合成双链cdna分子.pptVIP

第二讲　目的基因的制备 一、从基因文库中获得目的基因 The DNA molecules of an organism in interest are isolated. The DNA molecules are then partially digested by an endonuclease restriction enzyme. Sometimes, the DNA molecules are digested to different lengths of time in order to ensure that all the genes have been digested to manageable sizes. The hosts are kept in liquid media and can be frozen at -80°C for a long period of time. Usually the hosts are bacteria that do not contain any plasmids so as to be sensitive to antibiotics. ⑵制备全部基因组的DNA片断 ①机械断裂法 用超声波或高速搅拌DNA溶液，断裂片段两端没有与克隆载体匹配的粘末端, 因此插入载体之前还需要进行修饰加工，少用 ②限制性内切酶降解（鸟枪法） 过去用EcoRⅠ,现在用Sau3A，断裂片段两端有与克隆载体匹配的粘末端，可以直接与处理过的载体连接。 常用载体：粘性质粒，λ噬菌体，酵母人工染色体 ①基因组DNA +粘性质粒——重组DNA——转化细菌 ②基因组DNA + λ噬菌体 ——重组DNA—包装成噬菌体——感染细菌 　从人基因组DNA（总长度为3×109bp）的文库中筛选到长约1.5kb目的基因的可能性为99％，那么这个基因组文库要多大？ 理论克隆数：3×109 / 1.5 ×103＝2×106 　　　　　　　　　　　　　　In（1－0.99） 实际克隆数 ： N = ——————————————— = 9.2×106 　　　　　　　　In （1－1.5×103／3×109） 用质粒（pBR322）克隆目的基因（1.5kb）需要有9×106克隆才能汇集人基因组全部DNA序列;若 用λ噬菌体载体可构建含15kb目的基因的人基因组DNA文库，按上式计算所需要的重组体克隆数可以减少到9×105, 而要用柯斯质粒将40kb目的基因片断所需要重组体克隆数可降到3.5×105左右,均可满足建库要求。 2. cDNA library In genetics, complementary DNA (cDNA) is DNA synthesized from a mature mRNA template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to clone eukaryotic genes in prokaryotes. The central dogma of molecular biology outlines that in synthesizing proteins, DNA is transcribed into mRNA, which is translated into protein. One difference between eukaryotic and prokaryotic genes is that eukaryotic genes can contain introns (intervening sequences), which are not coding sequences, and must be spliced out of the RNA primary transcript before it becomes mRNA and can be translated into protein. Prokaryotic genes have no introns, so their RNA is not subject to splicing. Often it is desirable to express eukaryotic genes in prokaryotic cells. A simplified method of doing so would include the addition of eukaryotic DNA to a prokaryotic