WO2001009378A1

WO2001009378A1 - Method for detecting nucleic acid in nucleic acid sample using micro well plate with various dna probes

Info

Publication number: WO2001009378A1
Application number: PCT/KR2000/000839
Authority: WO
Inventors: Hee Tae Kim
Original assignee: Hee Tae Kim
Priority date: 1999-08-02
Filing date: 2000-08-01
Publication date: 2001-02-08
Also published as: AU6187200A; KR20010016720A

Abstract

The present invention is directed to a method for detecting a specific nucleic acid in a nucleic acid sample and, more particularly, to a method for detecting the presence and quantity of a specific nucleic acid in a nucleic acid sample, the method comprising the steps of adhering various DNA probes onto the surface of one tube in a micro well plate, injecting a nucleic acid sample to be tested into the tube so as to cause a coupling reaction between the DNA probe and the nucleic acid, and verifying the result of the coupling reaction with a reading apparatus. Advantageously, the method of the present invention enables a precise analysis of a plurality of samples in a simultaneous manner for a short time, compared to the conventional methods using a membrane or a glass plate.

Description

METHOD FOR DETECTING NUCLEIC ACID IN NUCLEIC ACID SAMPLE USING MICRO WELL PLATE WITH VARIOUS DNA PROBES

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for detecting a nucleic acid in a nucleic acid sample and, more particularly, to a method for detecting the presence and quantity of a specific nucleic acid in a nucleic acid sample that contains various nucleic acids. 2. Description of the Related Art

Some methods to detect a specific gene contained in a nucleic acid sample in a variety of gene-related tests, e.g., HLA/HPV typing and oncogene identification, and disease analyses use the DNA probe of the gene.

Recently, there has been used a method for detecting a nucleic acid using a specific DNA probe adhered to the surface of a micro well plate, or different DNA probes adhered to a membrane or a glass plate. The features and drawbacks of such a method are defined as follows.

In the first method using a micro well plate, streptavidin is coated on the surface of the micro well plate and a DNA probe labeled with biotin is added to the micro well plate, after which a nucleic acid sample to be tested is denatured by heat or a chemicals and injected into the micro well plate in order to cause a coupling reaction between the DNA probe and the nucleic acid. Then, the result of the coupling reaction is verified by an enzymatic assay. Disadvantageously, this method is incapable of analyzing various nucleic acids contained in the sample at the same time because basically only one DNA probe is adhered to the surface of the micro well plate, and takes much time (at least two hours) in regard to the enzymatic assay so as to verify the result of the coupling reaction.

In the second method using a membrane, various DNA probes are adhered to the surface of the membrane, after which a labeled nucleic acid sample to be tested is denatured by chemicals or the like and added to the membrane together with a nucleic acid coupling solution, while maintaining the temperature constant. Then, the labeling agent is read to determine the result of the coupling reaction between the nucleic acids and the DNA probes. This method is however disadvantageous in that it involves an extremely complicated testing procedure requiring much time (at least two hours) and is incapable of analyzing different samples in a simultaneous manner. Meanwhile, in the third method using a glass plate, various DNA probes adhered to the surface of the glass plate(hereinafter, referred to as "DNA chips") are brought in contact with a denatured nucleic acid sample to be tested, after which the result of the coupling reaction between the nucleic acids and the DNA probes is read with a laser beam. Disadvantageously, this method is incapable of analyzing different samples at once because it tests only one sample per a glass plate, although various probes are adhered to the single glass plate. This method also has a limitation on the number of times of using one glass plate in testing the nucleic acid coupling reaction.

Therefore, the conventional methods for detecting a nucleic acid are problematic in that the testing procedure is too complicated and takes much time and that only one sample is detectable each time for the test.

SUMMARY OF THE INVENTION

To overcome the drawbacks of the conventional methods for detecting a nucleic acid, the inventor has contrived to a novel nucleic acid detecting method that is efficient and preferable in the aspect of time and cost. Accordingly, it is an object of the present invention to provide a method for detecting a plurality of nucleic acid samples precisely in a simultaneous and simple manner for a short time relative to the conventional methods.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows side view and cross-sectional bottom view of a tube in a micro well plate to which DNA probes are adhered;

Fig. 2 is a cross-sectional view showing the bottom side of the tube in a micro well plate for explaining the result of Example 1; Fig. 3 is a cross-sectional view showing the bottom side of the tube in a micro well plate for explaining the result of Example 2;

Fig. 4 is a cross-sectional view showing the bottom side of the tube in a micro well plate for explaining the result of Example 3;

Fig. 5 is a cross-sectional view showing the bottom side of the tube in a micro well plate for explaining the result of Example 4;

Fig. 6 is an illustration of a membrane with DNA probes in a conventional method of Comparative Example 1; and

Fig. 7 is an illustration of a glass plate with DNA probes in a conventional method of Comparative Example 2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is directed to a method for detecting a specific nucleic acid in a nucleic acid sample and, more particularly, to a method for detecting the presence and quantity of a specific nucleic acid in a nucleic acid sample, the method comprising the steps of adhering various DNA probes onto the surface of one tube in a micro well plate, injecting a nucleic acid sample to be tested into the tube so as to cause a coupling reaction between the DNA probe and the nucleic acid, and verifying the result of the coupling reaction with a reading apparatus.

Specifically, the present invention provides a method for detecting a specific nucleic acid in a nucleic acid sample, comprising the steps of:

(A) adhering various DNA probes onto the surface of one tube in a micro well plate;

(B) injecting a labeled nucleic acid into the tube and heat-denaturing the labeled nucleic acid; (C) causing the heat-denatured nucleic acid to be coupled to the DNA probes in the tube;

(D) washing out non-specific nucleic acids not coupled to the DNA probes in the preceding step; and

(E) measuring the label of the nucleic acid coupled to the DNA probes in the tube to read the result of the coupling reaction between the specific nucleic acid and the DNA probes.

The step (A) of adhering various DNA probes onto the surface of the tube in the micro well plate may use a per se known DNA adhering technique, e.g., a streptavidin- biotin technique (See. "Methods in Enzymology", Green. N. M., Vol. XVIII, pl48 (1970)), a EDC (l-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide) technique (See. Anal Biochem., Rsamussen SR et al., 198:138-142 (1991)), or a polylysine technique.

To simultaneously read the coupling result of the nucleic acids with various DNA probes in a plurality of tubes of the individual micro well plate, there is used a per se known labeling and reading technique, e.g., a fluorescent labeling and reading method in which the nucleic acids are labeled with a fluorescent substance and the fluorescence of the substance is measured with a CCD camera or a laser beam.

The micro well plate as used in the present invention method may be a commercial plate that consists of 96 tiny tube wells for different samples so as to enable a simultaneous test of at least 90 samples. The present invention will be described below in further detail with reference to the following examples, which illustrate but are not intended to limit the present invention. Example 1 : HPV Nucleic Acid Typing using the Present Invention Method.

(1) A nucleic acid sample was extracted from a patient presumably HPV-infected and suffering from cervical cancer and a PCR procedure was performed by a known method using a labeled HPV primer containing interposed probe sequences shown in Table 1 (See. U.S. Patent Nos. 4,683,202; 4,683,195; and 4,964,188).

(2) The DNA probes specific to the respective types of HPV shown in Table 1 were adhered onto the surface of the tube of the micro well plate by a known DNA adhering technique, as shown in Fig. 1. Table 1

(3) The resultants of the PCR reaction in step (1) (i.e., amplified nucleic acid fragments) together with a coupling solution, e.g., 50% formamide, 5X SSPE, 2X Denhardt's reagent and 0.1% SDS were injected into one tube of the micro well plate with the DNA probes.

(4) The tube of step (3) was heated at 94 °C for 5 minutes to cause a heat denaturation of the nucleic acids in the sample, and cooled to 42 °C for 10 minutes of the coupling reaction between the DNA probes on the surface of the tube and the nucleic acids in the sample.

(5) Non-specific nucleic acids not coupled to the DNA probes in step (4) were washed out with a washing solution (2X SSC, 0.1% SDS) and the label was detected to measure the degree of the coupling reaction of the nucleic acid.

The result is presented in Fig. 2. It can be seen from Fig. 2 and Table 1 that the nucleic acid sample from the patient contains both HPV type 16 and 45 nucleic acids. Example 2 A nucleic acid was detected from a nucleic acid sample extracted from a second patient presumably HPV-infected and suffering from cervical cancer, and a coupling reaction between the nucleic acid and the DNA probe was performed in the same manner as described in Example 1. Then, the result of the coupling reaction was read to measure the degree of the coupling reaction between the nucleic acid and the DNA probe. The result is presented in Fig. 3. It can be seen from Fig. 3 and Table 1 that the nucleic acid sample from the patient contains both HPV type 18 and 33 nucleic acids. Example 3

A nucleic acid was detected from a nucleic acid sample extracted from a third patient presumably HPV-infected and suffering from cervical cancer, and a coupling reaction between the nucleic acid and the DNA probe was performed in the same manner as described in Example 1. Then, the result of the coupling reaction was read to measure the degree of the coupling reaction between the nucleic acid and the DNA probe.

The result is presented in Fig. 4. It can be seen from Fig. 4 and Table 1 that the nucleic acid sample from the patient contains both HPV type 31 and 6 nucleic acids. Example 4

A nucleic acid was detected from a nucleic acid sample extracted from a fourth another patient presumably HPV-infected and suffering from cervical cancer and a coupling reaction between the nucleic acid and the DNA probe was performed in the same manner as described in Example 1. Then, the result of the coupling reaction was read to measure the degree of the coupling reaction between the nucleic acid and the DNA probe.

The result is presented in Fig. 5. It can be seen from Fig. 5 and Table 1 that the nucleic acid sample from the patient contains both HPV type 6 and 11 nucleic acids. Comparative Example 1

: Comparison with the Conventional Measurement using Membrane.

(1) Various DNA probes were adhered onto the surface of a membrane.

(2) The membrane with the DNA probes was added to a nucleic acid coupling solution as shown in Fig. 6. (3) To the membrane was added a nucleic acid labeled with a radioactive isotope or a similar substance (e.g., DIG, biotin) by a PCR method or the like.

(4) The membrane was heat-denatured.

(5) After about one hour of reaction, the non-specific nucleic acids were removed with a washing solution. (6) An adequate measurement for the coupling reaction of the nucleic acid and the DNA probe was performed according to the type of the label on the washed membrane.

The present invention method can simply analyze more than 90 samples at the same time, whereas the membrane technique of Comparative Example 1 involves a complicated procedure limited on the number of samples to be tested in a simultaneous manner. Also, the required time for the testing is more than 3 hours in the membrane technique but no more than one hour in the present invention. Comparative Example 2

: Comparison with the Conventional Measurement using DNA Chip. (1) Various DNA probes were adhered onto the surface of a glass plate, as shown in Fig. 7, according to the target HPV type listed in Table 1.

(2) A nucleic acid labeled by the PCR method or the like was heat-denatured and applied to the glass plate with the DNA probes.

(3) After about 1 to 2 minutes of reaction, the non-specific nucleic acids were removed with a washing solution.

(4) The washed glass plate was read with a reader using a laser beam.

The conventional DNA chip technique of Comparative Example 2 requires about

5 to 10 minutes per a sample and 7 to 15 hours in total for analyzing all of the 90 samples, whereas the present invention method can analyze more than 90 samples at once for no more than 5 to 10 minutes because various DNA probes are adhered to each tube in 96 micro well plates.

As described above, compared to the conventional methods using a membrane or a DNA chip, the method of detecting a nucleic acid according to the present invention makes it possible to precisely analyze more than 90 nucleic acid samples in a simultaneous manner for a short time and uses a micro well plate, thereby facilitating utilization of the conventional automated testing facility such as a reader. Thus the present invention provides an efficient testing method in the aspects of time and cost.

It is to be noted that like reference numerals denote the same components in the drawings, and a detailed description of generally known function and structure of the present invention will be avoided lest it should obscure the subject matter of the present invention.

Claims

WHAT IS CLAIMED IS:

1. A method of detecting a specific nucleic acid in a nucleic acid sample, comprising the steps of: (A) adhering various DNA probes onto the surface of one tube in a micro well plate;

(B) injecting a labeled nucleic acid sample into the tube and heat-denaturing the labeled nucleic acid;

(C) causing the heat-denatured nucleic acid to be coupled to the DNA probes in the tube;

(E) measuring the label of the nucleic acid coupled to the DNA probes in the tube to determine the specific coupling result of the nucleic acid and the DNA probes.