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

1.快速合成寡核苷酸我们探索了制备足以用于人类基因组PCR的寡核苷酸（19mer）的方法。为了解决与PCR产物的纯度和产量相关的问题，我们引入了一种半固相合成方法，其中底物寡核苷酸在延伸时在溶液中是自由的，而当未反应的试剂被洗出时，它们与头结合。关于这一点，得到了一些结果。从各种生物体中制备模板dna，我们进行了超过100种基因组分析组合，其中包括随机PCR过程。通过这些实验，我们可以证实随机PCR可以广泛用于在基因组DNA上设置锚定位点。我们还达成了一个新的概念，即随机PCR锚定可以在基因组测序中发挥主要作用，因为它需要有限数量的寡核苷酸（基于随机PCR的快速ROSOR方法）。利用大肠杆菌基因组DNA (4.7Mb)，构建了限制性内切酶Sau3AI部分酶切产物文库。利用该文库成功进行特异性PCR，生成目标DNA片段。因此，该结果证明了PCR接力的总体方案。相关技术开发的PCR在一个微小的同质物（0.01 μ l）是可能的。聚丙烯酰胺凝胶形成多微细胞（30个细胞/cm2），可用于PCR反应。开发了计算机程序来帮助分析大量序列数据（PRS；序列的平面表示），并被证明对序列数据分析是有用的。综上所述，物理非分裂策略ROSOR对大量基因组DNA进行测序是非常可行的，此外，基于随机PCR的测序被证明是非常有效的。

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.