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Labeling DNA Probes03:31

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DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
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可调节的化DNA探针驱动快速和高分辨率的单分子光成像.

Mirjam Kümmerlin1,2, Qing Zhao1,2, Jagadish Hazra1,2

  • 1Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom.

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概括

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科学领域:

  • 生物物理学的生物物理.
  • 分子生物学分子生物学
  • 纳米技术纳米技术

背景情况:

  • 单分子光 (SMF) 测量受到"高度屏障"的限制,它限制了光物种的最大可用的度,以获得最佳的信号噪声比.
  • 现有的SMF技术在达到高度方面存在局限性,这阻碍了需要密集标签或快速事件获取的应用.

研究的目的:

  • 设计和设计新的化探针,以克服SMF中的高度障碍.
  • 调整探头设计以提高光和高效的杂交,以达到目标序列.
  • 为了证明这些探头在超分辨率显微镜等先进的成像技术中的实用性.

主要方法:

  • 开发基于短单链DNA序列的化探针,这些短单链DNA序列在杂交时会光.
  • 灭效率和光增强的工程通过选光剂-灭器组合,标签长度和序列图案.
  • 在全内部反射光显微镜 (TIRF) 和DNA-PAINT超分辨率成像协议中实施探针.

主要成果:

  • 在10μM光标签度下实现了SMF实验,比标准TIRF限制增加100倍.
  • 通过使用DNA-PAINT.成功地在约150秒内以20纳米分辨率对病毒基因组段进行超高分辨率成像.
  • 验证了探头设计的特殊可调性,以满足各种实验需求.

结论:

  • 开发的化探针有效地克服了SMF中的度限制.
  • 这些探测器显著提高了超高分辨率成像技术的速度和分辨率,如DNA-PAINT.
  • 探测器的设计为推进分子跟踪,smFRET和其他基于SMF的应用提供了广泛的应用.