目的基因的获得（Acquisition of target genes）.docVIP

目的基因的获得（Acquisition of target genes） In a genetic engineering, it is usually necessary to obtain the target gene first, and if the sequence of the gene is known, it can be synthesized by chemical methods, or amplified by a template using a polymerase chain reaction (PCR). In addition, the most commonly used and no known sequence method is to establish a gene library or cDNA library, from which the target gene is selected for cloning. (1) construction of gene library A gene library is a complete set of molecular clones obtained by inserting genomic DNA fragments into a cloning vector. Under ideal conditions, the gene library should contain all the genetic information of the genome. Usually include the following five steps: Fragmentation of the 1. chromosome DNA: random fragments of chromosome genomes are produced by restriction enzymes that recognize short sequences, producing numerous DNA fragments. 2. vector DNA: proper lambda phage vector was selected, and the left and right arms were cut by restriction endonuclease to connect the two ends of chromosome DNA fragment. 3. in vitro ligation and packaging: the chromosome DNA fragment is linked with the vector DNA fragment with T4DNA ligase, and then the recombinant DNA is packaged in vitro with the lambda bacteriophage coat protein 4. recombinant phage infection of Escherichia coli: recombinant phage infected cells recombinant DNA into cells, recombinant DNA proliferation and lysis of host cells in the cell lysates generated form recombinant phage clone libraries, namely gene library. Identification, amplification and preservation of 5. gene library: the library of the constructed gene library should identify its library capacity and amplify it if needed. The constructed gene library can be used many times. (two) establishment of cDNA library; The structure and expression control elements of eukaryotic genes are very different from those of prokaryotes. Because of the mosaic of exons and introns, the transcribed RNA m