BIOL 142 Lecture Notes - Lecture 14: Restriction Site, Plasmid, Recombinant Dna
Biology 142- Lecture 14- Recombinant DNA Technology
Review of Horizontal Gene Transfer
• In an F+ to F- transfer turns the F- into F+
o This occurs via conjugation
o In this transfer, only the fertility factor is being transferred
o Other traits are rarely passed on in this transfer
• In an HFr to F- transfer, the F- remains F-
o This occurs vis conjugation
o In this transfer, the fertility factor is not transferred, but other traits can be
transferred
o After this transfer, the recipient can still participate in transfers with Hfr and F+
• Hfr has a higher rate of transfer because a larger volume of traits are being passed on to
the recipient
• F+ to F- has a lower rate of transfer because the only thing being transferred is the
fertility factor
• Auxotrophs have a mutation that prevents them from doing something
• Prototrophs don’t have a specific point mutation that prevents them from doing
something
• The difference between auxotrophs and prototrophs is a metabolic change
• Auxotroph point mutations
o Ability to break down/metabolize carbon sources (lac, glu, gal)
▪ An organism might not be able to metabolize a specific carbon source,
but it won’t kill them
▪ An alternative carbon source must be provided
o Amino acid synthesis
▪ If an organism can’t synthesize an amino acid, it must be provided in
order for the organism to survive
o Antibiotic/Drug resistance
▪ If an organism is sensitive to a drug and the drug is present on the
environment, it will kill the organism
Recombinant DNA Technology
• Recombinant: exchanges of genetic information
• Recombinant DNA technology requirements/tools
o Restriction enzymes
▪ Restrictions are needed for many biological processes
▪ These enzymes are responsible for cutting out genes at specific sites
▪ These enzymes can be used to cut out genetic material from different
sources
o Vectors/inserts
▪ Vectors are the delivery systems for packaging
▪ The type of vector depends on the organism and species
▪ Example: the plasmid is a form of vector
o Cloning
▪ Cloning makes a direct copy of the vector
• What happens?
o The gene of interest must be identified, or isolated
o The gene of interest must be inserted in a delivery system, or packaged and sent
o A system must be created to allow for genetic material intake
o Must ensure genetic material is recognized and cloned/expressed
Bacterial Transformation
• Bacterial transformation: naked DNA taken up by a prokaryotic cell
• Bacterial transformation requires for the cell to become competent
o Competence: the physiological state that allows for the uptake of DNA
o Competence allows DNA to get through the bacterial cell wall and plasma
membrane
• DNA is taken up and incorporates into the chromosome
• If DNA is a plasmid, it can exist independently within the cell
• This process is harder in eukaryotes because in order to get to the genetic material of a
eukaryotic cell, the naked DNA must pass through the plasma membrane and the
nucleus
Restriction Enzymes
• Restriction enzymes (also called restriction endonucleases): Specialized nucleotide
enzymes that cleave nucleic acid polymers
• Endonucleases: cleave on the inside
• Exonucleases: remove nucleotides from the outside
• Plasmids require endonucleases
• Straight DNA can use either endonucleases or exonucleases
• Why are restriction enzymes needed?
o 2 meters of DNA is unmanageable, and restriction enzymes allow the DNA to be
cut down to a manageable size
• Restriction endonucleases are able to identify a recognition site (also called a restriction
site)
Document Summary
In an f+ to f- transfer turns the f- into f: this occurs via conjugation. In this transfer, only the fertility factor is being transferred: other traits are rarely passed on in this transfer. In an hfr to f- transfer, the f- remains f: this occurs vis conjugation. If a(cid:374) orga(cid:374)is(cid:373) (cid:272)a(cid:374)"t synthesize an amino acid, it must be provided in order for the organism to survive: antibiotic/drug resistance. If an organism is sensitive to a drug and the drug is present on the environment, it will kill the organism. If dna is a plasmid, it can exist independently within the cell eukaryotic cell, the naked dna must pass through the plasma membrane and the nucleus. Building a recombinant plasmid using a restriction enzyme, ecori: target gene identified: Image from: https://www. khanacademy. org/science/biology/biotech-dna-technology/dna- cloning-tutorial/a/restriction-enzymes-dna-ligase: restriction enzyme cuts at the restriction site of the plasmid and the target gene: Image from: https://www. khanacademy. org/science/biology/biotech-dna-technology/dna- cloning-tutorial/a/restriction-enzymes-dna-ligase: the target gene is incorporated into the new plasmid.
