怎样获取DNA序列？.ppt

怎样获取DNA序列？(如何享受HGP的成果） / NCBI: national center for biotechnology information. General procedure for cloning a DNA fragment in a plasmid vector. Although not indicated by color, the plasmid contains a replication origin and ampicillin-resistance gene. Uptake of plasmids by E. coli cells is stimulated by high concentrations of CaCl2. Even in the presence of CaCl2, transformation occurs with a quite low frequency, and only a few cells are transformed by incorporation of a single plasmid molecule. Cells that are not transformed die on ampicillin-containing medium. Once incorporated into a host cell, a plasmid can replicate independently of the host-cell chromosome. As a transformed cell multiplies into a colony, at least one plasmid segregates to each daughter cell. (Molecular Cell Biology) Cloning by cosmids. The cosmid is cut at a BglII site next to the cos site. Donor genomic DNA is cut by using Sau3A, which gives sticky ends compatible with BglII. A tandem array of donor and vector DNA results from mixing. Phage is packaged in vitro by cutting at the cos site. The cosmid with insert recircularizes after it is in the bacterial cell. (From J. D. Watson, M. Gilman, J. Witkowski, and M. Zoller, Recombinant DNA, 2d ed. Copyright ? 1992 by Scientific American Books.) (An Introduction to Genetic Analysis) 粘粒（cosmid）： 物理图谱的意义： 物理图谱是进行DNA序列分析和基因组结构研究的基础。 （三）转录图谱（transcription map）。 构建转录图的前提条件是获得大量基因转录的mRNA，通过逆向转录，即可得到cDNA，表达序列标鉴（expressed sequence tag，EST），为部分cDNA序列，300-500 bp。 将mRNA和EST与基因组DNA序列对比，就可找出同源序列，再将其进行标示即成转录图谱。 转录图谱的意义 1、能为估计人类基因的数目提供可靠的依据。 2、提供不同组织、不同时期基因表达的信息（数目、种类及结构功能）。 3、提供结构基因的标记，可以作为筛选基因的探针。 （四）序列图谱 最终目标：30亿bp的全序列图。 起始材料：跨叠克隆群（contig） 全序列测定的基本路线： 1、将所有染色体DNA片段一次性克隆进入YAC或BAC 人工染色体，建立相连片段群。 2、先用流式细胞仪将24条染色体逐一分开，然后将其分 别克隆，建立染色体特异的相连片段群。 3、利用STS或EST将各克隆排序、定位。 4、大规模DNA序列分析。 1、逐个克隆法（clony by clony）： 将YAC、BAC克隆逐一序列分析，然后排列组装。 2、鸟枪法（shotgun approach）： 随机挑取克隆测序，然后通过计算机排序并连接，Celera公司首创。 大规模测序的基本策略 （五）基因的鉴定和分析