Overview: DNA cloning
- DNA cloning is a molecular biology technique that makes many identical copies of a piece of DNA, such as a gene.
- In a typical cloning experiment, a target gene is inserted into a circular piece of DNA called a plasmid.
- The plasmid is introduced into bacteria via process called transformation, and bacteria carrying the plasmid are selected using antibiotics.
- Bacteria with the correct plasmid are used to make more plasmid DNA or, in some cases, induced to express the gene and make protein.
Overview of DNA cloning
Steps of DNA cloning
- Cut open the plasmid and "paste" in the gene. This process relies on restriction enzymes (which cut DNA) and DNA ligase (which joins DNA).
- Insert the plasmid into bacteria. Use antibiotic selection to identify the bacteria that took up the plasmid.
- Grow up lots of plasmid-carrying bacteria and use them as "factories" to make the protein. Harvest the protein from the bacteria and purify it.
1. Cutting and pasting DNA
- The plasmid, which has a single cut site
- The target gene fragment, which has a cut site near each end
2. Bacterial transformation and selection
3. Protein production
Uses of DNA cloning
- Biopharmaceuticals. DNA cloning can be used to make human proteins with biomedical applications, such as the insulin mentioned above. Other examples of recombinant proteins include human growth hormone, which is given to patients who are unable to synthesize the hormone, and tissue plasminogen activator (tPA), which is used to treat strokes and prevent blood clots. Recombinant proteins like these are often made in bacteria.
- Gene therapy. In some genetic disorders, patients lack the functional form of a particular gene. Gene therapy attempts to provide a normal copy of the gene to the cells of a patient’s body. For example, DNA cloning was used to build plasmids containing a normal version of the gene that's nonfunctional in cystic fibrosis. When the plasmids were delivered to the lungs of cystic fibrosis patients, lung function deteriorated less quickly.
- Gene analysis. In basic research labs, biologists often use DNA cloning to build artificial, recombinant versions of genes that help them understand how normal genes in an organism function.