臺灣博碩士論文加值系統

我們利用PCR的方式複製出片段的�趁174雙股DNA，並將其插入載體pYES3/CT/LacZ上（合稱為��/Z DNA）。然後將這段DNA當作質體轉殖到大腸桿菌中進行無性繁殖。純化後的��/Z DNA於兩端分別用digoxigenin與biotin這兩種分子修飾，讓DNA能分別與用anti-digoxigenin做表面修飾的polystyrene的小球（直徑為2�慆）及用streptavidin做表面修飾的polystyrene大球（直徑為20�慆）進行接合，DNA就會連結在大球與小球之間。實驗過程中，先靜待大球沈降於載波片的表面，再使用光鉗箝住小球。藉著PZT微調載波片的位置讓我們可以移動載波片上的大球，進而改變大球與小球之間的距離，使得做為連結的雙股DNA可被延展或收縮。當雙股DNA被拉扯至一定程度的緊繃狀態，小球就會同時受到雙股DNA的彈力與光鉗彈力的作用，藉此我們量測出雙股DNA彈性係數（KDNA），它的值是一個隨著DNA長度變化的函數。函數中第一段線性範圍的KDNA，其平均值從0.95pN/�慆 到8.73pN/�慆。這可能是因為作為連結的雙股DNA數量並非固定不變，所以致使KDNA的數值會隨著不同的DNA數量變動。然而，由於DNA的數量與KDNA有著線性增加的關係，因此所量測的數據最後將可推測出單一條雙股DNA的KDNA數值。

A �趁174 double-strand DNA fragment was generated using polymerase chain reaction (PCR) and then cloned into a pYES3/CT/LacZ vector, resulting in ��/Z. Plasmid ��/Z was used as the basis to generate single DNA molecular with digoxigenin on one end and biotin on the other end. The digoxigenin-labeled end is adhered to a small polystyrene particle coated anti-digoxigenin (2�慆 in diameter) in an optical trap, while the biotinylation end is adhered to a big polystyrene particle coated streptavidin (20�慆 in diameter). We made the big particle seating on a cover glass surface which was placed on a PZT stage. Thus, with the smaller particle trapped by optical tweezers, the DNA sample can be stretched (or held loose) by moving the PZT driven translational stage on which the cover glass was mounted. When the dsDNA is stretched tightly, it is supposed there are two elastic forces affect on the small particle at the same time: the DNA and optical tweezers. We have measured the elasticity of DNA as a function of its length. In addition, the value of DNA spring constant we obtained from our experiment was a range from 0.95pN/�慆 to 8.73pN/�慆. We assume KDNA become larger with the increasing number of double-strand DNA connected to both particles. The linear relationship is supposed to exist between KDNA and the increasing number of double-strand DNA and it would lead to know the KDNA of one double-strand DNA.

Chapter .1 Introduction 5
1.1 DNA 5
1.1.1 The role DNA plays in life 5
1.1.2 DNA structure 7
1.1.3 Significance of DNA elasticity 9
1.2 Optical tweezers 10
1.2.1 History of optical tweezers 10
1.2.2 Basics of optical tweezers 10
1.2.3 Applications of optical tweezers 13

Chapter .2 Material and Methods 15
2.1 DNA preparation 15
2.1.1 Preparation of the double strand DNA containing �趁174 fragment 16
2.1.2 Preparation of the biotin-labeled DNA fragment 22
2.1.3 Labeling of the ��/Z DNA fragments with digoxigenin 23
2.1.4 Verification of the DIG-labeling and biotinylation on DNA 24
2.1.5 Ligation of the labeled DNA fragments 26
2.2 Connect DNA with particles 27
2.3 Optical setup 28
2.3.1 Trapping system 28
2.3.2 Detection system 30
2.4 Experimental method 32

Chapter .3 Experimental Procedure and Results 33
3.1 Calibration 33
3.2 Verification that the sample particles were connected to each other via DNA 37
3.3 Measurement of the elasticity of DNA 39
3.3.1 Fluctuation displacement of trapped particle 39
3.3.2 The elasticity of DNA as a function of its length 40

Chapter .4 Discussion 46

Reference 50

Ashkin, A. (1970). "Acceleration and Trapping of Particles by Radiation Pressure." Physical Review Letters 24(4): 156-159.

Ashkin, A., J. M. Dziedzic, et al. (1986). "Observation of a single-beam gradient force optical trap for dielectric particles." Optics Letters 11(5): 288-290.

Baumann, C. G., S. B. Smith, et al. (1997). "Ionic effects on the elasticity of single DNA molecules." Proceedings of the National Academy of Sciences 94: 6185-6190.

Bockelmann, U. (2004). "Single-molecule manipulation of nucleic acids." Curr. Opin. Struct. Biol 14: 368–373.

Bustamante, C., J. F. Marko, et al. (1994). "Entropic elasticity of lambda-phage DNA." Science 265(5178): 1599-600.

Florin, E. L., A. Pralle, et al. (1998). "Photonic force microscope calibration by thermal noise analysis." Applied Physics A: Materials Science & Processing 66: 75-78.

Grier, D. G. (2003). "A revolution in optical manipulation." Nature 424: 810-816.

Hegner, M., S. B. Smith, et al. (1999). "Polymerization and mechanical properties of single RecA-DNA filaments." Proceedings of the National Academy of Sciences 96(18): 10109-10114.

Liphardt, J. (2001). "Reversible Unfolding of Single RNA Molecules by Mechanical Force." Science 292(5517): 733-737.

Marko, J. F. and E. D. Siggia (1994). "Bending and twisting elasticity of DNA." Macromolecules 27(4): 981-988.

Meiners, J. C. and S. R. Quake (2000). "Femtonewton Force Spectroscopy of Single Extended DNA Molecules." Physical Review Letters 84(21): 5014-5017.

Perkins, T. T., D. E. Smith, et al. (1995). "Stretching of a single tethered polymer in a uniform flow." Science 268(5207): 83.

Sasaki, K., M. Tsukima, et al. (1997). "Three-dimensional Potential Analysis Of Radiation Pressure Exerted On A Single Microparticle." Quantum Electronics and Laser Science Conference, 1997. QELS'97., Summaries of Papers Presented at the: 69-69.

Shivashankar, G. V., M. Feingold, et al. (1999). "RecA polymerization on double-stranded DNA by using single-molecule manipulation: The role of ATP hydrolysis." Proceedings of the National Academy of Sciences 96(14): 7916-7921.

Smith, D. E., X. Z. Wu, et al. (1992). "Viscous finger narrowing at the coil-stretch transition in a dilute polymer solution." Physical Review A 45(4): 2165-2168.

Smith, S. B., Y. Cui, et al. (1996). "Overstretching B-DNA: The Elastic Response of Individual Double-Stranded and Single-Stranded DNA Molecules." Science 271(5250): 795.

Wang, D., T. I. Meier, et al. (1995). "Discontinuous movements of DNA and RNA in RNA polymerase accompany formation of a paused transcription complex." Cell 81(3): 341-50.

Wang, M. D., H. Yin, et al. (1997). "Stretching DNA with optical tweezers." Biophysical Journal 72(3): 1335-1346.

Wuite, G. J. L., R. J. Davenport, et al. (2000). "An Integrated Laser Trap/Flow Control Video Microscope for the Study of Single Biomolecules." Biophysical Journal 79(2): 1155-1167.

Yin, H., M. D. Wang, et al. (1995). "Transcription against an applied force." Science 270(5242): 1653–1657.