표제지
목차
ABSTRACT 9
제1장 서론 11
제1절 진단에서 PCR의 사용 및 제한 사항 11
제2절 등온 핵산 증폭과 LAMP 13
제3절 기존 저온 LAMP의 제한 사항 16
제4절 연구 목적 18
제2장 실험 재료 및 방법 19
제1절 실험 재료 19
제2절 저온 LAMP의 반응 조성 최적화 25
제3절 저온 LAMP의 DNA probe 최적화 25
제4절 기존 LAMP 반응 26
제5절 저온 LAMP 반응 26
제6절 Heat block 에서 저온 LAMP 27
제7절 miR-21 검출을 위한 저온 LAMP 27
제3장 결과 및 고찰 28
제1절 저온 LAMP 28
1. 저온 LAMP의 개략도 28
2. 반응 조건 최적화 30
3. SLP 각 부분의 길이 최적화 35
4. Primer의 길이 및 농도 최적화 37
5. 기존 LAMP 와의 비교 39
6. 민감도 분석 43
제2절 일반적인 heat block 에서 분석 검증 45
제3절 miRNA 검출을 위한 저온 LAMP의 응용 47
제4장 결론 49
참고문헌 50
국문초록 56
Table 1. Sequences of oligonucleotides used in this study. Blue: left loop (LL); yellow: left stem (LS); black: center (C); red: right stem (RS); green: right loop (RL). The underlined and non-underlined sequences in the same color can hybridize.... 20
Table 2. Comparing conventional and low-temperature LAMPs in terms of reaction conditions, and SLP length. 41
Figure 1. Scheme of the polymerase chain reaction. 12
Figure 2. Schematic representation of the LAMP mechanism. 15
Figure 3. Scheme of a phosphorothioated loop-mediated isothermal amplification. 17
Figure 4. Schematic of a low-temperature LAMP operating at 37°C, using SLP as a model DNA template. LAMP (loop-mediated isothermal amplification);... 29
Figure 5. Selection of enzymes and buffers for low-temperature LAMP. (a) Tt values and (b) amplification curves for different combinations of DNA... 32
Figure 6. Contour plots of Tt value as a function of MgSO₄ and dNTPs concentrations. The concentration of SLP (C22) used was 5 pM.... 33
Figure 7. Optimization of reaction conditions for low-temperature LAMP. (a) Tt values for various concentrations of MgSO₄ and dNTPs. The concentration... 34
Figure 8. Optimization of the length of each part of the SLP for low-temperature LAMP. Tt values for different lengths of the (a) center (C), (b)... 36
Figure 9. Optimization of hybridization length between the primers and SLP. (a) Tt values for different combinations of forward inner primer (FIP) and... 38
Figure 10. Comparison of conventional and low-temperature LAMPs. Schematic illustrating the reaction results of (a) the conventional method and... 42
Figure 11. Sensitivity of low-temperature LAMP. (a) The amplification curve of low-temperature LAMP at various concentrations of SLP (RL15). (b)... 44
Figure 12. Fluorescence images taken at 5 minute intervals in a normal heat block. NTC indicates no template control. The concentration of SLP (RL15)... 46
Figure 13. Detection of miR-21 using low-temperature LAMP with a ligation reaction. (a) Schematic of DNA ligation catalyzed by SplintR ligase for... 48