1. Selection and Isolation of Target DNA: Target DNA is selected for gene cloning. Selected cells are slightly lysed. Cell membranes are broken down using enzymes lysozyme (bacterial cells), chitinase (fungal cells), cellulase (plant cells). The lysed cells are then centrifuged into test tubes. An amount of cesium chloride solution is introduced into a centrifuge test tube. The solution is then centrifuged. As a result, a band of DNA is formed on the test tube. DNA is mixed with other components to form a homogenate. Other components are separated from homogenate DNA using enzymes like protease (protein), ribonuclease (RNA), amylase (sugar), lipase (fat). The purified DNA is then precipitated as a thread by immersing in a cold ethanol solution. The desired DNA segment is selected from the degraded DNA.
2. Carrier Selection: The carrier is selected to carry the required portion of the target DNA. In this case, the bacterium Agrobacterium tumefaciens acts as a good carrier. The desired DNA segments are attached to the plasmid DNA located in the cytoplasm of these bacteria. In some cases carriers such as cosmids, phagemids, artificial chromosomes etc. are used.
3. Excision of the target DNA at a specific location: Several sections are cut from the target DNA using restriction enzymes. Corresponding segments are also excised from the carrier plasmid DNA using the same enzyme. This process is called restriction digestion.
4. Preparation of recombinant DNA: The target DNA is ligated to the carrier plasmid DNA with the help of an enzyme called DNA ligase. Recombinant DNA is produced by ligation of the desired DNA with the carrier plasmid DNA.
5. Introduction of Recombinant DNA into the Host: The host is selected to carry the recombinant DNA. In this case E. coli bacteria act as host. Recombinant DNA is introduced into host cells. But under normal conditions bacteria do not accept other plasmids. If the culture medium in which the bacterium is grown is heated and a suitable environment is created by adding calcium, the bacterium takes up another plasmid. The process of introducing recombinant DNA into the bacterial body is called transformation.
6. Amplification or Cloning of Recombinant DNA: After the recombinant DNA is introduced into the host, the host bacteria are grown in culture medium. Within a short period of time, the bacteria multiply in the culture medium to produce thousands of copies. Recombinant DNA is also produced. Thus increasing the number of recombinant DNA is called gene cloning.
