chap four isolation of aim gene.ppt

Chapter 4 preparation and isolation of aim gene Section one the preparation of aim gene 一、直接分离法 1、限性核酸内切酶酶切分离法 We have known the DNA sequence, cleave the DNA with RE, isolate the DNA segments and recombined into a vector and transfer into receptor cell. 2、物理化学法 密度梯度离心法：The DNA segments are separated according to their size, then select the aim gene with probe 单链酶解法：there are 3 bonds in G/C pairs and 2 bonds in A/T pairs，the double strands rich of A/T pairs will be separated at a suitable temperature, but the double strands rich of G/C pairs will not be separated, then cleave the single strands with enzyme and get the G/C dsDNA 分子杂交法：fix the aim gene by hybridizing it with probe. 3、双抗体免疫法 The protein has been purified, it’s antibody1 has been prepared, the antibody2 has been prepared with antibody1. mRNA was expressed then the protein was synthesized, at that time there is a polymer which consist of mRNA、protein。 二、construct the genome library Extract the total genome DNA from some tissue segmentate the genome DNA with some physics methods(ultrasonic, mixing) or with enzymes( cleave with RE) to the size we hoped. Recombine the DNA segments with a suitable vector (λphage, Cosmid ,YAC), then transfer into receptor cell. One receptor cell receive one recombination DNA molecule which consist of one DNA segment and the vector，all of the cells reproduced from one receptor cell contain the same DNA segment. 1、definition of genome library A collection of living bacteria colonies that have been transformed with different pieces of DNA from the organism that is the source of the gene of interest. 某种生物的基因组的遗传信息通过克隆载体贮存在一个受体菌的群体之中，这个群体即为这种生物的基因组DNA文库（genomic DNA library） If a library is to have a colony of bacteria for every gene, it will consist of tens of thousands of clones. 2、基因文库的大小 一个理想的基因组文库应该是在克隆群体中包含完整基因组的所有DNA序列。 DNA片段要有足够的长度，保证含有某个完整的基因序列。 估算：基因组文库的克隆数目=基因组DNA总长/DNA片段的平均长度。 如：3×106 kb/15kb=2×105 经验公式：N=ln(1-p)/ln(1-f) N为基因组文库的克隆数目，P为文库中含有的目的基因片段出现的概率，f是插入片段的大小与