Patent Application
20080051295
Preferred embodiments of the present invention will now be described in detail in accordance with the accompanying drawings.
A method of detecting a nucleic acid according to a first mode of the present invention includes the aforementioned steps (1) to (6). This method enables the universal PCR on a primer array.
In this case, if the base sequence to be detected is, for example, of a gene having a double-stranded nucleic acid, the sense base sequence is basically the same as the anti-sense base sequence. It is obvious that analysis of the anti-sense sequence reveals the sense base sequence complementary to the sequence (that is, the base sequence of the gene).
Each operation in each step can be performed using the known method. In the detection step as the final step, any procedure which is capable of detecting a formed hybridized product may be used. For example, a detection method using a fluorescent intercalator dye or fluorescent groove binder dye which interacts with a double-stranded nucleic acid to emit or enhance fluorescence may be utilized. Alternatively, there may also be adopted a procedure for labeling a nucleic acid having a base sequence complementary to a base sequence located in the position nearer 3′-end than that of the 3′-end of the base sequence to be detected of A-strand, which is used as a primer in the step (4) (which is a B-strand elongating primer and a primer derived from a segment nearer the 3′-end of A-strand; herein, the phrase “derived from a position nearer the 3′-end” means “derived from a segment located in the position nearer 3′-end than that of the base sequence to be detected which is nearest the 3′-end”), with a fluorescent dye such as fluorescein, tetramethylrhodamine, Cy3, or Cy5, or radioisotope. Note that, in the case of using the fluorescent dye, a fluorescent microscope may be used for observation, and a step of observing a label using, for example, a confocal fluorescent microscope may further be included.
In this method, one primer of a set of primers to be used PCR reactions is immobilized on a solid-phase, and the other (B-strand elongating primer) is dissolved in a reaction solution before use. The B-strand elongating primer has functions as a common primer for amplifying each base sequence to be detected. Further, the steps (2) and (3) of the first mode may be changed as follows:
(2) preparing as primers, nucleic acids each having one of the plural base sequences to be detected, immobilizing the respective primers independently in separate regions on a substrate, and preparing a primer array in which the respective base sequences to be detected are distributed in the primer-immobilized regions; and
(3) preparing a nucleic acid having a partial and sequential base sequence within the region between a 5′-end of the A-strand and the base sequence to be detected which is located nearest the 5′-end as a primer for elongating the A-strand and preparing a nucleic acid having a base sequence complementary to a partial and sequential base sequence within the region between a 3′-end of the A-strand and the base sequence to be detected which is located nearest the 3′-end as a primer for elongating the B-strand.
That is, in addition to the B-strand elongating primer, a nucleic acid having the same base sequence as a sequential and partial base sequence which is located in the position nearer 5′-end than that of the5′-end of the base sequence to be detected of A-strand (which is an A-strand elongating primer and a primer derived from a segment nearer the 5′-end of A-strand: herein, the phrase “derived from a position nearer the 5′-end” means “derived from a segment located in the position nearer 5′-end than that of the base sequence to be detected which is nearest the 5′-end”) may be dissolved in a reaction solution to be used concomitantly as the second common primer. In some situations, the amplifying efficiency in such concomitant use may be higher.
Note that, in some situations, this method may include a step of removing a substance other than the aforementioned hybridized product (such as a template nucleic acid, or a nucleotide monomer or enzyme used for PCR reactions) through a washing operation for removing a reaction solution on the substrate.
Those methods relate to the universal PCR as described above, and the method of detecting a nucleic acid according to a second mode of the present invention includes the above-described steps (1) to (6). This method enables the multiplex PCR.
In this case, the same detection method as that described above may be used for detecting a hybridized product. The aforementioned detection method may be used for plural single-stranded nucleic acids each having one or more partial and sequential base sequences to be detected (A-strand group: A1-strand An-strand: n≧2). Moreover, the steps (2) and (3) according to the second mode may be changed as follows:
(2) preparing nucleic acids as primers each having one of the plural base sequences to be detected, immobilizing the respective primers independently in separate regions on a substrate, and preparing a primer array in which the respective base sequences to be detected are distributed in the primer-immobilized regions; and
(3) preparing nucleic acids each having a partial and sequential base sequence within the region between a 5′-end of each strand of the A-strand group and the base sequence to be detected which is located nearest the 5′-end as primers for elongating the A-strand (PA-strand group: PA1-strand to PAn-strand: n≧2) and preparing nucleic acids each having a base sequence complementary to a partial and sequential base sequence within the region between a 3′-end of each strand of the A-strand group and the base sequence to be detected which is located nearest the 3′-end as primers for elongating the B-strand (PB-strand group: PB1-strand to PBn-strand: n≧2).
That is, in amplification reactions, plural nucleic acids each having the same base sequence as a sequential and partial base sequence which is located in the position nearer 5′-end than that of the 5′-end of the base sequence to be detected of the aforementioned A-strand group (PA-strand group: PA1-strand to PAn-strand: n≧2) may be dissolved in a reaction solution together with the PB-strand group to be used as common primers. In some situations, the amplification efficiency in this method is higher.
The aforementioned methods enable the solid- phase universal PCR, solid-phase multiplex PCR, or solid-phase universal multiplex PCR using the nucleic acid array.
Next, as in the third and forth modes, in order to simplify detection steps, a labeled monomer may be utilized as a substitute for a nucleotide monomer used in PCR reactions. The situation is shown in a schematic view of
Note that, in some situations, this method may include a step of removing a substance other than a nucleic acid which is elongated and amplified by the PCR reactions and corresponds to A-strand binding to a substrate, for example, a nucleic acid which is elongated and amplified and corresponds to B-strand, a template nucleic acid, a nucleotide monomer used in the PCR reactions, or an enzyme.
This method enables the solid-phase PCR capable of performing detection immediately after PCR, the solid-phase universal PCR capable of performing detection immediately after PCR, the solid-phase multiplex PCR, or the solid-phase universal multiplex PCR without performing two-tiered steps in which hybridization is performed after PCR, and was required to be improved in the aforementioned conventional technique.
An apparatus for realizing the detection methods described above fall within the scope of the present invention.
The above-described methods are preferred at least in points that PCR reactions and detection are performed in the form in which primer arrays are present in the same container, which is preferred in that an apparatus or the like can be simplified and in that, as described below, PCR reactions and detection can be performed using the same method simultaneously and continuously. In such case, when the PCR reactions and detection are performed using the same method simultaneously and continuously, the process of the PCR amplification can be monitored. Accordingly, this is a preferred mode in that so-called real-time PCR is realized, that is, highly quantitative detection can be performed. An apparatus that is equipped with a PCR reaction container and detection method enabling such a detection method is a preferred mode for carrying out the present invention.
The solution mentioned above is introduced from an inlet port (not shown) into the reaction chamber 3 to conduct PCR reactions while the temperature is controlled by the temperature controlling unit 4 in accordance with the aforementioned schedule.
Hereinafter, the present invention will be described in detail with reference to examples.
The following seven segments (Ec1 to Ec7) of sense base sequences of genome DNA of 16s ribosomal RNA (rRNA) of Escherischia coli (ATCC#11775) were defined as detection targets of a solid-phase universal PCR, which were selected as primers to be immobilized on solid-phase.
Those primers were synthesized by a synthesis company (BEX Co., Ltd.) on commission. The 5′-end of each of Ec1 to Ec7 was bound to a thiol (SH) group via a linker for binding to a solid-phase substrate as described below. The Ec1 structure to which a thiol group is bound is shown below as an example.
Moreover, the following base sequence (EcAP) was selected as a base sequence of a common primer of the anti-sense sequence:
ECAP was synthesized in a general manner. All the primers were deprotected and purified based on the conventional methods.
Firstly, an E. coli standard strain was cultured in accordance with the conventional method. 1.0 ml of the microorganism-cultured medium (OD600=0.7) was collected in a 1.5-ml microtube, and the bacterial cells were collected by centrifugation (8,500 rpm, 5 minutes, 4° C.). After the supernatant was discarded, 300 μl of Enzyme Buffer (50 mM Tris-HCl: p.H. 8.0, 25 mM EDTA) was added thereto, and the cells were resuspended using a mixer. The resuspended bacterial suspension was centrifuged again, to thereby collect bacterial cells (8,500 rpm, 5 minutes, 420 C.). After the supernatant was discarded, the following enzyme solutions were added to the collected bacterial cells, and the cells were resuspended using a mixer:
Lysozyme 50 μl (20 mg/ml in Enzyme Buffer)
N-Acetylmuramidase SG 50 μl (0.2 mg/ml in Enzyme Buffer).
Next, the resuspended bacterial suspension to which the enzyme solutions were added was left to stand in an incubator at 37° C. for 30 minutes, to thereby perform treatment for cell wall lysis. Subsequently, genome DNA was extracted using a kit for purifying a nucleic acid (Mag Extractor. Genome-: manufactured by TOYOBO Co., Ltd.).
Specifically, firstly, 750 μl of a dissolution/adsorption solution and 40 μmagnetic beads were added to the pretreated microorganism suspension, and the mixture was stirred vigorously for 10 minutes using a tube mixer (Step 1).
The microtube was set on a separatory stand (Magical Trapper) and allowed to stand for 30 seconds, to thereby collect the magnetic particles on the wall surface of the tube, and the supernatant was discarded while the tube was set on the stand (Step 2).
Next, 900 μl of a washing solution was added thereto, and the particles were resuspended by stirring using the mixer for about 5 seconds (Step 3).
Next, the microtube was set on the separatory stand (Magical Trapper) and allowed to stand for 30 seconds, to thereby collect the magnetic particles on the wall surface of the tube, and the supernatant was discarded while the tube was set on the stand (Step 4).
The procedures of Steps 3 and 4 were repeated, and the second washing was performed (Step 5). Subsequently, 900 μl of 70% ethanol was added thereto, and the particles were resuspended by stirring using the mixer for about 5 seconds (Step 6).
Next, the microtube was set on the separatory stand (Magical Trapper) and allowed to stand for 30 seconds, to thereby collect the magnetic particles on the wall surface of the tube, and the supernatant was discarded while the tube was set on the stand (Step 7).
The procedures of Steps 6 and 7 were repeated, and the second washing was performed with 70% ethanol (Step 8). Subsequently, 100 μl of pure water was added to the collected magnetic particles, and the mixture was stirred for 10 minutes using the tube mixer.
Next, the microtube was set on the separatory stand (Magical Trapper) and allowed to stand for 30 seconds, to thereby collect the magnetic particles on the wall surface of the tube, and the supernatant was collected in a new tube while the tube was set on the stand.
The collected genome DNA of Escherischia coli was subjected to agarose electrophoresis and absorbance measurement with the wavelength of 260/280 nm in accordance with the conventional methods to assay its quality (amount of contaminated low-molecular-weight nucleic acids, the degree of degradation) and collected amount. In this example, about 9 μg of the genome DNA was collected, and degradation of the genome DNA and contamination of rRNA were not observed. The collected genome DNA was dissolved in TE buffer so as to have a final concentration of 50 ng/μl, and the mixture was used in the following examples.
A glass substrate made of synthetic quartz (size: 25 mm×75 mm×1 mm, manufactured by Iiyama Tokusyu Glass Co. Ltd.) was placed in a heat-resistant and alkali-resistant rack and immersed in a washing solution for ultrasonic cleaning which had been adjusted to a predetermined concentration. The substrate was immersed in the washing solution overnight, and ultrasonic cleaning was then performed for 20 minutes. Subsequently, the substrate was taken out and lightly rinsed with pure water, followed by ultrasonic cleaning in ultrapure water for 20 minutes. Next, the substrate was immersed in a 1N sodium hydroxide solution heated to 80° C. for 10 minutes. Washing with pure water and washing with ultrapure water were performed again, to thereby prepare a quartz glass substrate for a DNA array.
A silane coupling agent KBM-603 (manufactured by Shin-Etsu Silicones) was dissolved in pure water so as to have a concentration of 1%, and the mixture was stirred for two hours at room temperature. Subsequently, the glass substrate that had been previously washed was immersed in the aqueous solution of the silane coupling agent and allowed to stand for 20 minutes at room temperature. The glass substrate was drawn up, and its surface was lightly washed with pure water. Subsequently, nitrogen gas was blown on both the sides of the substrate, and the substrate was dried. Next, the treatment with the coupling agent was completed by baking the dried substrate in an oven heated to 120° C. for 1 hour, to thereby introduce amino groups to the substrate surface. Next, N-(6-maleimidocaproyloxy)succinimido (hereinafter, abbreviated as EMCS; produced by Dojindo Laboratories) was dissolved in a mixed solvent of dimethylsulfoxide and ethanol (1:1) so as to have a final concentration of 0.3 mg/ml to prepare an EMCS solution. The baked glass substrate was left to cool and immersed in the prepared EMCS solution at room temperature for 2 hours. Through such treatment, the amino groups introduced onto the surface by using the silane coupling agent reacted with succinimido groups of EMCS, to thereby introduce maleimide groups onto the surface of the glass substrate. The glass substrate was drawn up from the EMCS solution, and the substrate was washed with the above-described mixed solvent and further washed with ethanol, followed by drying under nitrogen gas atmosphere.
(3-3) Primers The primers to be immobilized on solid-phase (Ec1 to Ec7) described in (1) were separately dissolved in pure water, and each mixture was dispensed so as to have a final concentration (when it was dissolved as described below) of 10 μM, followed by freeze-drying, to thereby remove water.
(3-4) Discharge of primer DNA by BJ printer, and binding to substrate An aqueous solution (mixed solvent) containing 7.5 wt % of glycerin, 7.5 wt % of thiodiglycol, 7.5 wt % of urea, and 1.0 wt % of Acetylenol EH (manufactured by Kawaken Fine Chemicals Co., Ltd.) was prepared. Subsequently, each of previously prepared 7 primers was dissolved in the aforementioned mixed solvent so as to have a prescribed concentration. The resultant DNA solution was filled into an ink tank for a bubble jet printer (trade name: BJF-850, manufactured by Canon Inc.), and the tank was attached to a print head.
Note that the bubble jet printer used herein had been modified so as to enable print on a flat plate. Moreover, the bubble jet printer can perform spotting of about 5 μl of a DNA solution at a pitch of about 120 μm by inputting a printing pattern in accordance with a predetermined file creation method.
Subsequently, a printing operation for one glass substrate was performed using the modified bubble jet printer for preparing a DNA array. After confirming that printing was performed without fail, the array was allowed to stand in a humidified chamber for 30 minutes to react maleimide groups on the surface of the glass substrate with thiol groups at the end of the nucleic acid primer.
After the reaction was performed for 30 minutes, the DNA solution remaining on the surface was washed out with 10 mM of phosphate buffer (pH 7.0) containing 100 mM of NaCl, to thereby yield a DNA array in which single-stranded DNA was immobilized on the surface of the glass substrate.
A method of amplifying Escherischia coli genome DNA was described below.
A microchamber in which a primer-binding site of the above-described primer array was covered and the temperature can be controlled was manufactured, and a reaction solution of the aforementioned composition was sealed in the chamber. Then, amplification reactions were performed in accordance with the following protocol. More specifically, after an incubation step at 95° C./10 minutes, a denaturation step at 92° C./45 seconds, an annealing step at 55° C./45 seconds, and an elongation step at 72° C./45 seconds were defined as one cycle, and the cycle was repeated 35 times, and finally an incubation step at 72° C./10 minutes was performed.
After the completion of the reactions, 1 μl of a solution prepared by previously diluting a dye of which (fluorescence is enhanced under coexistence of a double-stranded nucleic acid, SYBR (registered trademark) Green I (Molecular Probes: trade name SYBR (registered trademark) Green I nucleic acid gel stain 10,000×concentrate in DMSO) to 200-fold with pure water was added to the chamber, and the primer array was placed under the conditions of 65° C./3 minutes→92° C./2 minutes→45° C./3 hours for hybridization.
Next, the primer array was washed under the conditions of 2×SSC/0.1% SDS (25° C.)/2 minutes→2×SSC (20° C.)/2 minutes→pure water (10° C.)/2 minutes, and the array was taken out of the chamber and dried.
The DNA array after the completion of the hybridization reaction was subjected to fluorescence measurement using a fluorescence detecting apparatus for DNA array (manufactured by Axon Instruments, GenePix 4000B) (excitation wavelength: 532 nm, photomultiplier voltage: 400 V) . The measurement results are shown in Table 1. Note that, in this example, all the operations are performed twice, so that the results were separately shown in Table 1.
The numeric values of the fluorescent luminance in Table 1 represent pixel average luminance (resolution: 5 μm). As is clear from Table 1, the results obtained by amplifying genome DNA extracted from Escherischia coli by the solid-phase universal PCR can be detected with high reproducibility.
The solid-phase PCR and fluorescence detection were performed in accordance with the same method as that in Example 1 except that, in addition to the primers used for the solid-phase PCR in Example 1, a common primer (EcSP) of the sense sequence having the following base sequence was used at the same concentration as that of a common primer of the anti-sense sequence.
In Example 2, when the solid-phase PCR cycle was repeated 31 times, substantially the same results as those in Example 1 were obtained. The results reveal that the efficiency of the solid-phase PCR could be improved by letting both the common primers of the sense sequence and anti-sense sequence coexist in a solution.
In this example, genome DNA of rRNA of Pseudomonas aeruginosa (ATCC#10145) will be detected simultaneously with Escherischia coli detected in Example 1. For this purpose, firstly, 8 thiolized primers of the sense sequence for Pseudomonas aeruginosa (Pa1 to Pa8) were synthesized in the same way as that in Example 1. The base sequences of the primers are shown below.
Moreover, the following base sequence (PaAP) was selected as a base sequence of a common primer of the anti-sense sequence:
Extraction of Pseudomonas aeruginosa genome, manufacture of a DNA microarray immobilized with Escherischia coli primers and Pseudomonas aeruginosa primers, solid-phase PCR (Escherischia coli genome DNA and Pseudomonas aeruginosa genome DNA: 10 ng each, EcAP and PaAP: 20 pmole each), hybridization, washing, etc. were performed in the same way as that in Example 1, and fluorescence detection was performed. The results are shown in Table 2.
Escherischia
Pseudomonas
coli
aeruginosa
Table 2 shows that all the given primer sequences of rRNA genomes of both Escherischia coli and Pseudomonas aeruginosa were simultaneously detected by the solid-phase multiplex universal PCR. Example 4 (Solid-phase multiplex universal PCR II) The solid-phase PCR and fluorescence detection were performed in the same manner as that in Example 3 except that, in addition to the primers used for the solid-phase PCR in Example 2, common primers of the respective sense sequences of Escherischia coli and Pseudomonas aeruginosa (EcSP and PaSP) were used at the same concentration. The base sequence of PaSP is shown below.
In Example 4, under the condition that the cycle of the solid-phase PCR was repeated at given times fewer than those in Example 3 by about one or more, almost the same results as those in Example 3 were obtained. The results reveal that, in the solid-phase multiplex universal PCR, the efficiency of the solid-phase PCR could be improved by letting both the common primers of the sense sequence and anti-sense sequence coexist in a solution.
Extraction of Escherischia coli genome, synthesis of primers, and manufacture of a primer array were performed in completely the same manner as that in Example 1, and the solid-phase PCR was then performed using a PCR solution of the following composition.
Subsequently, the primer array was washed, without keeping the same under to hybridization conditions, under the conditions of 2×SSC/0.1% SDS (92° C.)/2 minutes→2×SSC/0.1% SDS (92° C.)/2 minutes→2×SSC/0.1% SDS (25° C.)/2 minutes→2×SSC (20° C.)/2 minutes→pure water (20° C.)/2 minutes, and the array was taken out of the chamber and dried.
Subsequently, fluorescence detection was performed in the same manner as that in Example 1. The results are shown in Table 3.
Escherischia coli
In accordance with the detection method of the present invention, fluorescence detection was performed adopting an incorporated label in the solid-phase universal PCR. As a result, fluorescence detection was easily accomplished as shown in Table 3. Accordingly, the results reveal that fluorescence detection can be performed without requiring the hybridization step.
The solid-phase universal PCR or solid-phase multiplex universal PCR in Examples 2 to 4 was performed using the incorporated label in Example 5. Although the respective fluorescence intensities were different from those in Examples 2 to 4, the results reveal that the rRNA genome DNA of Escherischia coli or the rRNA genome DNAs of both Escherischia coli and Pseudomonas aeruginosa can be detected simultaneously.
Detection of Escherischia coli genome DNA was performed by the solid-phase universal method in completely the same manner as that in Example 1 except that the common primer EcAP was labeled with tetramethylrhodamine, hybridization was performed without coexistence with a fluorescent dye such as SYBR green, and fluorescence detection was performed using the aforementioned tetramethylrhodamine. The structure of tetramethylrhodamine-labeled EcAP is shown below.
As a result, almost the same results as those in Example 1 were obtained although the differences in fluorescence intensities were observed.
A method of labeling the common primer of anti-sense sequence was performed in the methods of Examples 2 to 4. As a result, the results confirm that all the methods were effective were obtained.
The present invention is not limited to the above embodiments and various changes and modifications can be made within the spirit and scope of the present invention. Therefore to apprise the public of the scope of the present invention, the following claims are made.
This application claims priority from Japanese Patent Application No. 2004-070986 filed on Mar. 12, 2004, which is hereby incorporated by reference herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004-070986 | Mar 2004 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP05/04881 | 3/14/2005 | WO | 00 | 5/29/2007 |
