[IEEE 2012 IEEE 12th International Conference on Nanotechnology (IEEE-NANO) - Birmingham, United Kingdom (2012.08.20-2012.08.23)] 2012 12th IEEE International Conference on Nanotechnology (IEEE-NANO) - Effect of buffer concentration for the dynamics of DNA stretching with the molecular combing

2012 12th IEEE International Conference on Nanotechnology (IEEE-NANO) The International Conference Centre Birmingham 20-23 August 20112, Birmingham, United Kingdom Effect of Buffer Concentration for the Dynamics of DNA Stretching with the Molecular Combing * Yuanyuan Yang, Kaige Wangt ,Qiwei Qiao, Dan Li, Shuang Wang, Zhaoyu Ren, Jintao Bai Institute of Photonics and Photonic Technology Northwest University, National Key Laboratory of photoelectric technology and Functional materials, National photoelectric technology and Functional Materials & Application of Science and Technology International Cooperation Base, Xi'an, 7lO069, China Email: wangkg@nwu_edu_cn Abstract -This paper experimented with the A-DNA molecules which were successfully stretched on the hydrophobic surface of PMMA by the molecular combing method in a wide concentration range from 7mM to 20mM of tris buffer. The buffer concentration has a remarkable influence on the properties of the stretched DNA such as the length and uniformity. The results demonstrated the most applicable concentration of tris buffer for A-DNA stretching is lOmM. Index Terms - molecule combing, buffer concentration, dynamics, single DNA molecule I. INTRODUCTION Molecular combing technology (MCT) has become an efficient technique in biophysics and biotechnology since 1994 first reported by Bensimon [1]. It is a user-friendly technique for the direct visualization and analysis of single DNA molecule. DNA is stretched uniformly and attached irreversibly to a specially-treated solid surface. This technology improves the structural and functional study of DNA modifications and makes it possible to explore the whole genome at a high resolution in a single molecular analysis. Many attractive results of DNA molecule with this method have been achieved [2-5]. In recent years, combined with other technologies, molecular combing method is widely used in biophysics, biotechnology and micro/nano devices. It has been become groundwork for small scale researches. The worked principle of the molecule combing method can be described as the following three steps [6]. The first step is the adherence of one end of the coiled molecule to the hydrophobic substrate. In this step, one end of melting DNA molecule is adsorbed onto the hydrophobic surface. Secondly, the fluid flow results in the stretching of the rest of this one-end-fIxed DNA molecule. When the DNA molecule move accompanied as the buffer removing along the surface (the receding liquid meniscus), the fluid force stretched them in the liquid. Thirdly, the stretched DNA molecules , including the no-fully stretched and over­ stretched, deposited on the surface in a good arrangement as combing over. Only when the external applied force was stronger than the elastic force restoring in the molecules, the DNA molecules could be fully stretched. According to the previous works [7-9], the [mal DNA­ stretched results were determined by the experimental conditions, for example, PH value of DNA solution, the hydrophobic degree of the surface, the speed of receding meniscus, as well as the properties of the buffer solution. As we known, buffer is a class of solution which could be employed to balance the acidity with basicity, keeping the PH value of buffer constant, and accurately simulating the natural environment under laboratory conditions, buffer acts as an extremely pivotal component in biological research and application [13-14]. Thus, it is essential to study the concentration of the buffer solution. However, the buffer concentration-dependent DNA stretching has not been studied in details according to the reports. In this paper, we have carried out some experiments of DNA stretching with different concentration of tris buffer on PMMA substrates with the molecular combing method, demonstrating that the uniformity of single stretched DNA, and the proportion of fully stretched DNA in the whole DNA observed are strongly dependent on the concentration of tris buffer solution. II. EXPERIMENTS The A-phage DNA (48.5kbp, Sino-American Biotechnology Company, China) were stained with the intercalating fluorescent dye YOYO-l (Molecular Probes Company, USA). The base pair to dye molecules ratio was kept at lO: 1 (bp/dye=10). The complex solution was diluted with tris buffer to different concentration (5, 7, lO, 12, 15, 20, * The project supported by the Major Research Plan of the Natural Science Foundation of China National (Grant No. 91123030), the National Science and Technology Major Project of the Ministry of Science and Technology of U.S and China cooperation(Grant No.20 IlDF A 1220), the Nature Science Foundation of Shaanxi (Grant Nos.SJ08F08, 08JZ66), the Key Science and Technology Program of Xi' an (Grant No. CXYI019-1) 25mM, pH�8) respectively, with pH �8.0 at 25°C room temperature. The final DNA concentration of the complex solution is 3.9pM. After the DNAIYOYO-l solution incubated about 30min in dark, droplets of the stained DNA molecules in different concentration were successively put on the prepared PMMA hydrophobic surface (Quartz plate covered with 15% PMMA solution) by pipette (GILSON, France) and observed with an inverted optical epi-ftuorescence microscope (IX-70,Olympus, Japan) equipped with a 60x objective and with a 100 W mercury arc lamp for illumination. Fluorescence images and videos were obtained using EMCCD (Evolve 512, ROPER USA) in real time. III. RESULTS AND DISCUSSION A. The orientation movement We observed and divided the whole DNA-stretching process into two steps according to the velocity of the liquid meniscus. That is, the balance stage and the motion stage. During the former, the droplet edge remain motionless about 3-5 mins. During the latter, the liquid drop continuous spontaneous recession. During the long relaxation time of the balance stage, molecules in different part of the droplet had various actions. Close to the liquid-air surface, DNA molecules rotated with anticlockwise which were likely due to the Northern Hemisphere effect; within the drop, a great mass of coiled DNA molecules staying at thermal equilibrium suspend, acting as the Brownian motion [9]. Near the solid­ air-liquid interface, some DNA molecules migrated toward the edge with an obviously directional movement, which is described in Fig.1. The disequilibrium environment was caused by four reasons, including a perturbation by the evaporation of liquid drop surface, the affinity between the molecules, the low entropy at the Liquid-solid interface lead the molecules "conscious" movement, and one end of DNA molecules maybe adhere to the hydrophobic closely. Figure 1. The movement of the DNA molecules during the eraly stage of combing. The long arrow shows the fluidic recessionary direction, and the short arrow shows the molecule's directional movement. B. The Length and Proportion of Stretched DNA Fig.2 shows the stretched DNA molecules with the molecular combing method. As mentioned above, the [mal result is determined by the experimental conditions such as PH value of DNA solution, the hydrophobic degree of the surface. In order to the comparison, we performed experiments with the same freshly peeled PMMA film, and drop samples (i.e. several drops of DNAIYOYO-l) with different concentration ranged from 5 to 25mM, PH value is �8.0. Figure 2. The stretched DNA molecules with MCT We took images in a circle of the droplet and measured the length of all the stretched DNA molecules. Table.l gives the statistics of several parameters of the stretched molecules. Clearly, DNA molecules could be stretched in a wide buffer concentration range from 7mM to 20mM, but the stretching quality was not the same. In the table 1, the X defined the average length of stretched DNA; Ix - 221 represented the difference between the contour length (the theoretical length 22llm) and the experiment data; and the .; = -J"L. �i - X : stood for the n variance that describing the uniform quality of the stretching, the lower �, the better uniform. According to the previous researches concluded, that the DNA molecules' length between x - 3 and x + 3 were assumed to be well stretched, to some extent, the ratio of x ± 3 shows the actually qualified rate in the overall level. As described in the table. 1 , the degree of DNA stretching in the 10mM solution is best. Under the condition, the average length of stretched DNA (�19.6Ilm) is closest to the theoretical contour length (22Ilm). And more, the DNA­ stretched ratio is as high as 71.43%, obviously higher than others. And furthermore, the variance is 0.238493, still better than that under other concentration. It is that the DNA molecules in the IOmM buffer could be most stretched fully and homogeneously. It is noted that the stretched ratio in 15mM is also as high as 71.88%, but it was unacceptable because the average length (l4.31Ilm) is far from the theoretical value. The stretched DNA molecules didn't be observed when the buffer concentration was lower than 7mM or higher than 20mM. When the concentration is lower, the liquid evaporation is quickly, the DNA molecules may not only one end bonded to the substrate but deposited on the solid surface as wound a ball. When the polymer coiling rate is higher than the fluid stretching rate, the molecules couldn't be stretched. On the other hand, when the concentration is much higher, the liquid evaporation seems too slow, and which result in the stretching impossible. molecules easily reunite together and stick to the substrate, Table. I The statistics of the stretched molecules. 7mM 10mM xf./m � 17.0065 19.5894 Ix-221 4.9935 2.4106 �Hi-X� 0.32478 0.238493 q= n Ratio of X ± 3 41.15% 71.43% C. Conformation Changed The conformation of a polymer in elongational flow will be deformed when the force across the molecule exceeds the entropic elasticity, and the polymer tends to coil due to the hydrodynamic friction. DNA deformation in elongational flow can be described with Deborah number [9]. De = E:r , where, T is the characteristic time that is necessary for Brownian motion to globally rearrange the polymer's conformation, i is the velocity gradient of the fluid. DNA molecules show coil conformation in a free state, but in elongational flow, they will changed to several shapes, different conformation has different De. We record the whole process of the motion stage and show the velocity-time curve of the liquid meniscus as the Fig.3. The velocity-time variation curve appears as a "w" shape under every concentration. In the same droplet, T is a constant but E: is not (shown in Fig. 2), so the De is a variable, corresponding to the different conformation. Thus, we could conclude that with the meniscus continually receding, the DNA conformation has changed. D. Buffer concentration-dependent velocity The distinctive velocity of different buffer concentration is also can be found in the Figure 3. The recession velocity is vibration severely, the velocity curve can be divided into three stages: The initial stage was from O� 12.5s, the effective stage was from 12.5 � 25s and the last stage was after 25s. In the initial stage, there were a few molecules stretched, and in the last stage, no-fully stretched molecules and united-molecules were predominant. The DNA stretching process occurred mainly in the effective stage, the fully-stretched DNA molecules were numerous and homogeneous. From Fig.3 and Table. 1 , we can found that the ratio of x ± 3 is consistent with the velocity of the effective stage, the qualified rate range is as 7mM lOmM/l5mM. The velocity as well as its change in lOmM and 15mM was the slowest. IV. CONCLUSIONS A-DNA molecules can be successfully fully-stretched along the surface of the hydrophobic PMMA under a wide 12mM 15mM 20mM 16.6136 14.31 13.26206 5.3864 7.69 8.73794 0.44861 0.524318 0.493004 44.92% 71.88% 57.14% range of concentration of tris buffer which is from 7mM to 20mM with molecule combing technology. (i) E 1.2 o 0.9 o � E ::::l :;; 0.6 � o iii > 0.3 10 20 Time(S) ---7mM -. 10mM • 12mM -",-15mM 20mM Figure 3. Recession velocity of DNA solution drop. The buffer concentration has a remarkable influence on the uniformity, number and average length of stretched DNA. 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