한국보건산업진흥원 Korea Health Industry Development Institute
등록번호
TRKO201400018321
DB 구축일자
2014-11-29
초록▼
IV. 연구개발결과 ITS doning을 행하여 ITS 가 밝혀지지 않은 균주에서 PCR 및 직접염기서열 결정법으로 밝혔으며, 비결핵마이코박테리아검색을 위한 16S rRNA와 ITS의 보존적 지역에서 마이코박테리아 특이적 probe를 고안하고 비보존 지역에서 각종 특이적 probe 고안하였다. 그리고, DNA chip에 사용할 probe를 positive charged membrane을 이용한 역교잡법으로 비교실험을 함으로써 Membrane based probe test 및 설계하였으며, 개발된 DNA chip 이 임상에 이용
IV. 연구개발결과 ITS doning을 행하여 ITS 가 밝혀지지 않은 균주에서 PCR 및 직접염기서열 결정법으로 밝혔으며, 비결핵마이코박테리아검색을 위한 16S rRNA와 ITS의 보존적 지역에서 마이코박테리아 특이적 probe를 고안하고 비보존 지역에서 각종 특이적 probe 고안하였다. 그리고, DNA chip에 사용할 probe를 positive charged membrane을 이용한 역교잡법으로 비교실험을 함으로써 Membrane based probe test 및 설계하였으며, 개발된 DNA chip 이 임상에 이용되기 전에 충분한 임상 검체로 확인 및 이상시 교정을 실시하였다. 또한 차년도 연구인 주요 병원성 세균의 유전형 동정 probe 고안을 위하여 유전자 database 조사연구를 하였다. 미리 silylated된 유리를 사용하여 기존의 poly-L-lysine이 coating 된 유리보다 oligonucleotide들이 더 자유롭게 probe와 결합할 수 있는 방법을 개발하여 DNA chip 기반 기술을 확보하였으며, 이를 검정하기 위해 Laser confocal scanner를 이용하지 않고 실험용 oligonucleotide array를 96 well plate를 이용하여 개발하였다. 좁은 면적에 많은 수의 DNA 단편을 심기 위해서 3축 로봇 (robot) 을 제작하였으며, 정밀한 위치제어 알고리즘을 개발하였다. 로봇 동작에 있어서 진동을 방지하고 생산성을 향상시킬 수 있도록 이송 속도와 부가 장치의 배치 등을 최적화(optimization) 하였으며, Spotting head는 유체의 표면 장력을 이용하므로 정밀한 가공이 필요하며, 생산성을 위해서는 적절한 경도를 유지해야한다. 이같은 설계조건들을 만족시킬 수 있도록 SUS304 스테인리스강을 이용하여 제작하였다. 세척 장치는 spotting head의 청결도를 유지시킬 수 있도록 물 분사기, 진공 흡입기를 이용하여 수차려 반복시킨다. Microarrayer의 개별 모듈간의 통합관리 및 제어는 컴퓨터를 이용하여 행해지며, 이를 위한 소프트웨어를 개발하였다.
Abstract▼
DNA chip(or Microarray) technology is one of several developing approaches to comparatively analyze genome-wide patterns of mRNA expression. For mRNA expression studies, the ultimate goal is to develop arrays which contain everγ gene in a genome against which mRNA expression levels can be quantitati
DNA chip(or Microarray) technology is one of several developing approaches to comparatively analyze genome-wide patterns of mRNA expression. For mRNA expression studies, the ultimate goal is to develop arrays which contain everγ gene in a genome against which mRNA expression levels can be quantitatively assessed. The technology for cDNA based microarray technology is based on an approach where cDNA clone inserts are robotically printed onto a glass slide and subsequently hybridized to two differentially fluorescently labeled probes. The probes are pools of cDNAs which are generated after isolating mRNA from cells or tissues in two states that one wishes to compare. Resulting fluorescent intensities are produced using a laser confocal fluorescent microscope, and ratio information is obtained following image processing. Finally database development and design is critical for all aspects of this project. Tuberculosis, caused by members of the Mycobacterium tuberculosis complex, is one of the most common human infectious diseases, causing three million deaths a year worldwide. Mycobacterial infections due to the M. avium-intracellulare complex(MAC) are on the increase. Reduced and compromised immune function as found in newborns, infants, and immunosuppressed individuals allows opportunistic infections caused by mycobacteria other than tuberculosis(MOTT). The increasing number of mycobacterial infections has made it clinically important to quickly identify mycobacteria at the species level. For the rapid and accurate identification of mycobacteria, molecular methods are gaining increasing importance. DNA chip has considerable potential for identification of genus and species of microorganism at the molecular level. This is new and advanced method for assessing genetic diversity on a larger scale. ITS(Internal Transcribed Spacer region) sequence which was contained conserved sequence and polymorphic region was utilized to design probe of the DNA chip. ITS sequences of clinically important mycobacteria, M. fortuitum and M. chelonae, were obtained by the cloning and sequencing. We designed 68 probes with ITS sequences from Genbank and M. fortuitum and M. chelonae which we cloned. There are mycobacteria specific probes, TB complex specific probes, MAC specific probes, M. fortuitum specific probes, M. chelonae specific probes, M. gordonae specific probes and M. terrae specific probes. MYC probes(mycobacteria specific probe) differentiated mycobacteria from other pathogenic bacteria. Species specific probes showed specificity to each mycobacterial species. They are probe MTB for TB complex, MAC for M. avium-intracellulare, FOR for M. fortuitum, GOR for M. gordonae and SCR for M. scrofluceum. Probes designed with ITS sequences of mycobacteria in this study would be used as successful probes in DNA chips for differentiation of mycobacteria. Microarrayer makes DNA chip and microarray that contain hunders to thousands of immobilized DNA probes on surface of a microscope slide. Their principal components are a computer-controlled three-axis robot and a unique pen tip assembly. The key component of the arrayer is the print-head containing pens to immobilize cDNA, genomic DNA or similar biological material on glass surface. The robot is designed to automatically collect probes from two 96-well plates with up to 12 pens at the same time. Wash station and dryer are integrated into one module. The stationary wash basins contain flowing distilled water to enhance cleaning. The dryer is controlled by computer and accomplished rapid air flow around the tips and the partial vacuum. The objective of the microarray image analysis is to exσact probe intensities or ratios at each cDNA target location, and then cross-link printed clone target information so that biologists can easily interpret the outcomes and perform further high-level analysis. However, the microarray image sources are not only from one print-mode (i.e., number of printing tips) or one hybridization method (i.e., fluorescent or radioactive probe). Nevertheless, in order to simplify the presentation of information for this section we have chosen to model the microarray images below from two fluorescent probes as our main processing input images.
목차 Contents
표지 ... 1
제출문 ... 3
요약문 ... 4
SUMMARY ... 7
목 차 ... 9
제 1 장 서론 ... 10
제 2 장 국내ㆍ외 기술개발 현황 ... 12
제 3 장 연구개발 수행내용 및 결과 ... 13
제 1 절 제 1 세부과제 : Mycobateria 및 MOTT 진단용 DNA probe의 개발 ... 13
※ AI-Helper는 부적절한 답변을 할 수 있습니다.