KR20010016720A - Experimental method of execution experiment of DNA conjunction bonding hybridization in tube of micro well plate - Google Patents

Experimental method of execution experiment of DNA conjunction bonding hybridization in tube of micro well plate Download PDF

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KR20010016720A
KR20010016720A KR1019990031754A KR19990031754A KR20010016720A KR 20010016720 A KR20010016720 A KR 20010016720A KR 1019990031754 A KR1019990031754 A KR 1019990031754A KR 19990031754 A KR19990031754 A KR 19990031754A KR 20010016720 A KR20010016720 A KR 20010016720A
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김희태
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    • C12Q1/708—Specific hybridization probes for papilloma

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Abstract

PURPOSE: A method for detecting specific nucleic acids from a nucleic acid sample is provided which uses a micro well plate having various DNA probes in order to treat several samples at the same time. CONSTITUTION: A method for detecting specific nucleic acids from a nucleic acid sample is characterized by the next steps of: i) attaching various, specific DNA probes into a tube of a micro well plate; ii) adding nucleic acids to be measured into the tube and heating the nucleic acids for degeneration; iii) reacting the nucleic acids degenerated during step (ii) with DNA probes already attached on the tube; iv) washing non-specific nucleic acids not having reacted in step (ii) to remove the nucleic acids; and v) measuring the results of a reaction between the nucleic acids of step (ii) and DNA probes in the tube.

Description

여러 종류의 특이 핵산 탐침자( 이하 디엔에이 프로브 )를 마이크로 웰 플레이트의 한 튜브안에 함께 부착하여 핵산 결합 실험을 수행하는 실험기법.{Experimental method of execution experiment of DNA conjunction bonding hybridization in tube of micro well plate}Experimental method of execution experiment of DNA conjugation hybridization in tube of micro well plate by attaching several types of specific nucleic acid probes (hereinafter referred to as DNA probes) together in a tube of a microwell plate.

본 발명은 여러 종류의 DNA Probe를 micro well plate의 한 개의 tube 표면에 함께 부착하여 본 Tube내에서 부착된 다양한 Probe와 핵산 시료사이의 핵산 결합 반응을 실시하고 이를 Laser scanner등의 판독기구를 활용하여 실험 결과를 확인하는 실험기법이다.The present invention attaches several kinds of DNA probes together to the surface of one tube of a micro well plate to perform nucleic acid binding reaction between various probes and nucleic acid samples attached in the tube and by using a reading device such as a laser scanner. Experimental technique to check the experimental results.

그리고 본 기법을 활용할 경우 기존에 PCR과 Membrane 사용법으로 주로 시험하였던 HLA Typing이나 HPV Typing 그리고 암 유전자 확인 등의 실험시 Membrane 이나 Glass Plate( 또는 Plastic Plate )를 대신하여 사용할 수 있다.In addition, this technique can be used in place of Membrane or Glass Plate (or Plastic Plate) in experiments such as HLA Typing, HPV Typing, and cancer gene identification, which were previously tested mainly using PCR and Membrane.

기존의 기법은 DNA Probe를 효소 적인 방법으로 micro well plate 표면에 부착하거나 membrane또는 Glass Plate에 DNA probe를 부착하는 기법들이 주로 활용되고 있다. 각 방법의 특징과 단점은 아래와 같다.Conventional techniques are mainly used to attach DNA probes to the surface of micro well plates enzymatically or to attach DNA probes to membrane or glass plates. The characteristics and disadvantages of each method are as follows.

첫 번째 기법인 Micro well plate를 활용한 기법은 Micro well plate 표면에 streptavidin을 coating하여 준후 여기에 biotin이 부착된 DNA Probe를 처리하여 반응시킨 뒤 여기에 확인하고자 하는 핵산을 가열 또는 화학적인 방법으로 변성시켜 주입하여 결합반응을 시키고 효소 적인 방법 등을 활용하여 결합 반응의 결과를 확인하는 기법이다.The first technique, which uses a micro well plate, is coated with streptavidin on the surface of the micro well plate, and then treated with a biotin-attached DNA probe, followed by reaction to denature the nucleic acid to be identified by heating or chemical methods. It is a technique to check the result of the binding reaction by injecting it to make a binding reaction and enzymatic method.

단점은 micro well Plate의 표면에 기본적으로 1종류 이상의 핵산 Probe를 부착할 수 없으며 확인 과정도 효소 적인 확인 법이 주로 사용되기에 많은 시간이(최소 2시간 )소요 된다.Disadvantage is that it is not possible to attach more than one type of nucleic acid probe to the surface of the micro well plate, and the identification process takes a lot of time (at least 2 hours) because enzymatic identification is mainly used.

두 번째 기법인 Membrane을 활용한 기법은 Membrane 표면에 여러 종류의 DNA Probe를 부착하여 준후 여기에 확인하고자 하는 DNA를 화학적인 방법으로 변성시켜준후 이를 준비된 membrane에 특정한 핵산 결합용액과 함께 가하여준 뒤 온도를 일정하게 유지시켜 반응시킨 뒤 다양한 표식 확인 방법을 활용하여 핵산 결합 여부를 확인하는 기법이다.The second technique, using Membrane, attaches various types of DNA probes to the surface of Membrane, denatures the DNA to be identified by chemical method, and adds it with the specific nucleic acid binding solution to the prepared membrane. It is a technique to check whether the nucleic acid binding by using a variety of marker identification method after keeping the reaction constant.

단점은 Membrane을 사용하는 기법은 기본적으로 시험과정이 긴 시간(최소 2시간 )이 소요된다.The disadvantage is that the technique using Membrane basically takes a long time (at least 2 hours).

세 번째 기법인 Glass Plate표면에 DNA Probe를 부착하여 활용하는 기법( 이하 DNA Chip )은 glass 표면에 여러 종류의 DNA Probe를 부착하여 준 뒤 여기에 확인하고자 하는 변성된 핵산을 주입하여 반응시킨 뒤 이를 세척하고 핵산 결합 여부와 정도를 laser beam을 활용하여 읽어 내는 방법이다.The third technique, the DNA chip attached to the surface of the glass plate (hereinafter referred to as DNA Chip), attaches various types of DNA probes to the glass surface, injects the denatured nucleic acid to be confirmed, and reacts them. It is a method of washing and reading whether or not to bind nucleic acid using a laser beam.

단점은 1개의 Glass plate에 여러 개의 probe를 부착할 수 있지만 한번에 1개의 Plate에 하나의 시료만을 처리하여야 하기에 동시에 여러 시료를 처리 하는 것은 불가능하다. 그리고 Probe가 부착된 1개의 Glass Plate는 핵산 결합 시험에 사용될 수 있는 회수에 제한이 있다( 약 10회 이하 ).The disadvantage is that several probes can be attached to one glass plate, but it is impossible to process several samples at the same time because only one sample needs to be processed on one plate at a time. And one glass plate with a probe attached is limited in the number of times it can be used for nucleic acid binding test (about 10 times or less).

기존의 기법들은 각각의 불편한 점을 내포하고 있기에 새로운 기법의 발명이 요구되었으며 본 발명은 이러한 요구에 부합하는 경제적이며 효율적인 실험 기법이다.Since the existing techniques have their respective inconveniences, the invention of a new technique is required and the present invention is an economical and efficient experimental technique that meets these requirements.

따라서 본발명은 기존의 각 기법들이 가지고 있던 한계성과 단점, 그리고 개별 검출의 비효율성을 개선하기 위하여 개발 되었다.Therefore, the present invention was developed to improve the limitations and disadvantages of the existing techniques and the inefficiency of individual detection.

본 기법을 활용할 경우 기존에 PCR과 Membrane 사용법으로 주로 시험하였던 HLA Typing이나 HPV Typing 그리고 암 유전자 확인 등의 실험시 Membrane이나 Glass Plate( 또는 Plastic Plate )를 대신하여 사용할 수 있다.When using this technique, it can be used in place of Membrane or Glass Plate (or Plastic Plate) in experiments such as HLA Typing, HPV Typing, and cancer gene identification, which were mainly tested by PCR and Membrane.

(1) 동시에 여러 종류의 DNA Probe를 작은 Micro well Plate의 Tube well 표면에 부착하여 주는 기술의 개발.(1) Development of technology that attaches several kinds of DNA probes to tube well surface of small micro well plate at the same time.

(2) 상기 (1)에서 준비된 Tube well에 핵산시료를 가하여 준후 이를 열변성과 핵산 특이 결합을 시켜주는 기술을 개발한다.(2) After adding a nucleic acid sample to the tube well prepared in the above (1), develop a technology for thermal degradation and nucleic acid specific binding.

(3) 상기 (2)에서와 같이 각각의 micro plate 의 Tube well에 각각의 핵산 시료를 반응을 시켜 준 뒤 동시에 여러 well들의 핵산 결합여부를 판독 해낼 수 있는 기술을 개발한다.(3) As described in (2) above, each nucleic acid sample is reacted to the tube well of each micro plate, and then a technology capable of simultaneously reading the nucleic acid binding of several wells is developed.

(가). Micro well Plate는 96개의 작은 Tube well로 이루어져 있다.(end). Micro well plates consist of 96 small tube wells.

(나). Micro well Plate내의 모든 96개 tube well에는 아래의 그림 1.과 표 1.처럼 DNA Probe가 부착 고정된다.(I). The DNA probes are attached to all 96 tube wells in the micro well plate as shown in Figure 1 and Table 1.

(다). 본 기법의 활용예를 설명하기 위하여 HPV 핵산 Typing을 활용하였다.(All). HPV nucleic acid typing was used to illustrate the use of this technique.

그림 1. DNA Probe가 부착된 Micro well plate의 Tube측면과 바닥면.Figure 1. Tube side and bottom of Micro well plate with DNA probe attached.

표 1. 부착된 Probe의 위치별 결합 대상 HPV typeTable 1. Joining target HPV type by position of attached probe

[실시예 1]Example 1

(1) HPV에 감염되어 자궁경부암 발병이 의심되는 환자의 시료로부터 핵산을 추출한다.(1) A nucleic acid is extracted from a sample of a patient infected with HPV and suspected of developing cervical cancer.

(2) 상기 (1)의 핵산을 표 1.의 Probe의 서열들이 중간에 위치하는 HPV Primer를 사용하여 PCR을 수행한다.(2) PCR is performed using the HPV Primer in which the nucleic acids of (1) are located in the sequence of the probes of Table 1.

(3) 상기 (2)의 PCR반응 결과물( 증폭된 핵산 조각들 )을 그림 1.과 표 1.에서처럼 HPV의 각 Type에 특이적인 DNA Probe가 부착된 Micro well plate의 한 개의 Tube에 결합반응용액과 함께 주입하여 준다.(3) The PCR reaction product (amplified nucleic acid fragments) of (2) is bound to one tube of micro well plate attached with DNA probes specific to each type of HPV as shown in Figure 1. and Table 1. Inject with

(4) 상기 (3)의 Tube를 가열하여 PCR 결과물을 열변성시킨 뒤 온도를 낯추어 Tube의 표면에 고정된 Probe들과 PCR 결과물이 결합반응할 수 있도록 하여준다.(4) Heat the tube of (3) to heat-denature the PCR product and reduce the temperature so that the probes fixed on the surface of the tube and the PCR product can react.

(5) 상기 (4)의 핵산 결합반응 과정에서 결합되지 않은 비특이 핵산을 세척 용액을 사용하여 세척, 제거하고 판독기로 핵산 결합반응 결과를 읽어낸다.(5) The nonspecific nucleic acid not bound in the nucleic acid binding reaction of (4) is washed and removed using a washing solution, and the result of the nucleic acid binding reaction is read with a reader.

그림 2. 핵산 결합 실험이후의 Tube well.Figure 2. Tube well after nucleic acid binding experiment.

시험 결과 : 본 환자의 핵산에는 HPV type 16과 45의 핵산이 함께 존재하였다.Test Results: Nucleic acid of HPV type 16 and 45 was present in the nucleic acid.

[실시예 2]Example 2

(1) HPV에 감염되어 자궁경부암 발병이 의심되는 환자의 시료로부터 핵산을 추출한다.(1) A nucleic acid is extracted from a sample of a patient infected with HPV and suspected of developing cervical cancer.

(2) 상기 (1)의 핵산을 표 1.의 Probe의 서열들이 중간에 위치하는 HPV Primer를 사용하여 PCR을 수행한다.(2) PCR is performed using the HPV Primer in which the nucleic acids of (1) are located in the sequence of the probes of Table 1.

(3) 상기 (2)의 PCR반응 결과물( 증폭된 핵산 조각들 )을 그림 1.과 표 1. 에서처럼 HPV의 각 Type에 특이적인 DNA Probe가 부착된 Micro well plate의 한 개의 Tube에 결합반응용액과 함께 주입하여 준다.(3) The PCR reaction product (amplified nucleic acid fragments) of (2) is bound to one tube of micro well plate attached with DNA probes specific to each type of HPV as shown in Figure 1. and Table 1. Inject with

(4) 상기 (3)의 Tube를 가열하여 PCR 결과물을 열변성시킨 뒤 온도를 낯추어 Tube의 표면에 고정된 Probe들과 PCR 결과물이 결합반응할 수 있도록 하여준다.(4) Heat the tube of (3) to heat-denature the PCR product and reduce the temperature so that the probes fixed on the surface of the tube and the PCR product can react.

(5) 상기 (4)의 핵산 결합반응 과정에서 결합되지 않은 비특이 핵산을 세척용액을 사용하여 세척, 제거하고 판독기로 핵산 결합반응 결과를 읽어낸다.(5) The nonspecific nucleic acid not bound in the nucleic acid binding reaction of (4) is washed and removed using a washing solution, and the result of the nucleic acid binding reaction is read with a reader.

그림 3. 핵산 결합 실험이후의 Tube well.Figure 3. Tube well after nucleic acid binding experiment.

시험 결과 : 본 환자의 핵산에는 HPV type 18과 33의 핵산이 함께 존재하였다.Test Results: Nucleic acid of HPV type 18 and 33 were present in the nucleic acid.

[실시예 3]Example 3

(1) HPV에 감염되어 자궁경부암 발병이 의심되는 환자의 시료로부터 핵산을 추출한다.(1) A nucleic acid is extracted from a sample of a patient infected with HPV and suspected of developing cervical cancer.

(2) 상기 (1)의 핵산을 표 1.의 Probe의 서열들이 중간에 위치하는 HPV Primer를 사용하여 PCR을 수행한다.(2) PCR is performed using the HPV Primer in which the nucleic acids of (1) are located in the sequence of the probes of Table 1.

(3) 상기 (2)의 PCR반응 결과물( 증폭된 핵산 조각들 )을 그림 1.과 표 1. 에서처럼 HPV의 각 Type에 특이적인 DNA Probe가 부착된 Micro well plate의 한 개의 Tube에 결합반응용액과 함께 주입하여 준다.(3) The PCR reaction product (amplified nucleic acid fragments) of (2) is bound to one tube of micro well plate attached with DNA probes specific to each type of HPV as shown in Figure 1. and Table 1. Inject with

(4) 상기 (3)의 Tube를 가열하여 PCR 결과물을 열변성시킨 뒤 온도를 낯추어 Tube의 표면에 고정된 Probe들과 PCR 결과물이 결합반응할 수 있도록 하여준다.(4) Heat the tube of (3) to heat-denature the PCR product and reduce the temperature so that the probes fixed on the surface of the tube and the PCR product can react.

(5) 상기 (4)의 핵산 결합반응 과정에서 결합되지 않은 비특이 핵산을 세척 용액을 사용하여 세척, 제거하고 판독기로 핵산 결합반응 결과를 읽어낸다.(5) The nonspecific nucleic acid not bound in the nucleic acid binding reaction of (4) is washed and removed using a washing solution, and the result of the nucleic acid binding reaction is read with a reader.

그림 4. 핵산 결합 실험이후의 Tube well.Figure 4. Tube well after nucleic acid binding experiment.

시험 결과 : 본 환자의 핵산에는 HPV type 31과 6의 핵산이 함께 존재하였다.Test result: Nucleic acid of HPV type 31 and 6 were present in the nucleic acid of this patient.

[실시예 4]Example 4

(1) HPV에 감염되어 자궁경부암 발병이 의심되는 환자의 시료로부터 핵산을 추출한다.(1) A nucleic acid is extracted from a sample of a patient infected with HPV and suspected of developing cervical cancer.

(2) 상기 (1)의 핵산을 표 1.의 Probe의 서열들이 중간에 위치하는 HPV Primer를 사용하여 PCR을 수행한다.(2) PCR is performed using the HPV Primer in which the nucleic acids of (1) are located in the sequence of the probes of Table 1.

(3) 상기 (2)의 PCR반응 결과물( 증폭된 핵산 조각들 )을 그림 1.과 표 1.에서처럼 HPV의 각 Type에 특이적인 DNA Probe가 부착된 Micro well plate의 한 개의 Tube에 결합반응용액과 함께 주입하여 준다.(3) The PCR reaction product (amplified nucleic acid fragments) of (2) is bound to one tube of micro well plate attached with DNA probes specific to each type of HPV as shown in Figure 1. and Table 1. Inject with

(4) 상기 (3)의 Tube를 가열하여 PCR 결과물을 열변성시킨 뒤 온도를 낮추어 Tube의 표면에 고정된 Probe들과 PCR 결과물이 결합반응할 수 있도록 하여준다.(4) Heat the tube of (3) to denature the PCR product and lower the temperature so that the probes fixed on the surface of the tube and the PCR product can react.

(5) 상기 (4)의 핵산 결합반응 과정에서 결합되지 않은 비특이 핵산을 세척 용액을 사용하여 세척, 제거하고 판독기로 핵산 결합반응 결과를 읽어낸다.(5) The nonspecific nucleic acid not bound in the nucleic acid binding reaction of (4) is washed and removed using a washing solution, and the result of the nucleic acid binding reaction is read with a reader.

그림 5. 핵산 결합 실험이후의 Tube well.Figure 5. Tube well after nucleic acid binding experiment.

시험 결과 : 본 환자의 핵산에는 HPV type 6과 11의 핵산이 함께 존재하였다.Test result: Nucleic acid of HPV type 6 and 11 coexisted in the nucleic acid of this patient.

[비교예 1]Comparative Example 1

본 발명의 기법과 기존의 Membrane 사용 시험법과의 비교.Comparison of the techniques of the present invention with existing Membrane test methods.

(1) 핵산결합반응을 시켜보고자하는 여러 종류의 DNA Probe를 Membrane에 부착하여 고정 시켜준다.(1) It attaches and fixes various kinds of DNA Probes to Membrane to perform nucleic acid binding reaction.

그림 6. DNA Probe가 부착된 Membrane.Figure 6. Membrane with DNA Probe attached.

(2) 위의 그림 6.과 같이 DNA Probe가 부착, 고정된 Membrane을 결합반응 용액에 넣어 활성화시켜준다.(2) Activate the Membrane with DNA Probe attached and immobilized in the binding solution as shown in Figure 6.

(3) PCR등의 방법을 통해 방사선 동위원소나 이와 유사하게 사용되는 표식 인자( DIG, Biotin )를 부착한 핵산을 준비한다.(3) Prepare a nucleic acid to which a radioisotope or a similarly used labeling factor (DIG, Biotin) is attached by a method such as PCR.

(4) 상기 (3)에서 준비된 핵산을 열변성 시켜주어 핵산간의 특이결합이 이루어질 수 있도록 준비하여 주입하여 준다.(4) Prepare and inject the nucleic acid prepared in the above (3) to denature the nucleic acid to perform specific binding.

(5) 1시간 정도 반응을 시켜준후 세척 용액을 사용하여 결합반응을 이루지 못한 상기(3)의 표식된 핵산을 제거하여 준다.(5) After reacting for about 1 hour, remove the labeled nucleic acid in the above (3) by using the washing solution.

(6) 상기 (5)에서 세척된 Membrane을 핵산에 표식된 방법에 따라 각기 다른 판독 방법을 사용하여 판독한다.(6) The membrane washed in the above (5) is read using different reading methods according to the method labeled on the nucleic acid.

(가) 동위원소표식법 : 세척된 Membrane를 차광된 암실에서 X-ray film에 부착하고 이를 영하 70℃에 16시간 이상 보관하였다가 X-ray film를 떼어내어 이를 현상하여 시험 결과를 판독한다.(A) Isotope labeling method: Attach the washed membrane to the X-ray film in the shaded dark room, store it at minus 70 ℃ for more than 16 hours, remove the X-ray film, develop it, and read the test result.

(나) DIG, Biotin 표식법 : 이두가지 물질은 핵산표식에 사용되는 대표적인 물질이며 이들과 특이적으로 결합하는 항체나 단백질에 발색 또는 형광 효소를 부착하여 상기(6)의 세척된 Membrane에 가하여 핵산에 부착된 DIG, Biotin에 결합 시켜주고 여기에 발색 시약 등을 주입하여 반응시켜주고 이를 판독한다.(B) DIG, Biotin Labeling: These two substances are representative of nucleic acids and are added to the washed membrane of (6) by attaching color or fluorescent enzymes to antibodies or proteins that specifically bind them. It is bound to DIG and Biotin attached to it, and it is reacted by injecting color development reagents.

비교 결과 : 본 발명의 기법은 1번의 시험시 최고 90개의 시료를 분석할 수 있다.이에 비하여 기존 Membrane 기법은 과정의 복잡함에의 하여 동시 시험 가능 시료의 수량이 제한된다 그리고 시험 시간도 본 발명의 경우 1시간 이하이나 기존 Membrane기법은 3시간 이상이 소요된다.Comparative results: The technique of the present invention can analyze up to 90 samples in one test. In contrast, the conventional Membrane technique limits the number of samples that can be tested simultaneously due to the complexity of the process. In the case of less than 1 hour, the existing Membrane technique takes more than 3 hours.

[비교예 2]Comparative Example 2

본 발명의 기법과 기존의 DNA Chip사용 시험법과의 비교.Comparison of the technique of the present invention with the existing DNA Chip test method.

(1) 핵산결합반응을 시켜보고자하는 여러 종류의 DNA Probe를 표 1.의 위치별 결합대상 HPV type에 준하여 Glass Plate 표면에 부착하여 고정 시켜준다.(1) The DNA probes of various types to be subjected to the nucleic acid binding reaction are attached to the glass plate surface and fixed according to the binding target HPV type in Table 1.

그림 6. DNA Probe가 부착된 Glass Plate.Figure 6. Glass Plate with DNA Probe Attached.

(2) 위의 그림 6.과 같이 DNA Probe가 부착, 고정된 Glass Plate에 PCR등의 방법을 통해 얻어진 시험대상 핵산을 열변성 시켜준 뒤 이를 도포 하여 준다.(2) Heat-denature the test target nucleic acid obtained by PCR method on glass plate to which DNA probe is attached and fixed as shown in Figure 6.

(3) 1-2분 정도 반응을 시켜준후 세척 용액을 사용하여 결합반응을 이루지 못한 핵산을 제거하여 준다.(3) After reacting for 1-2 minutes, remove the nucleic acid that did not bind by using the washing solution.

(4) 세척된 Glass Plate는 laser beam을 사용하는 판독기를 사용하여 판독한다.(4) Clean the glass plate using a reader using a laser beam.

(5) 판독이 이루어진 이후 Glass Plate는 다음 시험 대상 시료의 처리를 위하여 가열과 세척 등의 방법에 의하여 부착된 핵산을 세척, 제거한다.(5) After the reading is made, the glass plate washes and removes the attached nucleic acid by heating or washing method for processing the next test sample.

비교 결과 :Comparison result:

(1) 본 발명의 기법은 96개의 Micro tube well에 DNA Probe를 동일하게 부착, 고정하여 사용하기에 1번의 시험시 최고 90개의 시료를 분석할 수 있으며 소요시간은 약 5-10분이다. 이에 비하여 기존 DNA Chip 기법은 위와 같은 방법과 순서로 시험이 진행되기에 1개의 시료당 5-10분 정도의 시간이 소요되며 90개의 시료를 분석할 경우 7-15시간이 소요된다.(1) In the technique of the present invention, DNA probes can be attached and fixed to 96 micro tube wells in the same manner, and up to 90 samples can be analyzed in one test. The time required is about 5-10 minutes. On the other hand, the existing DNA Chip technique takes about 5-10 minutes for each sample and 7-15 hours when analyzing 90 samples.

(2) DNA Chip의 경우 핵산 결합 시험의 회수가 반복되어질수록 부착된 DNA Probe의 양이 감소하여 1개의 Glass Plate가 분석에 활용될 수 있는 시료의 수량이 5-10개로 제한된다. 이러한 이유로 다량의 핵산 시료가 분석 되어야할 경우에는 필요에 따라 Glass Plate를 교체하여 주어야 한다.(2) In the case of DNA chip, the number of DNA probes attached decreases as the number of nucleic acid binding test is repeated, limiting the number of samples that one glass plate can be used for analysis. For this reason, if a large amount of nucleic acid samples should be analyzed, the glass plate should be replaced as necessary.

Claims (1)

(가) 여러 종류의 특이 DNA Probe를 micro well plate의 한 개의 Tube안에 함께 부착하고(A) Attach several kinds of specific DNA probes together in one tube of a micro well plate. (나) 상기 (가)단계의 여러 종류의 Probe가 부착된 한 개의 Tube안에 시험하고자 하는 핵산을 주입하여 열변성 시켜주고,(B) Inject the nucleic acid to be tested into one tube to which various types of probes are attached in step (a) and thermally denature it. (다) 열변성된 시험대상 핵산과 Tube내에 이미 부착 고정된 DNA Probe 사이의 다양한 핵산 결합반응이 이루어지도록 하고,(C) allow various nucleic acid binding reactions between the denatured test nucleic acid and the DNA probes already attached and immobilized in the tube; (라) 상기 (나)단계에서 결합반응을 이루지 않은 비특이 핵산을 세척용액을 사용하여 세척하여 제거하고,(D) non-specific nucleic acid that did not form a binding reaction in step (b) by washing with a washing solution to remove (마) 상기 (나)단계에서 이루어진 시험대상 핵산과 Tube내 DNA Probe간의 특이 핵산 결합반응의 결과를 판독하는 실험기법.(E) Experimental technique for reading the result of specific nucleic acid binding reaction between the nucleic acid under test in step (b) and the DNA probe in the tube.
KR1019990031754A 1999-08-02 1999-08-02 Experimental method of execution experiment of DNA conjunction bonding hybridization in tube of micro well plate KR20010016720A (en)

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KR100382703B1 (en) * 2000-03-15 2003-05-09 주식회사 바이오메드랩 diagnosis kit for genotyping of Human Papillomavirus and manufacturing method for thereof
KR100437626B1 (en) * 2001-06-21 2004-06-26 주식회사 마이진 Methods for Dectecting Human Papillomavirus and Detection Kit Thereof
KR100453207B1 (en) * 2001-11-19 2004-10-15 킹 카 푸드 인더스트리얼 콤파니 리미티드 Method and detector for detecting and identifying subtypes of human papilloma viruses

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SG96246A1 (en) * 2001-10-18 2003-05-23 King Car Food Ind Co Ltd Method and detector for identifying subtypes of human papilloma viruses

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US4358535A (en) * 1980-12-08 1982-11-09 Board Of Regents Of The University Of Washington Specific DNA probes in diagnostic microbiology
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KR100382703B1 (en) * 2000-03-15 2003-05-09 주식회사 바이오메드랩 diagnosis kit for genotyping of Human Papillomavirus and manufacturing method for thereof
KR100437626B1 (en) * 2001-06-21 2004-06-26 주식회사 마이진 Methods for Dectecting Human Papillomavirus and Detection Kit Thereof
KR100453207B1 (en) * 2001-11-19 2004-10-15 킹 카 푸드 인더스트리얼 콤파니 리미티드 Method and detector for detecting and identifying subtypes of human papilloma viruses

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