- Me… I’ve always wanted a twin…
- My dog… I don’t want to share her with my twin afterall…
- Goose that lays the golden egg…
- PCR the gene you are interested in so you have a pure sample of lots of copies of it
- Get some plasmid to plop it into
- Cut (aka digest) both the PCR and plasmid DNA to give you sticky ends so you can…
- Glue (aka ligate) them together to make a circular plasmid
- Put the DNA into E. coli bacteria (aka transformation)
- Express the protein in E. coli
Each of these steps depends upon tools developed by people studying bacteria.
Cloning List Item #1: PCR Depends Upon a Bacterial PolymeraseIf bacteria didn’t live in extreme environments, we wouldn’t have PCR. PCR depends upon a thermostable DNA polymerase. In PCR cycles, the reaction mix is repeatedly raised to near-boiling temperatures in order to disassociate the DNA strands and enable a fresh cycle to begin. DNA polymerase from humans, or mesophilic bacteria for that matter, would be obliterated by this process. It is only because bacteria did the work for us of evolving a thermostable polymerase that we are able to do PCR at all.
Cloning List Item #2: Plasmids Come From BacteriaBacteria carry some of their DNA on plasmids, which are small bits of DNA. While the E. coli genome is 4,639 kb (kilobases) long, plasmids are typically less than about 15 kb long. Plasmids tend to contain DNA for surviving in specific environments, such as antibiotic resistance genes.
Cloning List Item #3: Restriction Enzymes Are a Primitive Bacterial Immune SystemRestriction enzymes, which are essential for cloning, come from bacteria. Restriction enzymes are used in bacteria as primitive immune systems. Like us, bacteria are susceptible to viruses, except in bacteria these viruses are called bacteriophage. Bacteria produce restriction enzymes to cut foreign, viral DNA.
Bacteria are able to differentiate their own DNA from foreign DNA by adding a small chemical modification to their genomic DNA. This modification is called methylation. Methylated DNA isn’t cut by restriction enzymes. Methylation means the addition of a methyl group (-CH3) to something—in this case genomic DNA. Eukaryotes methylate DNA too, but they do this in order to control gene expression, not to protect it from restriction enzymes cutting.