In vivo gene cloning Flashcards
What is meant by in vivo gene cloning
Transferring the fragments to a host cell using a vector
Why are restriction endonucleases important in recombiant DNA
It allows sections of DNA cut with restriction endonucleases to bind with other DNA fragments that have been cut with the same restriction endonuclease, provided they both produce sticky ends and thus have complementary recongition sites.
5 stages of gene transfer and cloning
Isolation
Insertion
Transformation
Identification
Growth/Cloning
Preparation of the DNA fragement for insertion process
Promoter and terminator need to be added to the DNA fragment
What is a promoter and why is it needed for in vivo gene cloning
A region of DNA that is the binding site of RNA polymerase.
Allows the transcription of the gene to take place by allowing RNA polymerase to synthesise mRNA
What is a terminator and why is it needed in in vivo gene cloning
The region of DNA that releases RNA polymerase and ends transcription. Needs to stop transcription at the appropriate point when the desired protein is made
Insertion of DNA fragment into a vector process
Vectors are used to transport DNA into the host cell.
Plasmid is commonly used
Restriction endonuclease is used to break the plasmid loop
Restriction endonuclease used is the same one used to cut out the DNA fragement, ensuring sticky ends of plasmid are complementary to DNA fragment.
When they are incorparated, DNA ligase is used to form the phosphodiester bonds between fragment and plasmid
What is DNA ligase
An enzyme that forms phosphodiester bonds between DNA nucleotides after using restriction endonuclease.
How is the plasmid transformed into the bacterial host?
Plasmids and bacterial cells are mixed in a calcium ion containing medium with temp changes.
This alters permeability of the cell membrane, allowing plasmids to pass into the cytoplasm
Why do some bacterial cells not possess the DNA fragments?
Only a few bacterial cells take up the plasmid when mixed.
Some plasmids may have closed up without incorporating the fragment
Some DNA fragments may have formed their own plasmid
How are the host cells containing desired gene identified?
Can be identified using marker genes
HOw are bacterial cells that have taken up a plasmid identified, use ampicillin example.
All bacterial cells grown on medium containing ampicillin antiobiotic
Bacterial cells that take up plasmic aquire ampicillin resistance
Bacterial cells that have taken up plasmid therefore will not die, others will.
What are the types of marker genes
Antibiotic resistance markers
Fluorescent markers
Enzyme markers
How do antibiotic resistant markers work?
Uses another antibiotic resistant gene in the plasmid. If the antibiotic gene was cut in order to incorporate the new gene, the resistance will no longer work.
Bacteria that die in the antibiotic medium are the host bacteria. Replica plating is used to ensure the whole population is not killed off.
How do fluroescent markers work?
Organisms that contain genes that cause fluorescence are chosen. The desired gene is incorporated in the centre of the fluorescing gene.
When organisms are viewed under microscope, those that do not have fluorescent characteristics have take up the gene
