there is little variation in the stability of each hybrid formed in the fixed region of different probes, and the same level of detection sensitivity can be obtained in each hybrid. It has been desired to provide a probe set.

Therefore, the object of the present invention is to enable detection of single-stranded nucleic acid by a probe with high sensitivity that eliminates variations in detection values caused by the stability of the hybrid of the probe and single-stranded nucleic acid to be detected. to provide a probe carrier which is formed by using a probe set that.

  As a result of intensive research to solve the above problems, probe design that considers the stability of the hybrid of the nucleic acid to be detected and the probe is performed, and a probe set composed of probes having the same level of hybrid stability is obtained. It has been found that the above-described non-uniformity among a plurality of probes can be eliminated by supplying.

The probe carrier of the present invention comprises
Probe sets for detection of amplification products of single-stranded nucleic acids to be detected and single-stranded nucleic acid amplification products having complementary sequences thereof are provided in different regions on the solid phase that can be distinguished for each probe. A fixed probe carrier ,
The probe set has probes for detecting at least one different region of each single-stranded nucleic acid to be detected,
When each single strand to be detected and each probe form a hybrid, the number of bases of the portion including the 5 ′ end of the detection target strand among the single-stranded portions each of the hybrid has respectively L1 When the number of bases in the portion including the 3 ′ end is represented as L2,
Each hybrid body
L2 ≧ L1
Each probe is configured to satisfy the above relationship .

In addition, according to one aspect of the method for detecting a nucleic acid according to the present invention, a probe carrier configured as described above is reacted with a single-stranded nucleic acid to be detected and its complementary strand, and the probe is immobilized on the probe carrier. It is a nucleic acid detection method comprising a step of detecting a formed double-stranded hybrid .
Furthermore, another aspect of the method for detecting a nucleic acid according to the present invention is a detection method for detecting a target single-stranded nucleic acid using the probe carrier having the above-described configuration, wherein the target single-stranded nucleic acid and its complementary sequence are detected. A detection method comprising reacting a sample containing a complementary single-stranded nucleic acid having a probe carrier with the probe carrier .

According to the present invention, a probe set having the same level of hybrid stability can be obtained by using one or more probe sets for the nucleic acid to be detected and its complementary strand. A probe carrier to which these probe sets are bound can also be obtained. As a result, any probe setting required regardless of the stability of the hybrid body is possible.

It is a figure which shows L1, L2, and P when a probe is set with respect to the detection target nucleic acid. It is a figure when two types of probes from which a base length differs are set with respect to the detection target nucleic acid. It is a figure when two types of probes are set to the nucleic acid to be detected and its complementary strand.

Research has been conducted on the stability of hybrids having a partial double-stranded structure, and the applicant of the present application has applied for an invention focusing on this stability as Japanese Patent Application No. 2003-324647. According to those studies, the stability differs depending on the ratio of the base lengths of the 5 ′ side and the 3 ′ side of the single-stranded portion consisting of the nucleic acid to be detected present on both sides of the double-stranded structure part of the hybrid. Is clearly stated. More specifically, when the number of bases of the portion containing the 5 ′ end of the target strand of the two single-stranded portions is represented by L1, the number of bases of the portion containing the 3 ′ end is represented by L2,
L2 ≧ L1
It is specified that the hybrid is stable when it has the following relationship. Furthermore, it is known that the greater the magnitude of the relative ratio L2 / L1, the more stable the hybrid body.

  The knowledge about the stability of this hybrid is that even if multiple probes are set for the same nucleic acid to be detected and the Tm value of each probe is the same, the fluorescence intensity value obtained by solid-phase hybridization differs depending on the probe. It is shown that.

The binding force of the probe to the nucleic acid to be detected is one of the factors of probe design. As a measure of the binding strength of a complementary strand formed by binding double-stranded nucleic acids to each other, for example, a double strand formed from a probe sequence and a complementary strand to be detected, this dissociates into a single-stranded state Temperature, or Tm value, is generally used. The Tm value can be obtained by actually preparing a probe sequence and its complementary strand and actually measuring it in the same environment as when the sample nucleic acid and the probe are hybridized. The Tm value of the probe can also be calculated. Various calculation methods have been devised for calculating the Tm value using the salt concentration in hybridization as a parameter. As a calculation method that is generally used frequently, a nearest neighbor base pair method or the like can be used, and a probe set in which a sequence is set based on the Tm value calculated thereby can be set. As another calculation method, a Wallace (Wallace) method can also be used, and a probe set in which an array is set based on the calculated Tm value can be set. Furthermore, the GC% method can also be used, and a probe set in which the sequence is set based on the calculated Tm value can be set.

  As a method of adjusting the Tm value for optimizing the binding force of the probe, there are a method of changing the base length of the probe and a method of changing the GC%, and a probe constituted by a probe whose binding force is adjusted thereby. Set can be set.

  In the present invention, the binding force of each probe is set according to the number of bases from the 5 ′ end of the nucleic acid to be detected at the detection position of each probe, and more specifically, the base from the 5 ′ end to the detection position of the probe. Adjust the probe according to the size of the number.

  As a probe for detecting nucleic acid, DNA is generally used because of its ease of synthesis, but PNA (peptide nucleic acids) that have been attracting attention in recent years can also be used. It is possible to configure the probe set of the present invention by appropriately setting. If necessary, a probe set in which DNA and PNA are mixed may be used.

The probe set and probe carrier will be described in more detail.
(About the first probe set)
The first probe set is particularly effective when a plurality of probes are desired in the sequence of the nucleic acid to be detected. A hybrid body having a partially double-stranded structure composed of a nucleic acid to be detected and a probe has single-stranded portions on both sides of the double-stranded structure portion. Of these, when the portion present on the 5 ′ end side of the nucleic acid to be detected is the A chain, and the portion present on the 3 ′ end side is the B chain, depending on the ratio of the length of the A chain to the B chain, The stability of the hybrid is very different. When the number of bases of the A chain is L1, and the number of bases of the B chain is L2,
L2 ≧ L1
It is known that the hybrid body is stable when the relationship is satisfied, and that the stability increases as the value of L2 / L1 increases. When the position of the region to which each probe binds in the detection target nucleic acid is expressed by the number of bases P from the 5 ′ end of the detection target nucleic acid, if the value of P decreases, that is, if L2 / L1 increases, the hybrid The stability of the body increases and the binding force of the probe increases objectively.

  Therefore, when setting a plurality of probes for the nucleic acid to be detected, supplying a probe set in which the binding force of the probe is adjusted according to the size of P makes the stability of the hybrids the same level. be able to. As a reference, the relationship between L1, L2, and P is simply shown in FIG.

  When the probe constituting the probe set is DNA, the most common method for evaluating and expressing the binding force is the Tm value, which is the temperature at which the double strand is dissociated into a single strand. The Tm value can be determined directly by preparing a probe and its complementary strand as described above and measuring the absorption according to a conventional method. When measuring, it is desirable to put it under actual hybridization conditions as much as possible, that is, conditions for hybridization between the probe set and the sample nucleic acid containing the nucleic acid to be detected. When measurement under the same conditions is difficult, this is not necessarily the case, and the measurement may be performed under approximate conditions.

If the Tm value is not actually measured, it can also be obtained by calculation. Various methods for calculating the Tm value have been devised, and any method can be used without any particular limitation, but the closest base pair method, the Wallace method, the GC% method, etc. are particularly included in the present invention. It is suitably used for probes.

  As for the closest base pairing method, Breslauer KJ, Frank R., Blocker H., Markey LA (1986) Predicting DNA duplex stability from the base sequence quence-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, Schildkraut C., Lifson S. (1965) Dependence of the melting temperature of DNA on salt concentration-Biopolymers 3,195-208, etc., for the Wallace method, Wallace, RB; Shaffer, J .; Murphy. F.; Bonner, J .; Hirose, T .; Itakura, K .; (1979) Nucelic Acid Res. 6,3543, etc., and the GC% method, Dependence of the Melting Temperature of DNA on Salt Concentration, Schildkraut C., Lifson S. (1965) BIOPOLYMERS 3.195-208, Optimization of the annealing temperature for DNA amplification in vitro, W. Rychlik, Nucl. Acids Res. (1990) 18 (21) 6409-6412, and the like.

  When the Tm value of the probe constituting the probe set does not reach the desired value, it is necessary to change the design of the sequence and the like for each probe and adjust the Tm value. There are a method for changing the base length of the probe and a method for changing the GC%. In addition, a method for adding some chemical modification can be used as long as the specificity in sequence recognition is not significantly reduced.

  Although DNA is generally used as a probe, PNA is known to have the same function as DNA, and can be used as a probe by adjusting the Tm value in the same manner as a DNA probe.

  An example in which a plurality of probes are set for the nucleic acid to be detected according to the present invention is shown in FIG. In the probe set shown in FIG. 1, the probe A set on the 5 'side is 25 bp, while the probe B set on the 3' side is a 60 bp probe in order to increase the binding force.

(About the second probe set)
The second probe set is a probe set according to the present invention , and has a probe for detecting at least one different region of each of a single-stranded nucleic acid to be detected and a complementary strand having a complementary sequence. Is a probe set characterized by

  When the sequence region that requires detection is present at the 3 ′ end of the nucleic acid to be detected, that is, when the value of L2 / L1 is small, the hybrid formed by the probe and the nucleic acid to be detected is extremely low in stability. It becomes. In that case, as a result, not only the sensitivity is greatly lowered compared to other probes, but also the detection sensitivity may be lowered and impossible to detect in some cases. In order to avoid this in the second probe set, the complementary strand of the nucleic acid to be detected is to be detected. That is, by making the detection target sequence its complementary strand, the values of L1 and L2 are reversed, and as a result, the value of L2 / L1 is increased, and a probe and the detection target strand can form a stable hybrid. It is possible.

  The probe setting for the complementary strand can be particularly preferably applied when a sample nucleic acid is prepared by PCR. In the normal symmetric PCR method, the same amount of complementary strand nucleic acid as the target nucleic acid is prepared, and even if sample nucleic acid is prepared without changing the preparation method, it can be detected with a probe corresponding to the complementary strand. is there. Therefore, the second probe set is a probe set in which at least one probe is set from each of the detection target strand and its complementary strand in consideration of hybrid stability.

Since the single-stranded nucleic acid to be detected often needs to be amplified and labeled, it is often amplified and prepared in advance by a PCR method. The complementary strand is also synthesized at the same time. Therefore, the second probe set can be preferably used particularly when PCR products are to be detected. The probe of the second probe set only needs to be set for each of the detection target nucleic acid and its complementary strand, and it is possible to set two or more probes for the same detection target strand. The binding force of each probe constituting the probe set is determined according to the number of bases from the 5 ′ end of the nucleic acid to be detected of each probe. Specifically, the number of bases from the 5 ′ end increases. The probe configuration has a relationship that increases accordingly.

  An example in which a plurality of probes are set for the nucleic acid to be detected in the second probe set is shown in FIG. The probe set shown in FIG. 3 is composed of probes C and D for detecting two regions having different detection target strands. The probe C set on the 5 ′ side is 25 bp, whereas the probe set is on the 3 ′ side. The probe D set to 25 is also set to 25 bp, and these binding forces are set to be almost the same. In the probe design in the second probe set, the binding force to the single-stranded nucleic acid can be set based on the Tm value as in the case of the first probe set.

  Here, when a probe that binds to a portion corresponding to the recognition region of the probe D 1 of the detection target strand is set, L1 ≧ L2, and stability when the hybrid is formed is lowered. Therefore, by setting the region recognized by the probe to the complementary strand shown in FIG. 3 and selecting the probe D, in the hybrid body in which the complementary strand and the probe D are combined, L2 ≧ L1 and stability can be ensured. . Therefore, according to the second probe set, when setting different probes for recognizing different regions of the detection target strand, when setting the recognition region for the probe in the region on the 3 ′ side of the detection target strand, By setting the probe binding position on the complementary strand side, the stability of the hybrid body with the probe that recognizes the 3 ′ side region can be ensured without increasing the probe binding force, and the requirements for probe design can be relaxed. it can. In addition, it is preferable to set the position which a probe recognizes from the range from which L1 / L2 is set to 0-1.5, and also from the range from 0 to 1 for the probe set to the complementary strand side.

(About probe carrier)
Each probe constituting at least one of the first and second probe sets can be individually bonded onto a solid phase to form a probe carrier. For example, one probe is set in a region having a common sequence among the family genes listed above, and conversely, one probe is set in each unique region having a sequence different between the family genes. It can be suitably used for analysis. Therefore, the number of types of probes constituting each probe set is a predetermined number of 2 or more depending on the purpose of analysis.

  In order to fix each probe constituting the probe set to the carrier, it is necessary to be able to identify the type of each probe, and any method of spatial, optical, temporal, etc. can be used as the identification method. . For example, a DNA microarray in which probes are immobilized for different types in different areas separated on the same plane is a suitable representative example of the probe-binding carrier of the present invention.

  As a probe binding method, various bonding methods such as adsorption, ionic bonding, hydrogen bonding, and covalent bonding can be applied. In order to bind the probe in a preferable manner, it is desirable to introduce a desired functional group into the probe and bond it via it. It is relatively easy and common to introduce a functional group as a fixing site at the 5 'end of the probe nucleic acid. Similarly, it is relatively easy and common to introduce a functional group as a fixing site at the 3 'end. In addition, the probe can also be immobilized via a functional group as a site for immobilization introduced into the probe sequence.

  As the material of the solid phase carrier, any material that can fix the probe, such as metal, glass, plastic, metal thin film, fiber, etc., can be used without particular limitation. Regarding the form, it can be used without particular limitation, such as a flat shape, a bead, and a strand.

  A preferred example of the method for immobilizing probe DNA is the method disclosed in JP-A-11-187900. The immobilization method disclosed in this published patent publication is a technique in which a maleimide group is introduced onto a glass substrate and a probe DNA having a thiol group bonded to the 5 ′ end is bonded. Probe DNA is immobilized on a glass substrate which is a solid phase carrier. Moreover, the probe is identified by immobilizing it in a different area for each probe.

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, and the technical scope of the present invention is not limited to these examples.
( Reference Example 1)
I. pUC118 EcoRI / BAP PCR
(1) Primer design As a model of a specimen having a sequence to be examined, a commercially available vector pUC118 EcoRI / BAP (full length 3162 bp) manufactured by Takara is selected. From the base sequence, forward primer F1 having the following sequence and Two types of reverse primer R1 were designed. In addition, pUC118 EcoRI / BAP base sequence information is provided by Takara and is also available from public databases.

In designing the primer, pay careful attention to the sequence, GC%, and melting temperature (Tm value) so that the desired partial nucleotide sequence in pUC118 EcoRI / BAP is specifically and efficiently amplified by PCR amplification. Designed. The calculation conditions for the Tm value were 50 mM Na +, 1.5 mM Mg2 ⁺ , and 0.5 μM primer concentration.

  A PCR product of 1324 bp (PCR product 1) is amplified by PCR amplification using the designed primer and pUC118 EcoRI / BAP as a template.

(2) Primer synthesis
Two types of primers designed in (1) of Reference Example 1 were synthesized. For synthesis of each primer, DNA strands having the respective base sequences were synthesized with a DNA synthesizer according to a standard method. Purification was performed by cartridge purification to obtain two kinds of primers. The obtained primer was diluted with TE buffer to a concentration of 10 μM.

(3) PCR amplification reaction
PCR amplification reaction was carried out using the three primers synthesized in (2) of Reference Example 1, Takara vector pUC118 EcoRI / BAP as template gene DNA, and QIAGEN PCR kit HotStarTaq Master Mix. Master Mix contains four types of deoxynucleotides, dATP, dCTP, dTTP, and dGTP. In order to label the PCR product with a fluorescent label, Cy3dUTP manufactured by Amersham Pharmacia is added, and the PCR product is added with Cy3. Labeled.

  PCR reaction conditions were as follows. A reaction solution having the composition shown in Table 2 below was prepared according to the following protocol.

  About the prepared reaction liquid, PCR amplification reaction was performed according to the temperature cycle protocol of the following Table 3 using the commercially available thermal cycler. That is, after holding at 95 ° C./15 minutes (for enzyme activation), 92 ° C. (denaturation) / 15 seconds, 55 ° C. (annealing) / 30 seconds, and 72 ° C. (extension) / 60 seconds as one cycle 25 cycles, finally 72 ° C./10 min hold.

  After the amplification reaction, the PCR amplification product 1 was purified using a purification column (Qiagen QI Aquick PCR Purification Kit). After purification, the volume of the PCR amplification product solution was adjusted to 50 μl. A portion of the resulting purified PCR amplification product 1 solution was taken and subjected to electrophoresis according to a conventional method to confirm that the target PCR product was synthesized.

II. Preparation of DNA microarray (1) Probe design Three types of probes were designed for the PCR product 1 described above. As with the primer design, the design was performed with due consideration so that the partial base sequence designed by each probe could be specifically recognized. In this probe design, it is the DNA strand extended from the R1 primer that hybridizes with the probe to form a hybrid with the probe. The sequence of each probe was designed with due consideration to the stability of the hybrid and adjusting the base length.

  Table 4 shows the nucleotide sequences and Tm values of the designed probes.

  In the hybrid of the target strand for detection (extended strand from R1) in PCR product 1 and each probe, the chain length of the target strand 5 ′ from the hybrid portion is L1, and the strand length is 3 ′. Table 5 shows the values of L1 and L2 of each probe for each PCR product.

(2) Probe synthesis and DNA microarray production Probe synthesis and DNA microarray production were performed according to the DNA microarray production method disclosed by Canon Inc. That is, with respect to the substrate process, silane coupling agent treatment and EMCS were bonded to quartz glass, thereby introducing maleimide groups on the surface. As for probe synthesis, a probe having a thiol group introduced at the 5 ′ end was synthesized and purified by HPLC. In preparing the DNA microarray, a bubble jet printer (trade name: manufactured by BJF-850 Canon Inc.) is used and a glass substrate (size (W × L × T): 25 mm × 75 mm × 1 mm) is used. A DNA microarray in which 16 probes were spotted was prepared.

III. Hybridization reaction
Hybridization on the microarray was performed using the DNA microarray prepared in II and the PCR amplification product 1 prepared in I as the sample nucleic acid specimen.

(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% by weight, and the DNA microarray prepared in II was added to this solution at room temperature. And soaking for 2 hours to block the glass substrate surface. After completion of blocking, in 2 × SSC 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 washing, rinsing with pure water was performed. Thereafter, the DNA microarray was drained with a spin dry apparatus.

(2) Preparation of hybridization solution Each PCR product was prepared to be equimolar to each other, and then a hybridization solution was prepared using 2 microliters of the PCR amplification product solution so that the final concentration was as follows.

<Composition of hybridization solution>
6 × SSPE / 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)
(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 according to the following procedure and conditions.

<Hybridization conditions and procedures>
The hybridization solution was heated to 65 ° C. and held for 3 minutes, then further maintained at 92 ° C. for 2 minutes and then at 55 ° C. for 4 hours. Thereafter, washing was performed at 25 ° C. using 2 × SSC and 0.1% SDS. Further, the plate was washed with 2 × SSC at 20 ° C., rinsed with pure water according to a normal manual as required, and finally drained with a spin dryer to dry.

(4) Fluorescence measurement After the completion of the hybridization reaction, the fluorescence measurement derived from the hybrid was performed on the spin-dried DNA chip using a fluorescence detection device for DNA microarray (Genepix 4000B, manufactured by Axon). The results measured for each probe are shown in Table 7 below.

  In calculating the luminance, the fluorescence intensity observed in the portion of the DNA chip where there is no probe DNA spot is used as the background value, and the value obtained by subtracting the background value from the apparent fluorescence intensity from each spot is the fluorescence intensity. It was set as the actual measurement value. Moreover, a measurement is implemented twice and the average value is shown.

  In this example, three probes are set in the detection target regions having different hybrid stability. However, since the probe sequence is designed for each probe in consideration of the ratio of L1 and L2, the stability of the hybrid is improved. In spite of the difference, fluorescence luminance values of approximately the same number of levels from 2423 to 4054 were obtained.

(Example 1 )
(1) Probe design
One kind of probe was newly designed for the PCR product 1 synthesized in Reference Example 1. The newly designed probe is a probe set to detect the extended chain from the F1 primer at the portion corresponding to the P1 region of Reference Example 1. In designing the probe, the design was performed with sufficient consideration so that the designed partial base sequence could be specifically recognized. Table 8 shows the nucleotide sequences and Tm values of the designed probes.

The ratio of L1 and L2 is shown in Table 9 as in Reference Example 1.

(2) From DNA microarray production to hybridization
As in Reference Example 1, probe synthesis, chip preparation, and hybridization using the same PCR product 1 were performed. In addition to P4, P3 designed and used in Reference Example 1 was spotted on the DNA microarray, and everything else was performed in the same manner as Reference Example 1.

(3) Fluorescence measurement
The results of measuring the fluorescence brightness in the same manner as in Reference Example 1 are shown in Table 10 below.

  In this example, two probes were set in the detection target regions having different hybrid stability. Probe P4 in the region with low stability is set to the complementary strand, so the stability is high, and the fluorescence luminance value is almost the same level as P3 despite the probe having a lower Tm value than P1. It was.

(Comparative Example 1)
(1) Probe design
Two types of probes P5 and P6 were newly designed for the PCR product 1 synthesized in Reference Example 1. The newly designed probe is a probe set to detect the extended chain from the R1 primer in the portion corresponding to the P1 and P2 regions of Reference Example 1. The probes are designed so that both probes have the same binding force (Tm value) as P3. Table 11 shows the base sequence and Tm value of the designed probe.

The ratio of L1 and L2 is shown in Table 12 as in Reference Example 1.

(2) From DNA microarray production to hybridization
As in Reference Example 1, probe synthesis, chip preparation, and hybridization using the same PCR product 1 were performed. In addition to P5 and P6, P3 which was designed and used in Reference Example 1 was spotted on the DNA microarray, and everything else was performed in the same manner as Reference Example 1.

(3) Fluorescence measurement
The results of measuring the fluorescence brightness in the same manner as in Reference Example 1 are shown in Table 13 below.

  In this example, three probes were set in the detection target regions having different hybrid stability. Without considering the stability, each probe had the same level of binding force (Tm value). As a result, the luminance value obtained from each probe reflected the stability of the hybrid, and a large difference occurred despite the same specimen.

Claims (8)

Probe sets for detection of amplification products of single-stranded nucleic acids to be detected and single-stranded nucleic acid amplification products having complementary sequences thereof are provided in different regions on the solid phase that can be distinguished for each probe. A fixed probe carrier ,
The probe set is have a probe for detecting at least one different regions each of the single-stranded nucleic acid to be detected,
When each single strand to be detected and each probe form a hybrid, the number of bases of the portion including the 5 ′ end of the detection target strand among the single-stranded portions each of the hybrid has respectively L1 When the number of bases in the portion including the 3 ′ end is represented as L2,
Each hybrid body
L2 ≧ L1
Probe carrier, characterized in that each probe so as to satisfy the relation is constructed. The probe carrier according to claim 1, wherein the probe is DNA. The probe carrier according to claim 1, wherein the probe is PNA. Wherein each probe constituting the probe set is immobilized on the solid phase via a fixing site introduced at the end of 5 'end, the probe carrier according to any one of claims 1 to 3. Wherein each probe constituting the probe set, 3 'via a fixing site introduced at the end of the side is fixed to the solid phase, the probe carrier according to any one of claims 1 to 3. Probe carrier of any of claims 4-5 wherein the probe support is planar microarray. Two probes formed on a probe fixed to the probe carrier by reacting the probe carrier according to any one of claims 1 to 6 with a single-stranded nucleic acid to be detected and its complementary strand. A method for detecting a nucleic acid, comprising a step of detecting a hybrid of a chain. A detection method for detecting a target single-stranded nucleic acid using the probe carrier according to any one of claims 1 to 6 ,
Detection wherein the <br/> to Ru a sample containing complementary single-stranded nucleic acid having the sequence complementary to said target single-stranded nucleic acid is reacted with the probe carrier.

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