Lecture 5 Flashcards
What is gene cloning?
Cloning a single gene of interest and inserting it into a vector so that it can be copied quickly and its function and structure can be studied
What is gene cloning not like?
It is not like somatic cell nuclear transfer (what everyone thinks of when people say cloning eg dolly the sheep)
What is the first step when gene cloning?
Isolating the gene of interest from the chromosome in the host cell, this can be done using restriction enzymes or by using PCR
What happpens with the gene once it has been isolated from the chromosome in gene cloning?
The fragment of DNA needs to be placed into a vector
This vector is usually a bacterial plasmid
What does both the desired DNA fragment and the plasmid have to have been cut with to allow the joining of the two structures?
They both have to have been cut with the SAME restriction enzyme
What happens to the plasmid after the gene has been inserted into it?
It is placed into a bacterial cell such as e.coli that can then reproduce and produce many of the bacterial cell containing the desired gene
When can a gene be isolated using PCR?
If the gene has bee well studied and if it is under 5kb you can use PCR to amplify the DNA
What can you use to isolate a specific gene that cannot be amplified via PCR?
Restriction enzymes are used to digest the genome and then individual segments are cloned and reassembled
To act as a cloning vehicle what must something be able to do?
- The molecule must be capable of entering a host cell with the DNA molecule
- Once inside the cell they must be able to replicate and produce multiple copies of itself
What are the 2 types of naturally occurring types of DNA molecule that can be used as vectors?
- Plasmids
- Bacteriophages
What are plasmids and why are they useful during gene cloning?
- Extrachromosomal elements, double stranded and capable of replication
- Has an origin of replication and can multiply independent of the chromosome
- Easily isolated
- Easily manipulated
- High reproduction rate
What are the basic properties that a plasmid MUST have?
- An origin of replication - it is required to replicate independently
- A dominant selectable marker - this is required so that when screening etc the bacteria carrying the desired plasmid can be distinguished
- Unique restriction endonuclease cleavage sites - essential to allow the insertion of foreign DNA fragments into the plasmid
What happens when creating recombinant DNA molecules and what is it called?
It is called ligation
- Nucleases are used to cut, shorten or degrade nuclei acids
- Ligases are used to join nucleic acid molecules together
Why are restriction endonucleases used during gene cloning?
- Because they are very precise and and produce reproducible results (they are used because each vector molecule has to be cut at exactly the same position on the circle)
- They cleave the vector at a single position opening up the circle so that new DNA can be inserted
What do the restriction endonucleases and DNA lipase do when producing recombinant DNA?
- The restriction enzyme recognises a specific sequence in the DNA that is 4-8bp long
- On recognition of this site both strands of DNA become cleaved at that point because the restriction sites are short palandromic sequences
- The DNA is cut in a staggered manner leaving ‘sticky ends’ which can form H bonds with complementary sticky ends
- DNA ligase comes in and permanently binds the 2 complementary sticky ends together instead of them just being held by H bonds
How many types of restriction enzyme are there and which is the most commonly used?
There are 4 types of restriction enzyme and they type II restriction enzyme is the most commonly used
What can the palindromic sequence and the sticky ends also be referred to as?
- Axis of symmetry
- Two fold rotation of symmetry
How does DNA ligase bind the two molecules together?
It creates phosphodiester bonds between them
How is the recombinant plasmid inserted into the host cell?
- The bacterium and the plasmid are incubated together
- They are then chemically treated with CaCl2 which helps the transfer of the plasmid into the cell because the positively charged CaCl2 attracts the plasmid because of the negative charge on the DNA (the cell is now said to be a competent cell as it has the ability now to uptake the plasmid)
- The cells are then subjected to a heat shock which creates pores in the cell membrane allowing the plasmid to enter
- After the short heat shock the cells are subject to a low temperature to reseal the pores in the cell membrane (this cell is now said to be a transformed cell)
How do we screen the cells to make sure that the plasmid has been taken up and the transformation has been successful?
- The plasmids that were chosen as a vector must have had a selective marker, which usually is an antibiotic resistance gene
- An experiment is done then with the cells on an agar plate treated with that specific antibiotic and any cells that haven’t taken up the new plasmid will NOT grow on the plate (you will only see bacteria grow that has successfully taken up the plasmid)
How do we check that the ligation of the gene has been successful?
- We do this using another type of marker because the gene that is inserted into the plasmid is usually inserted into the middle of a second selective marker interrupting its function
- We can use the LacZ gene for blue-white screening (LacZ functions to break down lactose to glucose and galactose)
- The experiment to monitor this is to plate the cells alongside a chemical called X-Gal if the LacZ gene is still functioning then Beta galactosidase can break down X-Gal forming a deep blue colour - blue colonies therefore mean the plasmid is present but your gene isn’t
- A white colony will be present if the plasmid and the DNA have been successfully ligated
