CAS OpenIR  > 中科院上海应用物理研究所2004-2010年
Alternative TitleOrdered Sequencing Strategy Based on the Single Molecule Manipulation Technology
Thesis Advisor胡钧
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核技术及应用
Keyword基因组测序策略 有序化测序 单分子pcr反应
Abstract随着DNA测序技术的发展和测序策略的完善,大规模基因组测序已不再是人们的梦想。目前的基因组测序策略大都建立在上世纪80年代美国科学家Sanger发明的鸟枪法基础之上,鸟枪法的随机性使它们都不可避免地带有测序冗余度高,片段拼接困难等症结。本研究提出一种基于单分子操纵技术的新型测序策略:有序化基因组测序策略。本策略彻底消除了Sanger的化学方法带来的随机性。首先将单个靶DNA序列拉直进行有序切割,再对每个片段分别进行PCR扩增并测序得到片段信息,之后将所有片段顺序组合,最终还原出整个目标基因组的一致序列。有序化测序使得后期的片段拼装工作高度简化,工作量随之大大降低。在整个测序过程中,单分子定位切割拾取技术以及单分子PC(Polymerase Cycle Reaction)技术都是其中非常关键的步骤,因此本研究同时也对单分子PCR反应的产物错误率以及扩增效率进行了计算及讨论。通过计算单分子PCR产物错误率,我们发现只要很好地控制反应条件,它的数值是在一个可以接受的范围之内,能够保证侧序的成功;而单分子PCR扩增效率我们认为它很可能要高于常规PCR的扩增效率,这一点还需要实验的进一步验证。
Other AbstractGenome sequencing has come true with the development of the DNA sequencing technologies and sequencing strategies. Current sequencing strategies almost are based on the Shotgun Strategy which was invented in 1980's by American scientist Sanger. Because of its high randomness, the Shotgun method's redundancy is very large and assembly work is very difficult and laborious. So we put forward a new strategy which bases on single molecule manipulation technology. It is Ordered Sequencing Strategy. Our strategy eliminates the randomicity of Sanger's chemical method. Firstly, single target DNA sequence is stretched and dissected into small segments orderly, then every segment is amplied by single molecule PCR experiment and sequenced to get sequence information. At last, all segments are combined into a consistent sequence in order. Ordered Sequencing Strategy greatly simplify the assembly work, so the workload of the genome sequencing project is reduced obviously. Single molecule dissection and picking-up technology and single molecule PCR technology are all crucial steps in the whole process. So we also have computed and discussed the error rate of single molecule PCR's product and its amplification efficiency. After the computation, we release that if the experiment conditions are controlled very well, the error rate is low enough to ensure the accomplishment of sequencing project. At the same time, we conside the amplification efficiency maybe is higher than general PCR's, but this conclusion must be proved by next step experiments.
Document Type学位论文
Recommended Citation
GB/T 7714
王国华. 基于单分子操纵的有序化测序策略研究[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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