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Pilot Experiment to Demonstrate the Feasibility of a Strategy ROSOR for Human Genome Project.

展示人类基因组计划 ROSOR 策略可行性的试点实验。

基本信息

批准号：
06454661
负责人：
NISHIGAKI Koichi
金额：
$ 4.1万
依托单位：
Saitama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06454661/
关键词：
ROSOR method Rondom PCR Tetramer Library Enzymatic Block Synthesis Human Genome Project Base Sequencing PCR Relay Planar Representation of Sequence 酵素的ブロック合成配列平面表示法酵素合成ダイレクトシーケンシングオリゴヌクレオチド

项目摘要

1.Rapid synthesis of oligonucleotides We have explored the way to make oligonucleotide (19mer) which are sufficient to be used for PCR applied to human genome. In order to settle the problem related to purity and yields of the PCR products, we introduced a semi-solid phase synthesis in which substrate oligonucleotides are free in solution when they are extended while they are bound to a bead when unreacted reagents are washed out. Some results were obtained as to this.2.Random PCR Preparing template DNAs from various organisms, we performed more than 100 combinations of genome profilings which include a random PCR process. Through these experiments, we can obtain confirmation that random PCR can be widely used for the purpose of setting anchoring sites on a genome DNA.We also reached to a new concept that anchoring by random PCR can play the major role in genome sequencing since it needs a limited number of oligonucleotides (Random PCR-based fast ROSOR method).3.Demonstration of PCR relay Using E.coli genome DNA (4.7Mb), a library of restriction enzyme Sau3AI partial digestion products was contructed. Specific PCR was successfully carried out with this library, generating the aimed DNA fragments. Thus, overall scheme of PCR relay was demonstrated by this result.4.Related techniques developed PCR in a tiny aliquot (0.01mul) was possible. Multi-microcells (30 cells/cm2) were formed out of polyacrylamide gel and were usable for PCR reactions. Computer programs to help analyze voluminous sequence data were developed (PRS ; Planar representation of sequence)and were shown to be useful for sequence data analyzes.In conclusion, physically-non-dividing strategy, ROSOR,to sequence voluminous genome DNA was demonstrated to be quite feasible, and, in addition, random PCR based-sequencing proved to be highly effective.

1.寡核苷酸的快速合成我们探索了制备足以用于人类基因组PCR的寡核苷酸（19聚体）的方法。为了解决与PCR产物的纯度和产率相关的问题，我们引入了半固相合成，其中当底物寡核苷酸延伸时，它们在溶液中是游离的，而当未反应的试剂被洗出时，它们结合到珠上。2.随机PCR从各种生物体中制备模板DNA，我们进行了100多种组合的基因组分析，其中包括随机PCR过程。通过这些实验，我们证实了随机PCR可以广泛地应用于基因组DNA锚定位点的设计，并得出了一个新的概念，即随机PCR锚定技术可以在基因组测序中发挥重要作用，因为它所需的寡核苷酸数量有限（基于随机PCR的快速ROSOR法）3. PCR接力的证明利用大肠杆菌基因组DNA（4.7Mb）构建限制性内切酶Sau 3AI部分酶切产物文库。利用该文库成功进行了特异性PCR扩增，得到了目的DNA片段。4.相关技术的发展使微量PCR（0.01 μ l）成为可能。由聚丙烯酰胺凝胶形成多个微细胞（30个细胞/cm 2），并可用于PCR反应。计算机程序（PRS ; Planar representation of sequence）被开发用于分析大量的序列数据，并被证明是有用的序列数据分析。总之，物理非分割策略（ROSOR）被证明是非常可行的，并且，另外，基于随机PCR的测序被证明是非常有效的。