Quantitative high-resolution sensing of DNA hybridization using magnetic tweezers with evanescent illumination.
Quantitative high-resolution sensing of DNA hybridization using magnetic tweezers with evanescent illumination.
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DOI：
10.1039/c0nr00479k
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发表时间：
2011-02
期刊：
影响因子：
6.7
通讯作者：
Vezenov D
中科院分区：
文献类型：
--
作者：
Oliver PM;Park JS;Vezenov D
我们采用了渐逝纳米和力谱的结合方法，使用磁镊子来量化一个单一的合成单链DNA寡聚体的杂交程度，以接近一个单一的碱基的分辨率。在该设置中，将200个核苷酸长的DNA在一端共价连接到光学透明固体支持物的表面，并在另一端共价连接到超顺磁性荧光微球（力探针）的表面。使用电磁铁向探针施加力。从用全内反射显微镜观察到的探针荧光图像的强度确定端到端分子距离（即，力探针的图像平面外位置）。张力下的单链DNA分子的状态方程（可延伸的自由连接链）被用来推导渐逝场的穿透深度，并校准力探针的磁性。从状态方程获得的力探头的磁响应的参数保持不变时，改变穿透深度，表明一个强大的校准程序。这样的校准的结果也证实了使用独立测量的探针表面的距离安装到一个原子力显微镜的杠杆上的探针。在互补的50个核苷酸长的寡聚体的表面结合的200聚体的杂交，力-距离曲线的变化是一致的定量转化的25%的原始单链DNA的双链形式，这是模拟为一个弹性棒。这里提出的用于定量单个DNA分子的杂交状态的方法具有以高精度确定单个分子阵列中的单个分子的杂交程度的潜力。
We applied the combined approach of evanescent nanometry and force spectroscopy using magnetic tweezers to quantify the degree of hybridization of a single synthetic single-stranded DNA oligomer to a resolution approaching a single-base. In this setup, the 200 nucleotide long DNA was covalently attached to the surface of an optically transparent solid support at one end and to the surface of a superparamagnetic fluorescent microsphere (force probe) at the other end. The force was applied to the probes using an electromagnet. The end-to-end molecular distance (i.e. out-of-image-plane position of the force probe) was determined from the intensity of the probe fluorescent image observed with total-internal reflectance microscopy. An equation of state for single stranded DNA molecules under tension (extensible freely jointed chain) was used to derive the penetration depth of the evanescent field and to calibrate the magnetic properties of the force probes. The parameters of the magnetic response of the force probes obtained from the equation of state remained constant when changing the penetration depth, indicating a robust calibration procedure. The results of such a calibration were also confirmed using independently measured probe-surface distances for probes mounted onto cantilevers of an atomic force microscope. Upon hybridization of the complementary 50 nucleotide-long oligomer to the surface-bound 200-mer, the changes in the force-distance curves were consistent with the quantitative conversion of 25% of the original single-stranded DNA to its double-stranded form, which was modeled as an elastic rod. The method presented here for quantifying the hybridization state of the single DNA molecules has potential for determining the degree of hybridization of individual molecules in a single molecule array with high accuracy.
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影响因子：
56.9
作者：
Rief, M;Oesterhelt, F;Gaub, HE
通讯作者：
Gaub, HE
DOI：
10.1146/annurev.anchem.111808.073558
发表时间：
2010-01-01
期刊：
ANNUAL REVIEW OF ANALYTICAL CHEMISTRY, VOL 3
影响因子：
--
作者：
Efcavitch, J. William;Thompson, John F.
通讯作者：
Thompson, John F.
DOI：
10.1073/pnas.94.22.11935
发表时间：
1997-10-28
影响因子：
11.1
作者：
EssevazRoulet, B;Bockelmann, U;Heslot, F
通讯作者：
Heslot, F
单链 DNA 的单链弹性建模：三种模型的比较
影响因子：
4.6
作者：
Cui, Shuxun;Yu, You;Lin, Zhangbi
通讯作者：
Lin, Zhangbi
影响因子：
3.4
作者：
Abels, JA;Moreno-Herrero, F;Dekker, NH
通讯作者：
Dekker, NH