Title Page
Contents
국문 초록 9
ABSTRACT 11
I. Introduction 13
1. Background 16
A. DNA Methylation 16
B. Polydiactylene 17
2. Research Objective 18
II. Materials and Methods 19
1. Preparation of 5-methylcytosine antibody-decorated diacetylene monomer (5-mC Ab-PCDA) 19
A. Materials 19
B. Synthesis of a NHS-terminated diacetylene monomer (PCDA-NHS) 19
C. Preparation of 5-methylcytosine antibody-decorated diacetylene monomer (5-mC Ab-PCDA) 20
2. Preparation of 5-methylcytosine antibody-functionalized PDA (5-mC Ab-PDA) 21
3. Methylation specific polymerase chain reaction (PCR) 22
A. Culture of cancer cells (HT-29) and normal cells (CCD-18Co) 22
B. DNA extraction and bisulfite treatment 22
C. Methylation specific PCR 22
4. Characterization of 5-mC Ab-PDA 24
5. Colorimetric detection method of methylated DNA 25
III. Results 26
1. ¹H NMR spectra of NHS-terminated diacetylene monomer(PCDA-NHS) 26
2. Characterization of 5-mC Ab-PDA 27
3. Methylated specific PCR 30
4. Colorimetric detection of methylated DNA 32
5. Specific detection of methylated DNA 34
IV. Discussion 36
V. Conclusion 38
VI. Reference 39
Figure 1. (a) Synthesis steps of 5-methylcytosine antibody-functionalized polydiacetylene (5-mC Ab-PDA) and (b) procedure for colorimetric... 15
Figure 2. DNA methylation occurs by conversion to 5-methylcytosine by adding a methyl group to the carbon-5 position of the cytosine ring. 16
Figure 3. Figure 3. Schematic diagram of polydiacetylene synthesis and its blue to red color transition upon stimuli. 17
Figure 4. ¹H-NMR spectrum of PCDA-NHS in CDCl₃. ¹H NMR (500 MHz, CDCl₃): δ=0.881 (t, 3H, 6), 1.311-1.384 (br, 26H, 7, 8), 1.554 (tt, 2H,... 26
Figure 5. UV-vis spectra of (a) PDA and (b) 5-mC Ab-PDA. 28
Figure 6. Zeta potential of 5-mC Ab (green), PDA (blue), and 5-mC Ab-PDA (orange). 28
Figure 7. SEM image of (a) PDA and (b) 5-mC Ab-PDA. 29
Figure 8. 1.5% Agarose gel electrophoresis image of methylated and unmethylated DNA (Lane 1: amplified DNA via dNTP mix from HT-29;... 31
Figure 9. (a) UV-vis spectra (Inset: Photographs of each sample) and (b) CR (%) values of 5-mC Ab-PDA after addition of various... 33
Figure 10. A calibration curve between concentration of methylated DNA and CR (%) values of 5-mC Ab-PDA. 33
Figure 11. CR (%) values of 5-mC Ab-PDA after the addition of amplified DNA (50 nM) via dNTP and 5-mC dNTP mix from HT-29 and... 35