Steps In Genetic Engineering Lecture PDF

Steps in Genetic Engineering or Recombinant DNA Technology 1. Selection and Isolation Of DNA Insert First step in recombinant DNA technology (rDNA) is the selection of a DNA segment of interest which is to be cloned. This desired DNA segment is then isolated enzymatically. This DNA segment of intere...

Steps in Genetic Engineering or Recombinant DNA Technology 1. Selection and Isolation Of DNA Insert First step in recombinant DNA technology (rDNA) is the selection of a DNA segment of interest which is to be cloned. This desired DNA segment is then isolated enzymatically. This DNA segment of interest is termed as DNA insert or foreign DNA or target DNA or cloned DNA. 2. Selection of Suitable Cloning Vector A cloning vector is a self-replicating DNA molecule, into which the DNA insert is to be integrated. A suitable cloning vector is selected in the next step of rDNA technology. Most commonly used vectors are plasmids and bacteriophages. 3. Introduction of DNA-Insert into Vector to Form rDNA Molecule The target DNA or the DNA insert which has been extracted and cleaved enzymatically by the selective restriction endonuclease enzymes in step 1 are now ligated (joined) by the enzyme ligase to vector DNA to form rDNA molecule which is often called as cloning-vector-insert DNA construct. 4. Recombinant DNA Molecule is introduced into a Suitable Host Suitable host cells are selected and the rDNA molecule so formed step 3 is introduced into these host cells. This process of entry of rDNA into the host cell is called transformation. Usually selected hosts are bacterial cells like E. coli, however yeast and fungi may also be utilized. 5. Selection of Transformed Host Cells Transformed cells (or recombinant cells) are those host cells which have taken up the rDNA molecule. In this step the transformed cells are separated from the non-transformed cells by using various methods making use of marker genes. 6. Expression and Multiplication of DNA Insert in the Host Finally, it is to be ensured that the foreign DNA inserted into the vector DNA is expressing the desired character in the host cells. Also, the transformed host cells are multiplied to obtain sufficient number of copies. If needed, such genes may also be transferred and expressed into another organism.