Recombinant DNA Flashcards
What is recombinant DNA?
DNA molecules formed by human intervention in the lab or genetic recombination of genetic material from different sources.
Resulting sequences are not found in nature in these combos.
What is DNA cloning?
The isolation of a DNA fragment (from any species) and its propagation without alteration of the original DNA sequence.
You can use this for protein production etc. as it is in a real life cell.
What are the basics steps of DNA cloning?
- Isolation of target DNA fragments (often referred to as inserts)
- Ligation (addition) of inserts into an appropriate cloning vector (e.g., plasmids - amplifying the DNA in this way), creating recombinant molecules
- Transformation of recombinant plasmids into bacteria (these then amplify the vectors by division) or other suitable host for propagation
- Screening/selection of hosts containing the intended recombinant plasmid (e.g., can add in antibiotic resistance)
What is a vector in genetic cloning?
A DNA molecule that can be used to transfer a DNA molecule into a host cell (often bacterial cells are used as hosts) - e.g., bacterial plasmids
Why are bacterial plasmids typically used for genetic cloning?
- They have specific controls for elements for replication and expression (autonomous replication)
- Plasmids can be transferred to other bacteria asexually (this spreads properties throughout a colony)
What do plasmids used for cloning normally contain (include the components necessary for protein expression too)?
- Replication origin
- Cloning sites (MCS): sequences that are recognised by restriction enzymes
- Selectable markers to show which hosts contain the recombinant plasmid (e.g., antibiotic resistance)
- Promoter region (upstream of the MCS) - expression vectors for when protein production is required
- A start codon
- Polyhistidine-coding sequences (these facilitate protein purification and detection)
- A stop codon
What are the purpose of multiple cloning sites (MCSs)?
Sequences which are engineered to have a known pattern of restriction enzymes, allowing the vector to be cut open in a defined manner for insert addition.
How can you control protein production in recombinant plasmids?
A promoter region can be added to “trick” the bacteria into producing this protein.
This protein can then be purified and detected using polyhistidine amino acids (also known as His tags)
How can proteins produced via recombinant plasmids be selectively purified?
By adding a histidine tag after the start codon (ATG).
This histidine tag (6 histidine amino acids) binds to metal ions, which can then be used to “pull” the protein of interest out of the mixture. This process is known as Immobilises Metal Affinity Chromatography (IMAC)
How are the target DNA and plasmid digested?
Using restriction enzymes. This cuts the strands, forming either:
- Sticky ends with overhangs at the 3’ or 5’ (which make annealing easier as no covalent bonds are required)
- Blunt ends (these mean that the fragment can be inserted in either direction, but annealing is harder)
How does ligation occur?
The enzyme ligase directs the DNA fragments to the open vector (digested using restriction enzymes), connecting at the sticky or blunt ends.
How does transformation occur?
Now that the recombinant plasmid has been formed, it is ready to be added into the host cell.
There are two main techniques:
1. Chemical transformation (heat shock, heating from frozen to overheated and causing membrane rupture, allowing plasmids at high concentrations outside of the cell to diffuse into the bacterial cells)
2. Electroporation (a small amount of bacterial solution is pulsed with an electric current, once again rupturing the membrane and allowing the plasmids to diffuse down the concentration gradient into the bacterial cells)
The cells are then allowed to recover.
How are bacterial clones containing the required recombinant DNA selected for?
- Using an antibiotic (the cells with antibiotic resistance will survive and these are the ones containing the recombinant DNA)
This produces a colony composed of only bacterial cells with the recombinant plasmid in.
- Depending on the function of the recombinant DNA, some can either produce different visible characteristics, or change the function of the cell (e.g., bacterial cells which produce blue pigments, but this is disrupted upon recombinant protein addition and therefore stay white) -> this is known as Blue/white colony screening
How can plasmids for ligation be produced?
- Plasmid mini-prep can be used to purify and digest the required plasmid
- Success can be determined using gel electrophoresis
