《园艺植物生物技术》第二节 基因克隆操作.ppt

主要参考资料 基因 VIII 基因操作原理 孙明 华中农业大学 植物基因工程 王关林 方宏均 科学出版社 2002 Molecular Cloning 分子克隆实验指南（E3,2001） Principles of Gene Manipulation (S Primrose, R Twyman, Blackwell scientific publication) 在已知基因组序列的基础上可以不构建物理图谱。仅仅进行精细定位，最后用PCR的方法克隆基因。 * 水稻 1cM=270kb, 人类 1cM=1000kb * RIL: recombinant inbrid line. DH: doubled haploid NIL: near isogenic line PAC 载体以F 因子和噬菌体P1 为基础构建,兼有二者的特点，通过电激穿孔转化可将PAC 导入大肠杆菌。其特点是插入的外源DNA 没有明显的嵌合和缺失现象，PAC 载体可以插入约300 kb 的外源片段，可以稳定遗传及高效扩增。 YAC可以克隆 200-500kb 在RFLP作图中，连锁距离是根据重组率来计算的， 1cM（厘摩）相当于1%的重组率。人类基因组中， 1cM≈1000kb；拟南芥菜中，1cM≈290kb；小麦中， 1cM≈3500kb。 * 在RFLP作图中，连锁距离是根据重组率来计算的， 1cM（厘摩）相当于1%的重组率。人类基因组中， 1cM≈1000kb；拟南芥菜中，1cM≈290kb；小麦中， 1cM≈3500kb。 * The mRNA Differential Display technique works by resolving the 3 terminal portions of mRNAs on a DNA sequencing gel. Using a primer designed to bind to the 5 boundary of a poly A tail for the reverse transcription, followed by PCR amplification with additional upstream arbitrary primers, mRNA sub-populations are visualized by denaturing polyacrylamide gel electrophoresis. This allows direct side by side comparison of the mRNAs between two or more related cells. The Differential Display method has the potential to visualize all the expressed genes (about 10,000 to 15,000 mRNA species) in a mammalian cell by using multiple primer combinations. More importantly, the new method enables a researcher to recover their sequence information and use them as probes to isolate their cDNA and genomic DNA for further molecular characterizations. Because of its simplicity, sensitivity, and reproducibility, the mRNA Differential Display method is finding wide rang and rapid application in developmental biology, cancer research, pathology, plant physiology and other biological systems The mRNA Differential Display technique works by resolving the 3 terminal portions of mRNAs on a DNA sequencing gel. Using a primer designed to bind to the 5 boundary of a poly A tail for the reverse transcription, followed by PCR amplification with a