TARGET NUCLEIC ACIDS DETECTION METHOD USING QUANTUM DOT FOR DISPERSED LIGHT

• Main Authors: LEE, YOUNG, PARK, SEUNG KYO, CHOI, SIN YOUNG
• Format: Patent
• Language: English; Korean
• Published: 07.01.2019
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS OR TEST PAPERS THEREFOR; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL ORENZYMOLOGICAL PROCESSES; ENZYMOLOGY; INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIRCHEMICAL OR PHYSICAL PROPERTIES; MEASURING; MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEICACIDS OR MICROORGANISMS; METALLURGY; MICROBIOLOGY; MUTATION OR GENETIC ENGINEERING; PHYSICS; PROCESSES OF PREPARING SUCH COMPOSITIONS; SPIRITS; TESTING; VINEGAR; WINE

The present invention relates to a target nucleic acid detection method. In detecting a target nucleic acid on the basis of fluorescent characteristics of a fluorescent dye which changes in state when a detecting probe is hybridized with the target nucleic acid, the fluorescent dye is excited by quantum dots used as a dispersed light source, thus eliminating the need for bioconjugation of the quantum dots, and enabling convenient preparation of a reagent for detecting the target nucleic acid. In addition, it is also possible to prevent a light for exciting the quantum dot and fluorescent light of the fluorescent dye from interfering each other, thus enabling accurate detection of the target nucleic acid through analyses of the fluorescent light. In particular, the target nucleic acid detection method comprises: a hybridization step of hybridizing the detecting probe having a base sequence complementary to the target nucleic acid with the target nucleic acid in an environment dispersed with quantum dots, to induce a state change of a fluorescent dye whose fluorescent characteristics change depending on the hybridization; and a detection step of exciting the dispersed quantum dots and detecting a fluorescent light of the fluorescent dye, which receives a light emitted from the quantum dots serving as the dispersed light source, so as to detect the target nucleic acid. Research relating to the present invention was funded by Fundamental Materials Development Program of South Korean Ministry of Trade, Industry and Energy (Development of PNA-nanohybrid hybridized material and molecular diagnostic system for diagnosis of cancer genes at a femtomole level, Project number: 10047831). In addition, the verification experiment in which the target nucleic acid detection method according to the present invention is applied to an actual clinical sample was conducted with a clinical specimen provided by the Catholic University of Korea Incheon St. Mary′s Hospital. 본 발명은 검출 프로브(detecting probe)가 표적 핵산에 혼성화될 시에 상태 변화하는 형광염료의 형광 특성을 기반으로 표적 핵산을 검출함에 있어, 양자점(QD :　quantum dot)을 분산광원으로 하여 형광염료를 여기시킴으로써, 양자점을 생접합(bioconjugation)하지 아니하여도 되어 표적 핵산의 검출을 위한 시약을 쉽게 제조 사용할 수 있고, 양자점을 여기시킬 빛과 형광염료의 형광 빛을 상호 간섭되지 아니하게 하며 형광 빛을 분석하여 표적 핵산을 정확하게 검출할 수 있는 표적 핵산 검출 방법에 관한 것으로서, 표적 핵산에 대해 상보적 염기서열을 갖는 검출 프로브를 양자점이 산재되어 있는 환경 하에 표적 핵산과 혼성화시켜 혼성화 여부에 따라 형광 특성이 달라지는 형광염료의 상태 변화를 유도하는 혼성화 단계; 및 산재되어 있는 양자점을 여기시킴에 따라 분산광원 역할을 하는 양자점의 방출 빛을 받는 형광염료의 형광 빛을 검출함으로써 표적 핵산을 검출하는 검출 단계; 를 포함한다. 본 발명과 관련된 연구는 산업통상자원부 핵심소재원천기술개발사업(펨토몰 수준의 암유전자 진단을 위한 PNA-나노하이브리드 융합소재 및 분자진단시스템 개발, 과제번호:10047831)의 지원을 받아 수행하였다. 또한, 본 발명에 따른 표적 핵산 검출 방법을 실제 임상샘플에 적용한 검증 실험은 인천성모병원 인체유래물질 자원은행에서 임상 검체를 제공받아 수행하였다.