Wize AP Biology Textbook > Biotechnology
Gene Cloning
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Overview of Gene Cloning
Gene cloning can be done when scientists want a gene to be expressed in a bacterial host. This is done in a series of steps:
- Amplify gene of interest
- Put gene in plasmid
- Put plasmid in bacteria
- Bacteria expresses gene
- Purify protein
Step 1: PCR with restriction sites
Restriction sites are sequences of DNA that are recognized by specific enzymes (endonucleases). These sites can be added to the gene of interest by designing primers.
- Primers partly match the DNA to be amplified.
- The "hanging end" has the sequence we want to introduce and ends up amplified during PCR.
Step 2: Digestion and ligation
Plasmid and PCR product are cut with restriction enzymes or endonucleases (REs).
- Digestion with restriction enzymes creates complementary sticky ends.
- DNA ligase sticks them together.
Steps 3 & 4: Introduction of DNA in bacteria and gene expression.
Plasmids are inserted in bacterial cells.
- Bacteria transcribe and translate the gene.
- Protein is produced and can be purified.
