CAS OpenIR  > 中科院上海应用物理研究所2004-2010年
面向分子手术的DNA单分子的纳米操纵研究
Alternative TitleTowards molecular surgery with nano-manipulation of single DNA molecules
吕军鸿
Subtype博士
Thesis Advisor胡钧
2004
Degree Grantor中国科学院上海应用物理研究所
Place of Conferral中国科学院上海应用物理研究所
Degree Discipline无机化学
Keyword分子手术 原子力显微镜 Dna单分子 纳米操纵 定位拾取 测序策略
Abstract“分子手术”是通过光学、力学或酶学原理在纳米尺度和单分子水平实现对生物分子的各种人工改造。本文旨在基于原子力显微镜(AFM)对DNA分子进行精确的纳米操纵,解决“分子手术”中的若干关键技术,并探索其在生物学中的应用。解决了“分子手术台”的相关问题。在对DNA样品进行进一步纯化后,分别以对DNA吸附强度比较弱的Ni+修饰衬底和以高温钝化处理来降低APS一云母吸附力,为下文的操纵做准备。为适合DNA样品以弱吸附力沉降在表面的要求,选择作用力较小的离心法作为拉直手段,拉直了长链入DNA分子和较短pBR322片段。建立了一种从基因组DNA中纳米定位切割和分离DNA片段的技术。基于AFM的interleave扫描中的抬高模式,在成功实现了对DNA分子进行切割、局域拉伸、诱导折叠等操纵之后,调节针尖与样品之间的距离来精细地控制针尖与样品针尖的相互作用,从而实现了对DNA分子的纳米切割和拾取,并对拾取的单个DNA分子进行了单分子PCR扩增。探讨了拾取的可能机制。提出了一种有序化纳米测序的新策略。基于所发展的单分子纳米操纵技术,结合常规的凝胶测序方法,提出了可对DNA单分子进行有序化测序的方法和两种组合策略。与传统的测序方法比较,具备用时少、操作简便等优点,并且在解决序列拼接、无法克隆片段等问题上有独特的优势。
Other Abstract"Molecular surgery" means " modifying biomolecules with nanometer resolution at the single molecule level in the principle of optics, mechanics or enzymology". Our motivation is to realize precise manipulation of single DNA molecules using atomic force microscopy, to develop some key techniques, and to explore its possible applications in biology. Some issues involved in "operating table" were figured out. After further purification of the DNA molecules, substrates for DNA manipulation were prepared by modifying the mica with nickel ion or further treating the 3-aminopropyl triethoxysilane modified mica under a temperature of 120 ℃ for more than 4 hours. To match the fact that DNA should be weakly deposited on the solid surface, centrifugal method was used to stretched long strands of A DNA as well as pBR322 DNA. A unique technique for the positioning dissection and isolation of single DNA molecules by using atomic force microscopy (AFM) has been demonstrated. Based on lift mode in the interleave scan, single DNA fragment was dissected from one of that genome DNA strands with the AFM tip at the desired position, and then isolated (or picked up) after a special operation called "kneading". All the operations including imaging, dissection and isolation could be carried out with one tip. The isolated DNA fragment on the AFM tip could be successfully amplified by single molecule PCR. Some of the physical and chemical mechanisms have been discussed. A new strategy for DNA sequencing in order was proposed. Ordered sequencing strategy. Combined with traditional sequencing methods based on gel electrophoresis, the nanomanipulation technique of single DNA molecules was applied to sequencing the genome sequences. Two hybrid strategies were also brought forward. Compared to traditional sequencing methods, our strategy has some obviously advantages, such as less time and cost, more simple, apt to assembly, and so on.
Language中文
Document Type学位论文
Identifierhttp://ir.sinap.ac.cn/handle/331007/7385
Collection中科院上海应用物理研究所2004-2010年
Recommended Citation
GB/T 7714
吕军鸿. 面向分子手术的DNA单分子的纳米操纵研究[D]. 中国科学院上海应用物理研究所. 中国科学院上海应用物理研究所,2004.
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