In Vivo Cloning Flashcards
What are the two methods of cloning DNA fragments?
In vivo - by transferring the fragments to a host cell using a vector or in vitro - using the polymerase chain reaction
Once the DNA fragments have been obtained, what is the next stage?
They must be cloned so that there is a sufficient quantity for medical or commercial use
How are sticky ends joined after the DNA is cut by restriction endonucleases?
Using DNA ligase
What are the sequences of DNA that are cut by restriction endonucleases called?
Recognition sites
If the same restriction endonuclease is used to cut DNA, what will the fragments that are produced be?
Complementary to one another
If the fragments that are produced are complementary, what does this mean?
It means that the single stranded end of any one fragment can be joined to the single stranded end of any other fragment
Why are ‘sticky ends’ important, provided that the same restriction endonuclease is used?
We can combine the DNA of one organism with that of any other organism
Once the DNA fragment has been cut from the rest of the DNA, what must it be joined into in order to be transported into the host cell?
A carrying unit, known as a vector
What is the most commonly used vector?
Bacterial plasmids
What do all plasmids tend to contain?
Genes for antibiotic resistance
Where do restriction endonucleases break the plasmid loop?
At the antibiotic-resistance genes
What restriction endonuclease is used to cut open the plasmid loop, and why?
The same one that was used to cut out the DNA fragment to ensure that the sticky ends of the opened up plasmid are complementary to the sticky ends of the plasmid
What do the plasmids have once the DNA fragment is inserted into it?
Recombinant DNA
What is the name of the process by which the modified plasmid is reintroduced into bacterial cells?
Transformation
What does transformation involve?
The plasmids and bacterial cells are mixed together in a medium containing calcium ions
What makes the bacteria permeable, allowing the plasmids to pass through the cell membrane and into the cytoplasm?
The calcium ions and changes in temperature
Why will not all of the bacterial cells possess the DNA fragments?
Only a few of the bacterial cells take up the plasmids when the two are mixed together and some plasmids will have closed up again without incorporating the DNA fragment
What does the task of finding out which bacterial cells have taken up the plasmid involve?
It involves using a gene for antibiotic resistance, which is unaffected by the introduction of the new gene
Describe the process of using antibiotics to test for the uptake of a plasmid
All of the bacterial cells are grown on a medium that contains the antibiotic, the bacterial cells that have taken up the plasmids will have acquired the gene for antibiotic resistance, these bacterial cells are able to break down the antibiotic and therefore survive, the bacterial cells that have not taken up the plasmids will not be resistant to the antibiotic and therefore die
What is the purpose of gene markers?
They are used to identify whether a gene has been taken up by bacterial cells by using a second, separate gene
What makes the gene marker easily identifiable?
It may be resistant to an antibiotic, it may make a fluorescent protein that can be easily seen or it may produce an enzyme whose action can be identified
What is the name of the process where those cells that have taken up the new gene are identified?
Replica plating
What does replica plating involve the use of?
The other antibiotic resistance gene in the plasmid - the gene that was cut in order to incorporate the required gene
How does replica plating allow the bacterial cells that contain the DNA fragment to be identified?
The antibiotic resistant gene that had been cut will no longer produce the enzyme that breaks down that antibiotic, in other words the bacteria that have taken up the required gene will no longer be resistant to that antibiotic
What is the problem with replica plating?
The antibiotic that is used to identify the bacteria that contain the required gene will destroy those very cells
How does the process of replica plating identify the bacteria that contain the modified plasmid if the antibiotic kills those cells?
The cells that survive from the treatment with the first antibiotic are cultured and placed in agar plates that contain genetically identical colonies of which a sample is transferred to a replica plate that contains the second antibiotic
What is a more recent and more rapid method of identifying the successful uptake of a gene?
The transference of a gene from a jellyfish (GFP) which produces a green fluorescent protein into the plasmid
What is another gene marker that allows you to visually identify the successfully modified bacteria?
The required gene can be transplanted into a gene that makes lactase - an enzyme that will turn a particular colourless substrate blue
How does the use of enzyme markers such as lactase allow a successfully modified bacterial cell to be identified?
If the plasmid with the required gene is present in a bacterial cell, the colonies grown from it will not produce lactase, therefore, the colourless substrate will not be turned blue
