Patent Application
20050064488
This application claims priority from Korean Patent Application No. 2003-65218 filed on Sep. 19, 2003, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
1. Field of the Invention
The present invention relates to a method for detecting target nucleic acid in an efficient manner.
2. Description of the Related Art
Methods for amplifying a nucleic acid are potent techniques for rapidly detecting a specific target sequence and are known in the art. Examples of such methods include polymerase chain reaction (PCR) [U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159 and 4,965,188], strand displacement amplification (SDA) [U.S. Pat. No. 5,270,184], nucleic acid sequence based amplification (NASBA) [U.S. Pat. No. 5,130,238], transcription based amplification [D. Kwoh et al., 1989. Proc. Nat. Acad. Sci. USA 86, 1173-1177], self-sustained sequence replication (3SR) [J. Guatelli et al., 1990. Proc. Nat. Acad. Sci. USA 87,1874-1878], and Qβ replicase system [P. Lizardi et al., 1988. BioTechnology 6, 1197-1202].
Methods for identifying or quantifying the amplified nucleic acids on a microarray include a direct labeling method, a sandwich analysis method, and the like. Among direct labeling methods, a direct dye incorporation method comprises directly labeling PCR products by performing PCR with a PCR reaction solutions containing a monomer labeled with a label, such as fluorescence, for example, a fluorescent labeled dNTP, hybridizing the labeled PCR products to capture probes on a microarray and then reading signals generated from the labels to detect or quantify the target nucleic acid. This method has an advantage that labeling can be performed through PCR without a need for a separate labeling reaction. However, this method has the following drawbacks: labeling efficiency is low and it is difficult to obtain a quantitative relationship between the PCR products obtained by PCR and the signals generated from the PCR products. Further, amplification of the target nucleic acid by a multiplex PCR increases the amount of dyes used, thereby increasing costs. There is another direct labeling method in which the ends of amplification product are labeled with dyes using enzymes such as terminal transferases. However, this method needs a separate enzyme reaction and has a difficulty in maintaining a consistent activity of enzyme, thus resulting in a low reproducibility. An analysis method using stacking is described by C. Mirkin in U.S. Pat. No. 6,582,921, wherein capture probes immobilized on a microarray and labeled detection probes, which specifically bind to target nucleic acids, are used. Specifically, the method comprises amplifying target nucleic acids and hybridizing the amplified target nucleic acids with capture probes immobilized on a microarray and detection probes. The hybridization process is illustrated in FIG.1. Referring to
The inventors studied and developed a method for detection of target nucleic acid which can be performed in a simple process without separately bonding labels, such as dyes, to the targets during the amplification process, even when a plurality of target nucleic acids are amplified as in a multiplex PCR.
The present invention provides a method for detecting target nucleic acids in a simple and efficient manner using a detection probe which can be universally used for at least one target nucleic acid.
According to an aspect of the present invention, there is provided a method for detecting a target nucleic acid, comprising:
According to another aspect of the present invention, there is provided a method for detecting a target nucleic acid, comprising:
According to still another aspect of the present invention, there is provided a method for detecting a target nucleic acid, comprising:
The above and other features and advantages of the present invention will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings in which:
The term “target nucleic acid”, as used herein, refers to a nucleic acid sequence to be amplified. The term also refers to a nucleic acid sequence comprising a sequence region which may bind to a detection probe attached to a microarray. The term “tag sequence”, as used herein, refers to any sequence which is included in an amplification primer at a 5′ end, which after amplification is to be included in an amplified target nucleic acid; The term “detection probe”, as used herein, refers to a probe which may be hybridized with a specific sequence of a target nucleic acid so as to detect the target nucleic acid. With regard to the invention, the term means a nucleic acid having a sequence which may be hybridized with the tag sequence in the amplified target nucleic acid and when hybridized with the tag sequence, it is possible to detect the presence or absence of the target nucleic acids and the quantitative amount of the target nucleic acids. The detection probe may be labeled with a label. The term “primer”, as used herein, refers to a nucleic acid which may be hybridized to a target nucleic acid in a nucleic acid amplification reaction and act as a point of initiation of elongation reaction of a nucleic acid. The primer used in the present invention includes a tag sequence at its 5′ end and a sequence hybridized to the target nucleic acid in the downstream of the tag sequence. Among a set of primers used in the amplification of a nucleic acid in the present invention, one primer may include the tag sequence and another primer may not. Both forward and reverse primers may also include the tag sequence. If a PCR of a nucleic acid is performed using a reaction mixture containing a plurality of primer sets, as in the multiplex PCR, at least one of forward and reverse primers in each primer set includes a tag sequence. The term “a multiplex PCR”, as used herein, refers to a PCR method in which amplification is carried out with a PCR reaction mixture containing a plurality of primer sets in one tube.
According to an embodiment of the present invention, there is provided a method for detecting a target nucleic acid, comprising: amplifying a target nucleic acid from a nucleic acid sample using a set of primers comprising at least one primer having a tag sequence at its 5′ end and a target binding sequence at its 3′ end; hybridizing the resulting amplified products with detection probes labeled with a label, the detection probes being capable of specifically binding to the tag sequence; hybridizing the hybridization products with capture probes immobilized on a microarray, the capture probes being capable of specifically binding to the target nucleic acid and not to the tag sequence; and determining the results of hybridization.
In the method of the present invention, amplification may be performed by any amplification process using a primer, such as a PCR or SDA method. Preferably, a PCR or SDA method is used. More preferably, a multiplex PCR method is used. In the method of the present invention, the label may be selected from a fluorescent label, a radioactive, a receptor, and a ligand but is not limited thereto. Any substance can be used as a label so long as it generates signals and does not interfere with hybridization between the detection probe and the tag sequence. In the method of the present invention, the results of hybridization may be determined by a signal generated from the label of the detection probes. The signal may be in the form of light, such as fluorescence or radioactive rays, or an indirect signal derived from enzymatic activity.
According to another embodiment of the present invention, there is provided a method for detecting a target nucleic acid, comprising: amplifying a target nucleic acid from a nucleic acid sample using a set of primers comprising at least one primer having a tag sequence at its 5′ end and a target binding sequence at its 3′ end; hybridizing the resulting amplified products with capture probes immobilized on a microarray, the capture probes being capable of specifically binding to the target nucleic acid and not to the tag sequence; hybridizing the hybridization products with detection probes labeled with a label, the detection probes being capable of specifically binding to the tag sequence; and determining the results of hybridization.
In the method of the present invention, amplification may be performed by any amplification process using a primer, such as a PCR or SDA method. Preferably, a PCR or SDA method is used. More preferably, a multiplex PCR method is used. In the method of the present invention, the label may be selected from a fluorescent label, a radioactive, a receptor, and a ligand but is not limited thereto. Any substance can be used as a label so long as it generates signals and does not interfere hybridization between detection probe and tag sequence. In the method of the present invention, the results of hybridization may be determined by a signal generated from the label of the detection probes. The signal may be in the form of light, such as fluorescence or radioactive rays, or an indirect signal derived from enzymatic activity.
According to still another embodiment of the present invention, there is provided a method for detecting a target nucleic acid, comprising: amplifying a target nucleic acid from a nucleic acid sample using a set of primers comprising at least one primer having a tag sequence at its 5′ end and a target binding sequence at its 3′ end; hybridizing the resulting amplified products with detection probes labeled with a label and capture probes immobilized on a microarray, the detection probes being capable of specifically binding to the tag sequence and the capture probes being capable of specifically binding to the target nucleic acid and not to the tag sequence; and determining the results of hybridization.
In the method of the present method, amplification may be performed by any amplification process using a primer, such as a PCR or SDA method. Preferably, a PCR or SDA method is used. More preferably, a multiplex PCR method is used. In the method of the present invention, the label may be selected from a fluorescent label, a radioactive label, a receptor, and a ligand but is not limited thereto. Any substance can be used as a label so long as it generates signals and does not interfere hybridization between detection probe and tag sequence. In the method of the present invention, the results of hybridization may be determined by a signal generated from the label of the detection probes. The signal may be in the form of light, such as fluorescence or radioactive rays, or an indirect signal derived from enzymatic activity.
The length of the primer, tag sequence, detection probe, and capture probe is not specifically limited and may depend on the hybridization conditions, the objects of detection, and the like. The length may preferably be 10 to 200 nucleotides, more preferably 10 to 100 nucleotides, and most preferably 15 to 50 nucleotides.
The method of the present invention will be explained with regard to the drawings.
Hereinafter, the present invention will be described in more detail with reference to the following example. The example is given for the purpose of illustration and not intended to limit the scope of the invention.
In this example, a target, i.e., MODY 1 gene, was first amplified by PCR using a set of amplification primers containing a tag sequence at the 5′ end. The PCR products were purified and the concentration thereof was measured. The resulting PCR products were hybridized with labeled detection probes which are complementary to the tag sequence. Next, the hybridization products were hybridized with capture probes immobilized on a microarray, the capture probes having a sequence complementary to the target nucleic acid. After the hybridization products were washed, scanning was carried out and then the degree of hybridization was determined to detect the target nucleic acids. The experimental processes of Example 1 are specifically described below.
(1) Amplification of MODY 1 Gene by PCR Using Primers Containing a Tag Sequence
First, a set of primers targeted to seven exons of MODY 1 gene were designed (see, Table 1) (Bioneer, Taejeon, Korea). Each primer of the primer set contains tag sequences 1 and 2 (Sequence ID Nos. 1 and 2) at the 5′ end. The primer set was divided into two groups and subjected to a multiplex PCR using a genome of a normal person as a template. The conditions of the PCR reactions were as follows: the initial denaturation of primers at 94° C. for 3 minutes; the 1st to 7th cycles with one cycle including denaturation at 94° C. for 30 sec, annealing at 64° C. for 15 sec, and extension at 72° C. for 40 sec; the 8th to 29th cycles with one cycle including simultaneous annealing and extension at 72° C. for 3 minutes; and the 30th cycle of final extension at 72° C. for 3 minutes.
(2) Hybridization of the PCR Products with Detection Probes and then Hybridization of the Obtained Hybridization Products with the Capture Probes
After the PCR products were purified, the purified PCR products, detection probes 1 (SEQ ID No. 3), and detection probes 2 (SEQ ID No. 4) were mixed with a buffer for hybridization. The detection probes 1 and 2 contained Cy3 dye attached to the 5′ end, as seen in SEQ ID Nos. 3 and 4. The final concentrations of the PCR products and each of the detection probes were adjusted to 100 nM.
Then, the hybridization products of the PCR products and the detection probes were hybridized to capture probes complementary to the PCR products, the capture probes being immobilized on a microarray. The type of mutants observed in MODY1 gene, which may be detected on the microarray, and the capture probes capable of detecting each of the mutants are shown in Table 2. The probes were arranged on the microarray so that they had triple spots, respectively. The hybridization reaction on the microarray was performed in 6×SSPET buffer at 42° C. for 16 hours. After the completion of hybridization reaction, the products were washed with 6×SSPET buffer solution for 5 minutes and 3×SSPET buffer solution for 5 minutes. The hybridization results on the microarray were scanned using an Axon 4000B LASER scanner, and then an imaging process was performed.
The obtained images are shown in
(3) Hybridization of the PCR Products with Capture Probes and then Hybridization of the Obtained Hybridization Products with the Detection Probes
The PCR amplified products were first hybridized with the capture probes on the microarray, and then the obtained hybridization products were hybridized with the detection probes and scanned by a scanner.
The hybridization reaction on the microarray was performed in 6×SSPET buffer at 42° C. for 16 hours. After the completion of hybridization reaction, the products were washed with 6×SSPET buffer solution and 3×SSPET buffer solution for 5 minutes each. Next, to the hybridization products, the detection probes dissolved in the hybridization buffer solution (the final concentration of 20 nM) were secondly hybridized at room temperature for one hour. Then, washing was performed as described above. The hybridization results on the microarray were scanned using an Axon 4000B LASER scanner, and then an imaging process was performed.
The results are shown in
(4) PCR Amplification of the Target Products by a Direct Labeling and Hybridization of the Amplified Products
To compare the detection method of the present invention with a conventional detection method, the target products were amplified through a conventional PCR method by a direct labeling and then hybridized with capture probes on a microarray to detect the amplified products.
First, the composition of a direct labeling reagent used in a direct labeling method was as follows: water (dH2O) 27.9 μl; 10× buffer, 5 μl; dNTP(dA, dG, dC=200 μM, dT=40 μM), 0.5 μl; template DNA (200 ng/μl), 1 μl; Taq polymerase (3U), 0.6 μl; primer (200 nM), 14 μl; and cy3-dUTP (20 μM), 1 μl. The conditions of the PCR reactions were as follows: the initial denaturation at 95° C. for 5 minutes; the 1st to 40th cycles with one cycle including denaturation at 95° C. for 30 sec, annealing at 63° C. for 15 sec, and extension at 72° C. for 3 minutes; and then the final extension at 72° C. for 3 minutes and storage at 4° C.
The results are shown in
As illustrated in
Fluorescent signals of the capture probe spots were analysed for detecting mutation of a specific gene, using the resulting image data. A graph was plotted and analysed, in which the x-axis represents a log of a fluorescent signal (wp) of the capture probe spots for detecting a normal type and the y-axis of y represents a log of the ratio (mp/wp) of a fluorescent signal (mp) of the capture probe spots for detecting a mutant type to a fluorescent signal (wp) of the capture probe spots for detecting a normal type. For example, the analytical results of fluorescent signals for spots of 1454V mutant of exon 10 and capture probe for the detection of its normal type, i.e., MO1E10-01mp and MO1E10-01wp, are shown in
In
As described above, the method for detecting target nucleic acids according to the present invention may provide almost similar effects in terms of fluorescent intensity, compared with the conventional direct labeling method (see,
According to the method for detecting target nucleic acids of the present invention, even a plurality of target nucleic acids amplified by a multiplex PCR method may be detected using the same detection probes. Also, with regard to the increase in cost, which is proportional to the number of type of target nucleic acids, the present method has a relatively low cost increase rate than the conventional method. Further, the method of the present invention may be performed in a simpler process since it does not need a separate labeling reaction.
The amount of commercially available fluorescent dyes used in the method of the present invention is just about 2% of that in the conventional direct labeling method, which is advantageous in view of economy. Also, the method of the present invention may prevent the adverse effects of delaying or stopping the PCR as occurred in the direct labeling method.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2003-65218 | Sep 2003 | KR | national |
